WO2023092136A1

WO2023092136A1 - Effector proteins and uses thereof

Info

Publication number: WO2023092136A1
Application number: PCT/US2022/080278
Authority: WO
Inventors: Aaron DELOUGHERY; Benjamin RAUCH; Clarissa Oriel RHINES; Fnu YUNANDA; Stepan TYMOSHENKO
Original assignee: Mammoth Biosciences, Inc.
Priority date: 2021-11-22
Filing date: 2022-11-21
Publication date: 2023-05-25

Abstract

Provided herein are compositions, systems, and methods comprising effector proteins and uses thereof. These effector proteins may be characterized as CRISPR-associated (Cas) proteins. Various compositions, systems, and methods of the present disclosure may leverage the activities of these effector proteins for the modification, detection, and engineering of nucleic acids.

Description

EFFECTOR PROTEINS AND USES THEREOF

CROSS-REFERENCED APPLICATIONS

[0001] This application claims benefit of priority of U.S. Provisional Application No. 63/282, 111, filed on November 22, 2021, and U.S. Provisional Application No. 63/374,357, filed on September 1, 2022, the entire contents of each of which are incorporated herein by reference

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which has been submitted via Patent Center. The Sequence Listing titled 203477-70560 I PCT SL.xml, which was created on November 16, 2022 and is 197,307 bytes in size, is hereby incorporated by reference in its entirety.

FIELD

[0003] The present disclosure relates generally to polypeptides, such as effector proteins, compositions of such polypeptides and guide nucleic acids, systems and methods of using such polypeptides and compositions, including detecting and editing target nucleic acids.

BACKGROUND

[0004] Programmable nucleases are proteins that bind and cleave nucleic acids in a sequence-specific manner. A programmable nuclease may bind a target region of a nucleic acid and cleave the nucleic acid within the target region or at a position adjacent to the target region. In some instances, a programmable nuclease is activated when it binds a target region of a nucleic acid to cleave regions of the nucleic acid that are near, but not adjacent to the target region. A programmable nuclease, such as a CRISPR-associated (Cas) protein, may be coupled to a guide nucleic acid that imparts activity or sequence selectivity to the programmable nuclease. In general, guide nucleic acids comprise a CRISPR RNA (crRNA) that is at least partially complementary to a target nucleic acid. In some cases, guide nucleic acids comprise a trans-activating crRNA (tracrRNA), at least a portion of which interacts with the programmable nuclease. In some cases, a tracrRNA is provided separately from the crRNA and hybridizes to a portion of the crRNA that does not hybridize to the target nucleic acid. In other cases, a tracrRNA and a crRNA may be linked as a single guide RNA.

[0005] Programmable nucleases may cleave nucleic acids, including single stranded RNA (ssRNA), double stranded DNA (dsDNA), and single-stranded DNA (ssDNA). Programmable nucleases may provide cis cleavage activity, trans cleavage activity, nickase activity, or a combination thereof. Cis cleavage activity is cleavage of a target nucleic acid that is hybridized to a guide nucleic acid, wherein cleavage occurs within or directly adjacent to the region of the target nucleic acid that is hybridized to guide RNA. Trans cleavage activity includes cleavage of DNA or RNA that is near, but not hybridized to the guide RNA. Trans cleavage activity may be triggered by the hybridization of guide RNA to the target nucleic acid. Nickase activity is the selective cleavage of one strand of a dsDNA molecule.

[0006] Programmable nucleases may be modified to have reduced nuclease or nickase activity relative to its unmodified version, but retain their sequence selectivity. For instance, amino acid residues of the programmable nuclease that impart catalytic activity to the programmable nuclease may be substituted with an alternative amino acid that does not impart catalytic activity to the programmable nuclease.

[0007] While certain programmable nucleases may be used to edit and detect nucleic acid molecules in a sequence specific manner, challenging biological and sample conditions (e.g, high viscosity, metal chelating) may limit their accuracy and effectiveness. There is thus a need for systems and methods that employ programmable nucleases having specificity and efficiency across a wide range of biological and sample conditions.

SUMMARY

[0008] The present disclosure provides compositions, systems, and methods comprising effector proteins and uses thereof. Compositions, systems, and methods disclosed herein leverage nucleic acid modifying activities (e.g., cis cleavage activity and trans cleavage activity) of these effector proteins for the modification, detection, and engineering of target nucleic acids.

[0009] Provided herein are compositions comprising: an effector protein, or a nucleic acid encoding an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of sequences recited in TABLE 1; and a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid. In some embodiments, compositions provided herein comprise: an effector protein, or a nucleic acid encoding an effector protein, wherein the amino acid sequence of the effector protein is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of sequences recited in TABLE 1; and a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid. In some embodiments, compositions provided herein comprise: an effector protein, or a nucleic acid encoding an effector protein, wherein the effector protein comprises about 100, about 120, about 140, about 160, about 180, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 340, about 360, about 380, about 400, about 420, about 440, about 460, about 480, about 500, about 520, about 540, about 560, about 580, about 600, or about 620 contiguous amino acids of any one of sequences recited in TABLE 1; and a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid. In some embodiments, compositions provided herein comprise: an effector protein, or a nucleic acid encoding an effector protein, wherein the effector protein comprises the amino acid sequence located at positions 1-100, 150-250, 101-200, 250-350, 201-300, 350-450, 301-400, 350-450, 401-500, 450-550, 501-600, or 550-615 of any one of sequences recited in TABLE 1; and a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid. In some embodiments, compositions provided herein comprise: an effector protein, or a nucleic acid encoding an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to an equal length portion of any one of sequences recited in TABLE 1, wherein the portion of the sequence is about 30%, about 40% about 50%, about 60%, about 70%, about 80%, or about 90% of any one of sequences recited in TABLE 1; and a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid. In some embodiments, at least a portion of the guide nucleic acid binds the effector protein. In some embodiments, the portion of the guide nucleic acid that is bound by the effector protein comprises at least 10, at least 15, at least 20, at least 25, or at least 30 contiguous nucleotides of any one of sequences recited in TABLE 4. In some embodiments, the portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to an equal length portion of any one of sequences recited in TABLE 4. In some embodiments, the guide nucleic acid comprises a sequence that hybridizes to a target sequence of a target nucleic acid, and wherein the target nucleic acid comprises a protospacer adjacent motif (PAM) selected from any one of sequences recited in TABLE 3. In some embodiments, the PAM is located within 20, 40, 60, 80, or 100 nucleotides of the 5’ end of the target sequence. In some embodiments, the guide nucleic acid comprises a first sequence and a second sequence, wherein the first sequence is heterologous with the second sequence. In some embodiments, the first sequence comprises at least five nucleotides and the second sequence comprises at least five nucleotides. In some embodiments, at least one of the effector protein, the guide nucleic acid, and the combination thereof, are not naturally occurring. In some embodiments, at least one of the effector protein and the guide nucleic acid is recombinant or engineered. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of sequences recited in TABLE 4, TABLE 5, TABLE 6, or TABLE 7. In some embodiments, the guide nucleic acid comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 contiguous nucleotides of any one of sequences recited in TABLE 4, TABLE 5, TABLE 6, or TABLE 7. In some embodiments, the guide nucleic acid comprises at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, or at least 220 contiguous nucleotides of any one of sequences recited in TABLE 4, TABLE 5, TABLE 6, or TABLE 7. In some embodiments, the guide nucleic acid comprises a crRNA. In some embodiments, the guide nucleic acid comprises an intermediary RNA. In some embodiments, the crRNA is covalently linked to the intermediary RNA. In some embodiments, the guide nucleic acid comprises a sgRNA. In some embodiments, the sgRNA comprises a handle sequence. In some embodiments, the handle sequence comprises the intermediary RNA. In some embodiments, the guide nucleic acid comprises a tracrRNA. In some embodiments, the guide nucleic acid does not comprise a tracrRNA. In some embodiments, the guide nucleic acid comprises a crRNA covalently linked to a tracrRNA. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 1, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 75. In some embodiments, the PAM is of SEQ ID NO: 10. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 47. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 48. In some embodiments, the guide nucleic acid is sgRNA, and wherein the sgRNA is at least 90% identical to SEQ ID NO: 49 or 50. In some embodiments, the guide nucleic acid comprises at least one sequence that is at least 90% identical to a sequence selected from SEQ ID NOS: 19-22. In some embodiments, compositions described herein comprise a crRNA and a tracrRNA, wherein the crRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 19, and the tracrRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 20. In some embodiments, compositions described herein comprise a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is at least 95% or 100% identical to SEQ ID NO: 19, and the nucleotide sequence of the tracrRNA is at least 95% or 100% identical to SEQ ID NO: 20. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 2, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of sequence selected from SEQ ID NO: 76 - 79. In some embodiments, the PAM is of SEQ ID NO: 11. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 51. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 52 or 53. In some embodiments, the guide nucleic acid is sgRNA, and wherein the sgRNA is at least 90% identical to any one of SEQ ID NO: 54-56. In some embodiments, the guide nucleic acid comprises at least one sequence that is at least 90% identical to a sequence selected from SEQ ID NOS: 23-28. In some embodiments, compositions described herein comprise a crRNA and a tracrRNA, wherein the crRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 23, and the tracrRNA comprises a nucleotide sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 24 and SEQ ID NO: 25. In some embodiments, compositions described herein comprise a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is at least 95% or 100% identical to SEQ ID NO: 23, and the nucleotide sequence of the tracrRNA is at least 95% or 100% identical to a sequence selected from SEQ ID NO: 24 and SEQ ID NO: 25. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 3, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 80. In some embodiments, the PAM is of SEQ ID NO: 12. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 57. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 58. In some embodiments, the guide nucleic acid comprises at least one sequence that is at least 90% identical to a sequence selected from SEQ ID NOS: 29 and 30. In some embodiments, compositions described herein comprise a crRNA and a tracrRNA, wherein the crRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 29, and the tracrRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 30. In some embodiments, compositions described herein comprise a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is at least 95% or 100% identical to SEQ ID NO: 29, and the nucleotide sequence of the tracrRNA is at least 95% or 100% identical to SEQ ID NO: 30. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 4, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 81. In some embodiments, the PAM is of SEQ ID NO: 13. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 59. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNAis at least 90% identical to SEQ ID NO: 60. In some embodiments, the guide nucleic acid comprises at least one sequence that is at least 90% identical to a sequence selected from SEQ ID NOS: 31 and 32. In some embodiments, compositions described herein comprise a crRNA and a tracrRNA, wherein the crRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 31, and the tracrRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 32. In some embodiments, compositions described herein comprise a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is at least 95% or 100% identical to SEQ ID NO: 31, and the nucleotide sequence of the tracrRNA is at least 95% or 100% identical to SEQ ID NO: 32. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 5, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 82. In some embodiments, the PAM is of SEQ ID NO: 14. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 61. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 62 or 63. In some embodiments, the guide nucleic acid comprises at least one sequence that is at least 90% identical to a sequence selected from SEQ ID NOS: 33-35. In some embodiments, compositions described herein comprise a crRNA and a tracrRNA, wherein the crRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 33, and the tracrRNA comprises a nucleotide sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 34 and SEQ ID NO: 35. In some embodiments, compositions described herein comprise a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is at least 95% or 100% identical to SEQ ID NO: 33, and the nucleotide sequence of the tracrRNA is at least 95% or 100% identical to a sequence selected from SEQ ID NO: 34 and SEQ ID NO: 35. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 6, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 83. In some embodiments, the PAM is of SEQ ID NO: 15. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 64. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 65 or 66. In some embodiments, the guide nucleic acid is sgRNA, and wherein the sgRNA is at least 90% identical to any one of SEQ ID NO: 67-68. In some embodiments, the guide nucleic acid comprises at least one sequence that is at least 90% identical to a sequence selected from SEQ ID NOS: 36-40. In some embodiments, compositions described herein comprise a crRNA and a tracrRNA, wherein the crRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 36, and the tracrRNA comprises a nucleotide sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 37 and SEQ ID NO: 38. In some embodiments, compositions described herein comprise a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is at least 95% or 100% identical to SEQ ID NO: 36, and the nucleotide sequence of the tracrRNA is at least 95% or 100% identical to a sequence selected from SEQ ID NO: 37 and SEQ ID NO: 38. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 7, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 84. In some embodiments, the PAM is of SEQ ID NO: 16. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 69. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 70. In some embodiments, the guide nucleic acid comprises at least one sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 41 and 42. In some embodiments, compositions described herein comprise a crRNA and a tracrRNA, wherein the crRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 41, and the tracrRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 42. In some embodiments, compositions described herein comprise a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is at least 95% or 100% identical to SEQ ID NO: 41, and the nucleotide sequence of the tracrRNA is at least 95% or 100% identical to SEQ ID NO: 42. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 8, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 85. In some embodiments, the PAM is of SEQ ID NO: 17. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 71. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 72. In some embodiments, the guide nucleic acid comprises at least one sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 43 and 44. In some embodiments, compositions described herein comprise a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is at least 95% or 100% identical to SEQ ID NO: 43, and the nucleotide sequence of the tracrRNA is at least 95% or 100% identical to SEQ ID NO: 44. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 9, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 86. In some embodiments, the PAM is of SEQ ID NO: 18. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 73. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 74. In some embodiments, the guide nucleic acid comprises at least one sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 45 and 46. In some embodiments, compositions described herein comprise a a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is at least 95% or 100% identical to SEQ ID NO: 45, and the nucleotide sequence of the tracrRNA is at least 95% or 100% identical to SEQ ID NO: 46. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 87, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 118. In some embodiments, the PAM is of SEQ ID NO: 98. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 109. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 127. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 88, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 119. In some embodiments, the PAM is of SEQ ID NO: 98 or 99. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 110. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 128 or 129. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 89, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 120. In some embodiments, the PAM is of SEQ ID NO: 98 or 99. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 111. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 130 or 131. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 90, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 121. In some embodiments, the PAM is of SEQ ID NO: 98 or 100. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 112. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 132. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 91, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 122. In some embodiments, the PAM is of SEQ ID NO: 98 or 101. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 113. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 133. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 92, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 123. In some embodiments, the PAM is of SEQ ID NO: 98 or 102. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 114. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 134 or 135. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 93, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 124. In some embodiments, the PAM is of SEQ ID NO: 98 or 102. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 115. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 136 or 137. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 94, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 123. In some embodiments, the PAM is of SEQ ID NO: 103 or 104. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 114. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 138. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 95, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 125. In some embodiments, the PAM is of SEQ ID NO: 105 or 106. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 116. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 139. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 96, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 123. In some embodiments, the PAM is of SEQ ID NO: SEQ ID NO: 98. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 114. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 140. In some embodiments, the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 97, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 126. In some embodiments, the PAM is of SEQ ID NO: SEQ ID NO: 107 or 108. In some embodiments, the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 117. In some embodiments, the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 141. In some embodiments, the effector protein comprises a nuclear localization signal. In some embodiments, the length of the effector protein is at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, or at least 600 linked amino acid residues. In some embodiments, the length of the effector protein is less than about 700 linked amino acids. In some embodiments, the length of the effector protein is about 300 to about 400, about 350 to about 450, about 400 to about 500, about 450 to about 550, about 500 to about 600, or about 550 to about 650 linked amino acids. In some embodiments, compositions provided herein further comprise a donor nucleic acid. In some embodiments, compositions provided herein further comprise a fusion partner protein linked to the effector protein. In some embodiments, the fusion partner protein is directly fused to the N terminus or C terminus of the effector protein via an amide bond. In some embodiments, the fusion partner protein is directly fused to the N terminus or C terminus of the effector protein via a peptide linker. In some embodiments, the fusion partner protein comprises a polypeptide selected from a deaminase, a transcriptional activator, a transcriptional repressor, or a functional domain thereof. In some embodiments, the effector protein comprises at least one mutation that reduces its nuclease activity relative to the effector protein without the mutation as measured in a cleavage assay, optionally wherein the effector protein is a catalytically inactive nuclease.

[0010] Also provided herein are compositions comprising a nucleic acid expression vector, wherein the nucleic acid vector encodes at least one of the effector proteins and the guide nucleic acid of any one of the compositions provided herein. In some embodiments, compositions further comprise a donor nucleic acid, optionally wherein the donor nucleic acid is encoded by the nucleic acid expression vector or an additional nucleic acid expression vector. In some embodiments, the nucleic acid expression vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0011] Also provided herein are compositions comprising a virus, wherein the virus comprises a composition described herein.

[0012] Provided herein are pharmaceutical compositions comprising any one of the compositions provided herein, and a pharmaceutically acceptable excipient.

[0013] Provided herein are systems comprising any one of the compositions provided herein, and at least one detection reagent for detecting a target nucleic acid. In some embodiments, the at least one detection reagent is selected from a reporter nucleic acid, a detection moiety, an additional effector protein, or a combination thereof, optionally wherein the reporter nucleic acid comprises a fluorophore, a quencher, or a combination thereof. In some embodiments, systems provided herein comprise at least one amplification reagent for amplifying a target nucleic acid. In some embodiments, the at least one amplification reagent is selected from the group consisting of a primer, a polymerase, a deoxynucleoside triphosphate (dNTP), a ribonucleoside triphosphate (rNTP), and combinations thereof. In some embodiments, the system comprises a device with a chamber or solid support for containing the composition, target nucleic acid, detection reagent or combination thereof.

[0014] Provided herein are methods of detecting a target nucleic acid in a sample, comprising the steps of: contacting the sample with: any one of the compositions provided herein or any one of the systems provided herein, and a reporter nucleic acid comprising a detectable moiety that produces a detectable signal in the presence of the target nucleic acid and the composition or system; and detecting the detectable signal. In some embodiments, the reporter nucleic acid comprises a fluorophore, a quencher, or a combination thereof, and wherein the detecting comprises detecting a fluorescent signal. In some embodiments, methods further comprise reverse transcribing the target nucleic acid, amplifying the target nucleic acid, in vitro transcribing the target nucleic acid, or any combination thereof. In some embodiments, methods further comprise reverse transcribing the target nucleic acid and/or amplifying the target nucleic acid before contacting the sample with the composition. In some embodiments, methods further comprise reverse transcribing the target nucleic acid and/or amplifying the target nucleic acid after contacting the sample with the composition. In some embodiments, amplifying comprises isothermal amplification. In some embodiments, the target nucleic acid is from a pathogen. In some embodiments, the pathogen is a virus. In some embodiments, the target nucleic acid comprises RNA. In some embodiments, the target nucleic acid comprises DNA.

[0015] Provided herein are methods of modifying a target nucleic acid, the method comprising contacting the target nucleic acid with any one of the compositions provided herein or any one of the systems provided herein, thereby modifying the target nucleic acid. In some embodiments, modifying the target nucleic acid comprises cleaving the target nucleic acid, deleting a nucleotide of the target nucleic acid, inserting a nucleotide into the target nucleic acid, substituting a nucleotide of the target nucleic acid with an alternative nucleotide or an additional nucleotide, or any combination thereof. In some embodiments, methods provided herein further comprise contacting the target nucleic acid with a donor nucleic acid. In some embodiments, the target nucleic acid comprises a mutation associated with a disease. In some embodiments, the disease is selected from an autoimmune disease, a cancer, an inherited disorder, an ophthalmological disorder, a metabolic disorder, or a combination thereof. In some embodiments, the disease is any disease set forth in TABLE 9. In some embodiments, contacting the target nucleic acid comprises contacting a cell, wherein the target nucleic acid is located in the cell. In some embodiments, the contacting occurs in vitro. In some embodiments, the contacting occurs in vivo. In some embodiments, the contacting occurs ex vivo.

[0016] Provided herein is a cell comprising any one of the compositions provided herein. Provided herein is a cell modified by any one of the compositions provided herein. Provided herein is a cell modified by any one of the systems provided herein. Provided herein is a cell comprising a modified target nucleic acid, wherein the modified target nucleic acid is a target nucleic acid modified according to any one of the methods provided herein. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is an animal cell. In some embodiments, the cell is a T cell, optionally wherein the T cell is a natural killer T cell (NKT). In some embodiments, the cell is a chimeric antigen receptor T cell (CAR T-cell). In some embodiments, the cell is an induced pluripotent stem cell (iPSC).

[0017] Provided herein are populations of cells as described herein.

[0018] Provided herein are methods of producing a protein, the method comprising: contacting a cell comprising a target nucleic acid to any one of the compositions described herein, thereby editing the target nucleic acid to produce a modified cell comprising a modified target nucleic acid; and producing a protein from the cell that is encoded, transcriptionally affected, or translationally affected by the modified nucleic acid. [0019] Provided herein are methods method of treating a disease comprising administering to a subject in need thereof any one of the compositions described herein, or any one of the cells described herein. [0020] Provided herein is are systems for modifying or detecting a target nucleic acid, comprising at least two components each individually comprising one of the following: an effector protein, or a nucleic acid encoding an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of sequences recited in TABLE 1; and a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid, wherein the guide nucleic acid at least partially binds to the target nucleic acid.

[0021] Provided herein is are systems for modifying or detecting a target nucleic acid, comprising at least two components each individually comprising one of the following: an effector protein, or a nucleic acid encoding an effector protein, wherein the amino acid sequence of the effector protein is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of sequences recited in TABLE 1; and a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid, wherein the guide nucleic acid at least partially binds to the target nucleic acid.

[0022] Provided herein is are systems for modifying or detecting a target nucleic acid, comprising at least two components each individually comprising one of the following: an effector protein, or a nucleic acid encoding an effector protein, wherein the effector protein comprises about 100, about 120, about 140, about 160, about 180, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 340, about 360, about 380, about 400, about 420, about 440, about 460, about 480, about 500, about 520, about 540, about 560, about 580, about 600, or about 620 contiguous amino acids of any one of sequences recited in TABLE 1; and a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid, wherein the guide nucleic acid at least partially binds to the target nucleic acid. Provided herein is are systems for modifying or detecting a target nucleic acid, comprising at least two components each individually comprising one of the following: an effector protein, or a nucleic acid encoding an effector protein, wherein the effector protein comprises the amino acid sequence located at positions 1-100, 150-250, 101-200, 250-350, 201-300, 350-450, 301-400, 350-450, 401-500, 450- 550, 501-600, or 550-615 of any one of sequences recited in TABLE 1; and a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid, wherein the guide nucleic acid at least partially binds to the target nucleic acid. Provided herein is are systems for modifying or detecting a target nucleic acid, comprising at least two components each individually comprising one of the following: an effector protein, or a nucleic acid encoding an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to an equal length portion of any one of sequences recited in TABLE 1, wherein the portion of the sequence is about 30%, about 40% about 50%, about 60%, about 70%, about 80%, or about 90% of any one of sequences recited in TABLE 1; and a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid, wherein the guide nucleic acid at least partially binds to the target nucleic acid. In some embodiments, at least a portion of the guide nucleic acid binds the effector protein. In some embodiments, the portion of the guide nucleic acid that is bound by the effector protein comprises at least 10, at least 15, at least 20, at least 25, or at least 30 contiguous nucleotides of any one of sequences recited in TABLE 4. In some embodiments, the portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to an equal length portion of any one of sequences recited in TABLE 4. In some embodiments, the effector protein comprises a nuclear localization signal comprising any one of the amino acid sequences recited in TABLE 2. In some embodiments, the system further comprises a component comprising a donor nucleic acid. In some embodiments, the system further comprises a component comprising a fusion partner protein. In some embodiments, the fusion partner protein is fused to the effector protein. In some embodiments, the fusion partner protein is not fused to the effector protein. In some embodiments, the effector protein comprises a catalytic activity that is 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less, relative to a naturally occurring counterpart effector protein.

INCORPORATION BY REFERENCE

[0023] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety for any purpose and to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 shows the position frequency matrix (PFM) derived WebLogos that revealed the presence of enriched 5’ PAM consensus sequences for the various effector proteins by in vitro screening.

[0025] FIG. 2 shows the position frequency matrix (PFM) derived WebLogos that revealed the presence of enriched 5 ’ PAM consensus sequences for the various effector proteins by mammalian in vitro screening.

[0026] FIG. 3 shows PAM preferences for effector proteins disclosed herein. Frequency of nucleotides at each PAM position was independently calculated using a position frequency matrix (PFM) and plotted as a WebLogo. The numbers at the bottom of the plot denote the effector protein used, as well as the combination of crRNA and tracrRNA. DETAILED DESCRIPTION OF THE INVENTION

[0027] It is to be understood that both the foregoing general description and the following detailed description are exemplary, and explanatory only, and are not restrictive of the disclosure.

[0028] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Definitions

[0029] Unless otherwise indicated, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise indicated or obvious from context, the following terms have the following meanings:

[0030] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

[0031] Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

[0032] Use of the term “including” as well as other forms, such as “includes” and “included,” is not limiting.

[0033] As used herein, the term “comprise” and its grammatical equivalents specifies the presence of stated features, integers, steps, operations, elements, and/or components, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

[0034] As used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers +/- 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0035] The terms “percent identity,” “% identity,” and % “identical,” or grammatical equivalents thereof, as used herein refer to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X% identical to SEQ ID NO: Y” can refer to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y. Generally, computer programs can be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci. 1988 Mar;4(l): 11-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444-8; Pearson, Methods Enzymol. 1990;183:63-98) and gapped BLAST (Altschul et al., Nucleic Acids Res. 1997 Sep l;25(17):3389-40), BLASTP, BLASTN, or GCG (Devereux et al., Nucleic Acids Res. 1984 Jan 11;12(1 Pt l):387-95).

[0036] The terms “amplification” and “amplifying,” or grammatical equivalents thereof, as used herein refers to a process by which a nucleic acid molecule is enzymatically copied to generate a plurality of nucleic acid molecules containing the same sequence as the original nucleic acid molecule or a distinguishable portion thereof.

[0037] The term “base editing enzyme,” as used herein refers to a protein, polypeptide, or fragment thereof that is capable of catalyzing the chemical modification of a nucleobase of a deoxyribonucleotide or a ribonucleotide. Such a base editing enzyme, for example, is capable of catalyzing a reaction that modifies a nucleobase that is present in a nucleic acid molecule, such as DNA or RNA (single stranded or double stranded). Non-limiting examples of the type of modification that a base editing enzyme is capable of catalyzing includes converting an existing nucleobase to a different nucleobase, such as converting a cytosine to a guanine or thymine or converting an adenine to a guanine, hydrolytic deamination of an adenine or adenosine, or methylation of cytosine (e.g., CpG, CpA, CpT or CpC). A base editing enzyme itself may or may not bind to the nucleic acid molecule containing the nucleobase. [0038] The term “base editor,” as used herein refers to a fusion protein comprising a base editing enzyme fused to an effector protein. The base editor is functional when the effector protein is coupled to a guide nucleic acid. The guide nucleic acid imparts sequence specific activity to the base editor. By way of non-limiting example, the effector protein may comprise a catalytically inactive effector protein. Also, by way of non-limiting example, the base editing enzyme may comprise deaminase activity. Additional base editors are described herein.

[0039] The term “catalytically inactive effector protein,” as used herein refers to an effector protein that is modified relative to a naturally-occurring effector protein to have a reduced or eliminated catalytic activity relative to that of the naturally-occurring effector protein, but retains its ability to interact with a guide nucleic acid. The catalytic activity that is reduced or eliminated is often a nuclease activity. The naturally-occurring effector protein may be a wildtype protein. In some embodiments, the catalytically inactive effector protein is referred to as a catalytically inactive variant of an effector protein, e.g., a Cas effector protein.

[0040] The term “cis cleavage,” as used herein refers to cleavage (hydrolysis of a phosphodiester bond) of a target nucleic acid by an effector protein complexed with a guide nucleic acid refers to cleavage of a target nucleic acid that is hybridized to a guide nucleic acid, wherein cleavage occurs within or directly adjacent to the region of the target nucleic acid that is hybridized to the guide nucleic acid.

[0041] The terms “complementary” and “complementarity,” as used herein with reference to a nucleic acid refer to the characteristic of a polynucleotide having nucleotides that base pair with their Watson- Crick counterparts (C with G; or A with T) in a reference nucleic acid. For example, when every nucleotide in a polynucleotide forms a base pair with a reference nucleic acid, that polynucleotide is said to be 100% complementary to the reference nucleic acid. In a double stranded DNA or RNA sequence, the upper (sense) strand sequence is in general, understood as going in the direction from its 5'- to 3 '-end, and the complementary sequence is thus understood as the sequence of the lower (antisense) strand in the same direction as the upper strand. Following the same logic, the reverse sequence is understood as the sequence of the upper strand in the direction from its 3'- to its 5 '-end, while the ‘reverse complement’ sequence or the ‘reverse complementary’ sequence is understood as the sequence of the lower strand in the direction of its 5'- to its 3 '-end. Each nucleotide in a double stranded DNA or RNA molecule that is paired with its Watson-Crick counterpart called its complementary nucleotide.

[0042] The term “cleavage assay,” as used herein refers to an assay designed to visualize, quantitate, or identify cleavage of a nucleic acid. In some cases, the cleavage activity may be cis-cleavage activity. In some cases, the cleavage activity may be trans-cleavage activity.

[0043] The term “clustered regularly interspaced short palindromic repeats (CRISPR),” as used herein refers to a segment of DNA found in the genomes of certain prokaryotic organisms, including some bacteria and archaea, that includes repeated short sequences of nucleotides interspersed at regular intervals between unique sequences of nucleotides derived from the DNA of a pathogen (e.g., virus) that had previously infected the organism and that functions to protect the organism against future infections by the same pathogen.

[0044] The terms “CRISPR RNA” or “crRNA,” as used herein refer to a type of guide nucleic acid, wherein the nucleic acid is RNA, comprising a first sequence, often referred to herein as a “spacer sequence,” that hybridizes to a target sequence of a target nucleic acid, and a second sequence that either a) hybridizes to a portion of a tracrRNA or b) is capable of being non-covalently bound by an effector protein. In some embodiments, the crRNA is covalently linked to an additional nucleic acid (e.g., a tracrRNA) that interacts with the effector protein.

[0045] The term, “detectable signal,” as used herein refers to a signal that can be detected using optical, fluorescent, chemiluminescent, electrochemical, and other detection methods known in the art.

[0046] The term, “donor nucleic acid,” as used herein refers to nucleic acid that is incorporated into a target nucleic acid.

[0047] The term, “donor nucleotide,” as used herein refers to a single nucleotide that is incorporated into a target nucleic acid. A nucleotide is typically inserted at a site of cleavage by an effector protein. [0048] The term “effector protein,” as used herein refers to a protein, polypeptide, or peptide that non- covalently binds to a guide nucleic acid to form a complex that contacts a target nucleic acid, wherein at least a portion of the guide nucleic acid hybridizes to a target sequence of the target nucleic acid. In some embodiments, the complex comprises multiple effector proteins. In some embodiments, the effector protein modifies the target nucleic acid when the complex contacts the target nucleic acid. In some embodiments, the effector protein does not modify the target nucleic acid, but it is fused to a fusion partner protein that modifies the target nucleic acid. A non-limiting example of modifying a target nucleic acid is cleaving (hydrolysis) of a phosphodiester bond. Additional examples of modifying target nucleic acids are described herein and throughout.

[0049] The term, “functional domain,” as used herein refers to a region of one or more amino acids in a protein that is required for an activity of the protein, or the full extent of that activity, as measured in an in vitro assay. Activities include, but are not limited to nucleic acid binding, nucleic acid modification, nucleic acid cleavage, protein binding. The absence of the functional domain, including mutations of the functional domain, would abolish or reduce activity.

[0050] The term, “functional fragment,” as used herein refers to a fragment of a protein that retains some function relative to the entire protein. Non-limiting examples of functions are nucleic acid binding, protein binding, nuclease activity, nickase activity, deaminase activity, demethylase activity, or acetylation activity.

[0051] The term “fusion effector protein,” “fusion protein,” and “fusion polypeptide,” as used herein refer to a protein comprising at least two heterologous polypeptides. Often a fusion effector protein comprises an effector protein and a fusion partner protein. In general, the fusion partner protein is not an effector protein. Examples of fusion partner proteins are provided herein.

[0052] As used herein, the terms “fusion partner protein” or “fusion partner,” as used herein refers to a protein, polypeptide or peptide that is fused to an effector protein. The fusion partner generally imparts some function to the fusion protein that is not provided by the effector protein. The fusion partner may provide a detectable signal. The fusion partner may modify a target nucleic acid, including changing a nucleobase of the target nucleic acid and making a chemical modification to one or more nucleotides of the target nucleic acid. The fusion partner may be capable of modulating the expression of a target nucleic acid. The fusion partner may inhibit, reduce, activate, or increase expression of a target nucleic acid via additional proteins or nucleic acid modifications to the target sequence.

[0053] The term “guide nucleic acid,” as used herein refers to at least one nucleic acid comprising: a first nucleotide sequence that hybridizes to a target nucleic acid; and a second nucleotide sequence that is capable of being non-covalently bound by an effector protein. The first sequence may be referred to herein as a spacer sequence. The term, “guide nucleic acid,” may be used to refer to two separate nucleic acids, (e.g., a crRNA and tracrRNA), at least a portion of each hybridize to one another. In some instances, the first sequence is covalently linked to the second sequence, either directly (e.g., by a phosphodiester bond) or indirectly (e.g., by one more nucleotides). In some embodiments, the first sequence is located 5’ of the second nucleotide sequence. In some embodiments, the first sequence is located 3’ of the second nucleotide sequence.

[0054] The term, “handle sequence,” as used herein, in the context of a sgRNA refers to a portion of the sgRNA that is: 1) capable of being non-covalently bound by an effector protein, and 2) connects the portion of the sgRNA capable of being non-covalently bound by an effector protein to a nucleotide sequence that hybridizes to a target nucleic acid. The nucleotide sequence of a handle sequence may comprise all or a portion of an intermediary RNA. In such embodiments, at least a portion of an intermediary RNA non-covalently interacts with an effector protein. Additionally, or alternatively, the nucleotide sequence of a handle sequence may contain all or a portion of a repeat sequence. In such embodiments, at least a portion of an intermediary RNA or both, at least a portion of the intermediary RNA and at least a portion of repeat sequence, non-covalently interacts with an effector protein. In general, a single guide nucleic acid, also referred to as a single guide RNA (sgRNA), comprises a handle sequence. The term handle sequence when used in the context of a sgRNA refers to a portion of the sgRNA that is capable of being non-covalently bound by an effector protein.

[0055] The term “heterologous,” as used herein, means a nucleotide or polypeptide sequence that is not found in a native nucleic acid or protein, respectively. In some embodiments, fusion proteins comprise an effector protein and a fusion partner protein, wherein the fusion partner protein is heterologous to an effector protein. These fusion proteins may be referred to as a “heterologous protein.” A protein that is heterologous to the effector protein is a protein that is not covalently linked via an amide bond to the effector protein in nature. In some embodiments, a heterologous protein is not encoded by a species that encodes the effector protein. In some instances, the heterologous protein exhibits an activity (e.g., enzymatic activity) when it is fused to the effector protein. In some instances, the heterologous protein exhibits increased or reduced activity (e.g., enzymatic activity) when it is fused to the effector protein, relative to when it is not fused to the effector protein. In some instances, the heterologous protein exhibits an activity (e.g., enzymatic activity) that it does not exhibit when it is fused to the effector protein. A guide nucleic acid may comprise a first sequence and a second sequence, wherein the first sequence and the second sequence are not found covalently linked via a phosphodiester bond in nature. Thus, the first sequence is considered to be heterologous with the second sequence, and the guide nucleic acid may be referred to as a heterologous guide nucleic acid.

[0056] The term “in vitro, ” as used herein is used to describe an event that takes places contained in a container for holding laboratory reagents such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed. The term “in vivo” is used to describe an event that takes place in a subject’s body. The term “ex vivo” is used to describe an event that takes place outside of a subject’s body. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample is an “in vitro” assay.

[0057] The term “linked amino acids" as used herein refers to at least two amino acids linked by an amide bond.

[0058] The term “linker,” as used herein refers to a bond or molecule that links a first polypeptide to a second polypeptide. A “peptide linker” comprises at least two amino acids linked by an amide bond.

[0059] The term “modified target nucleic acid,” as used herein refers to a target nucleic acid, wherein the target nucleic acid has undergone a modification, for example, after contact with an effector protein. In some cases, the modification is an alteration in the sequence of the target nucleic acid. In some cases, the modified target nucleic acid comprises an insertion, deletion, or replacement of one or more nucleotides compared to the unmodified target nucleic acid.

[0060] The term “mutation associated with a disease,” as used herein refers to the co-occurrence of a mutation and the phenotype of a disease. The mutation may occur in a gene, wherein transcription or translation products from the gene occur at a significantly abnormal level or in an abnormal form in a cell or subject harboring the mutation as compared to a non-disease control subject not having the mutation.

[0061] The terms “non-naturally occurring” and “engineered,” as used herein are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid, refer to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid that is at least substantially free from at least one other feature with which it is naturally associated in nature and as found in nature, and/or contains a modification (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the naturally occurring nucleic acid, nucleotide, protein, polypeptide, peptide, or amino acid. The terms, when referring to a composition or system described herein, refer to a composition or system having at least one component that is not naturally associated with the other components of the composition or system. By way of a non-limiting example, a composition may include an effector protein and a guide nucleic acid that do not naturally occur together. Conversely, and as a non-limiting further clarifying example, an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes an effector protein and a guide nucleic acid from a cell or organism that have not been genetically modified by the hand of man.

[0062] The term “nucleic acid expression vector,” as used herein refers to a plasmid that can be used to express a nucleic acid of interest.

[0063] The term “nuclear localization signal,” as used herein refers to an entity (e.g., peptide) that facilitates localization of a nucleic acid, protein, or small molecule to the nucleus, when present in a cell that contains a nuclear compartment.

[0064] The term “nuclease activity,” as used herein refers to the enzymatic activity of an enzyme which allows the enzyme to cleave the phosphodiester bonds between the nucleotide subunits of nucleic acids; the term “endonuclease activity” refers to the enzymatic activity of an enzyme which allows the enzyme to cleave the phosphodiester bond within a polynucleotide chain. An enzyme with nuclease activity may be referred to as a “nuclease.”

[0065] The term, “prime editing enzyme,” as used herein refers to a protein, polypeptide, or fragment thereof that is capable of catalyzing the modification (insertion, deletion, or base-to-base conversion) of a target nucleotide or nucleotide sequence in a nucleic acid. A prime editing enzyme capable of catalyzing such a reaction includes a reverse transcriptase. A prime editing enzyme may require a prime editing guide RNA (pegRNA) to catalyze the modification. Such a pegRNA can be capable of identifying the nucleotide or nucleotide sequence in the target nucleic acid to be edited and encoding the new genetic information that replaces the targeted nucleotide or nucleotide sequence in the nucleic acid. A prime editing enzyme may require a prime editing guide RNA (pegRNA) and a single guide RNA to catalyze the modification.

[0066] The term “protospacer adjacent motif (PAM),” as used herein refers to a nucleotide sequence found in a target nucleic acid that directs an effector protein to modify the target nucleic acid at a specific location. A PAM sequence may be required for a complex having an effector protein and a guide nucleic acid to hybridize to and modify the target nucleic acid. However, a given effector protein may not require a PAM sequence being present in a target nucleic acid for the effector protein to modify the target nucleic acid.

[0067] The term “recombinant,” as used herein as applied to proteins, polypeptides, peptides, and nucleic acids, refers to proteins, polypeptides, peptides, and nucleic acids that are products of various combinations of cloning, restriction, and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. Generally, DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame uninterrupted by internal non translated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions and may act to modulate production of a desired product by various mechanisms (see "DNA regulatory sequences", below).

[0068] The term “recombinant” polynucleotide or “recombinant nucleic acid” refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. The term, “recombinant polypeptide” or “recombinant protein” refers to a polypeptide which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of amino sequences through human intervention. Thus, e.g., a polypeptide that comprises a heterologous amino acid sequence is a recombinant polypeptide.

[0069] The terms “reporter”, “reporter nucleic acid,” and “reporter molecule” are used interchangeably herein to refer to a non-target nucleic acid molecule that can provide a detectable signal upon cleavage by an effector protein. Examples of detectable signals and detectable moieties that generate detectable signals are provided herein. [0070] The term “sample,” as used herein generally refers to something comprising a target nucleic acid. In some instances, the sample is a biological sample, such as a biological fluid or tissue sample. In some instances, the sample is an environmental sample. The sample may be a biological sample or environmental sample that is modified or manipulated. By way of non-limiting example, samples may be modified or manipulated with purification techniques, heat, nucleic acid amplification, salts, and buffers.

[0071] The term “subject,” as used herein can be a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. In some instances, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.

[0072] The term “target nucleic acid,” as used herein refers to a nucleic acid that is selected as the nucleic acid for modification, binding, hybridization, or any other activity of or interaction with a nucleic acid, protein, polypeptide, or peptide described herein. A target nucleic acid may comprise RNA, DNA, or a combination thereof. A target nucleic acid may be single-stranded (e.g., singlestranded RNA or single-stranded DNA) or double-stranded (e.g., double-stranded DNA).

[0073] The term “target sequence,” as used herein in reference to a target nucleic acid refers to a sequence of nucleotides that hybridizes to an equal length portion of a guide nucleic acid. Hybridization of the guide nucleic acid to the target sequence may bring an effector protein into contact with the target nucleic acid.

[0074] The term “trans cleavage,” is used herein in reference to cleavage (hydrolysis of a phosphodiester bond) of one or more nucleic acids by an effector protein when that effector protein is complexed with a guide nucleic acid and a target nucleic acid. The one or more nucleic acids may include the target nucleic acid as well as non-target nucleic acids.

[0075] The term “trans-activating RNA (tracrRNA),” as used herein, refers to a nucleic acid that comprises a first sequence that is capable of being non-covalently bound by an effector protein. TracrRNAs may comprise a second sequence that hybridizes to a portion of a crRNA, which may be referred to as a repeat hybridization sequence. In some embodiments, a tracrRNA is covalently linked to a crRNA.

[0076] The term “transactivating” or “transactivate,” as used herein, refers to the ability of a tracrRNA to (1) hybridize to a crRNA, wherein the tracrRNA and the crRNA are not covalently linked, and wherein the crRNA comprises a region that hybridizes to a target nucleic acid; and (2) interact with an effector protein, thereby bringing the effector protein into the proximity of the target nucleic acid where the effector protein provides a modifying activity on the target nucleic acid. In general, a tracrRNA is a feature of a dual-guide system. [0077] The term “transcriptional activator,” as used herein, refers to a polypeptide or a fragment thereof that can activate or increase transcription of a target nucleic acid molecule.

[0078] The term “transcriptional repressor,” as used herein, refers to a polypeptide or a fragment thereof that is capable of arresting, preventing, or reducing transcription of a target nucleic acid.

[0079] The terms “treatment” or “treating,” as used herein, are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying, or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0080] The term “viral vector,” as used herein, refers to a nucleic acid to be delivered into a host cell via a recombinantly produced virus or viral particle. The nucleic acid may be single-stranded or double stranded, linear or circular, segmented or non-segmented. The nucleic acid may comprise DNA, RNA, or a combination thereof. Non-limiting examples of viruses or viral particles that can deliver a viral vector include retroviruses (e.g., lentiviruses and y-retroviruses), adenoviruses, arenaviruses, alphaviruses, adeno-associated viruses (AAVs), baculoviruses, vaccinia viruses, herpes simplex viruses and poxviruses. A virus containing a viral vector may be replication competent, replication deficient or replication defective.

I. Introduction

[0081] Disclosed herein are compositions, systems and methods comprising at least one of: a. a polypeptide or a nucleic acid encoding the polypeptide; and b. an engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid.

[0082] Polypeptides described herein may bind and, optionally, cleave nucleic acids in a sequencespecific manner. Polypeptides described herein may also cleave the target nucleic acid within a target sequence or at a position adjacent to the target sequence. In some embodiments, a polypeptide is activated when it binds a certain sequence of a nucleic acid described herein, allowing the polypeptide to cleave a region of a target nucleic acid that is near, but not adjacent to the target sequence. A polypeptide may be an effector protein, such as a CRISPR-associated (Cas) protein, which may bind a guide nucleic acid that imparts activity or sequence selectivity to the polypeptide. In some embodiments, when describing binding and interacting, and grammatical equivalents thereof, reference may be made to a non-covalent interaction between macromolecules (e.g., between two polypeptides, between a polypeptide and a nucleic acid; between a polypeptide/guide nucleic acid complex and a target nucleic acid; and the like). While in a state of noncovalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g. , when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner). Non-limiting examples of non-covalent interactions are ionic bonds, hydrogen bonds, van der Waals and hydrophobic interactions. Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequencespecific.

[0083] In some embodiments, compositions, systems, and methods comprising effector proteins and guide nucleic acids comprise a first sequence, at least a portion of which interacts with a polypeptide. In some embodiments, the first sequence comprises a sequence that is similar or identical to a repeat sequence. In some embodiments, compositions, systems, and methods comprising effector proteins and guide nucleic acids comprise a second sequence that is at least partially complementary to a target nucleic acid, and which may be referred to as a spacer sequence.

[0084] Effector proteins disclosed herein may cleave nucleic acids, including single stranded RNA (ssRNA), double stranded DNA (dsDNA), and single-stranded DNA (ssDNA). Polypeptides disclosed herein may provide cis cleavage activity, trans cleavage activity, nickase activity, nuclease activity, or a combination thereof.

[0085] Also disclosed herein are non-naturally occurring compositions, systems, and methods of use thereof, comprising an effector protein and an engineered guide nucleic acid, which may simply be referred to herein as a guide nucleic acid. An effector protein may be referred to herein as an engineered effector protein. In general, an engineered effector protein and an engineered guide nucleic acid refer to an effector protein and a guide nucleic acid, respectively, that are not found in nature. In some instances, systems, methods and compositions comprise at least one non-naturally occurring component. For example, compositions, methods and systems may comprise a guide nucleic acid, wherein the sequence of the guide nucleic acid is different or modified from that of a naturally-occurring guide nucleic acid. In some instances, compositions, methods and systems comprise at least two components that do not naturally occur together. For example, compositions, methods and systems may comprise a guide nucleic acid comprising a repeat sequence and a spacer sequence which do not naturally occur together. Also, by way of example, compositions, methods and systems may comprise a guide nucleic acid and an effector protein that do not naturally occur together. Conversely, and for clarity, an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “ found in nature” includes effector proteins and guide nucleic acids from cells or organisms that have not been genetically modified by a human or machine. [0086] In some instances, a guide nucleic acid comprises: first nucleotide sequence that hybridizes to a target nucleic acid; and a second nucleotide sequence that is capable of being non-covalently bound by an effector protein. The first sequence may be referred to herein as a spacer sequence. The second sequence may be referred to herein as a repeat sequence or a handle sequence. In some embodiments, the handle sequence comprises the repeat sequence. In some embodiments, the first sequence is located 5’ of the second sequence. In some embodiments, the first sequence is located 3’ of the second sequence. [0087] In some instances, the guide nucleic acid comprises a non-natural nucleotide sequence. In some instances, the non-natural sequence is a nucleotide sequence that is not found in nature. The non-natural sequence may comprise a portion of a naturally-occurring sequence, wherein the portion of the naturally-occurring sequence is not present in nature, absent the remainder of the naturally-occurring sequence. In some instances, the guide nucleic acid comprises two naturally-occurring sequences arranged in an order or proximity that is not observed in nature. In some embodiments, the guide nucleic acid comprises two or more heterologous sequences arranged in an order or proximity that is not observed in nature. In some instances, compositions, methods and systems comprise a ribonucleotide complex comprising an effector protein and a guide nucleic acid, sometimes reffered to herein as a ribonucleotide protein complex (or an RNP), that do not occur together in nature. Engineered guide nucleic acids may comprise a first sequence and a second sequence that do not occur naturally together. For example, an engineered guide nucleic acid may comprise a sequence of a naturally-occurring repeat sequence and a spacer sequence that is complementary to a naturally-occurring eukaryotic sequence. The engineered guide nucleic acid may comprise a sequence of a repeat sequence that occurs naturally in an organism and a spacer sequence that does not occur naturally in that organism. An engineered guide nucleic acid may comprise a first sequence that occurs in a first organism and a second sequence that occurs in a second organism, wherein the first organism and the second organism are different. The guide nucleic acid may comprise a third sequence located at a 3 ’ or 5 ’ end of the guide nucleic acid, or between the first and second sequences of the guide nucleic acid. For example, a composition comprising an engineered guide nucleic acid may comprise a naturally occurring CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) sequence, coupled by a linker sequence.

[0088] In some instances, compositions, methods and systems described herein comprise an engineered effector protein that is similar to a naturally occurring effector protein. The engineered effector protein may lack a portion of the naturally occurring effector protein. The effector protein may comprise a mutation relative to the naturally-occurring effector protein, wherein the mutation is not found in nature. The effector protein may also comprise at least one additional amino acid relative to the naturally-occurring effector protein. In some embodiments, the effector protein may comprise a heterolgoous polypeptide. For example, the effector protein may comprise an addition of a nuclear localization signal relative to the natural occurring effector protein. In certain embodiments, the nucleotide sequence encoding the effector protein is codon optimized (e.g., for expression in a eukaryotic cell) relative to the naturally occurring sequence. II. Polypeptide Systems

Effector Proteins

[0089] Provided herein, in certain embodiments, are compositions, methods and systems that comprise one or more effector proteins and uses thereof. Also provided herein are compositions, methods and systems that comprise a nucleic acid, wherein the nucleic acid encodes any of one the effector proteins described herein. The nucleic acid may be a nucleic acid expression vector. By way of non-limiting example, the nucleic acid expression vector may be a viral vector, such as an AAV vector. In general, effector proteins disclosed herein are CRISPR-associated (“Cas”) proteins.

[0090] An effector protein provided herein interacts with a guide nucleic acid to form a complex. In some embodiments, the complex interacts with a target nucleic acid, a non-target nucleic acid, or both. In some embodiments, an interaction between the complex and a target nucleic acid comprises one or more of: recognition of a protospacer adjacent motif (PAM) sequence within the target nucleic acid by the effector protein, hybridization of the guide nucleic acid to the target nucleic acid, modification of the target nucleic acid and/or the non-target nucleic acid by the effector protein, or combinations thereof. In some embodiments, recognition of a PAM sequence within a target nucleic acid may direct the modification activity of an effector protein.

[0091] Modification activity of an effector protein or an engineered protein described herein may be cleavage activity, binding activity, insertion activity, substitution activity, and the like. Modification activity of an effector protein may result in: cleavage of at least one strand of a target nucleic acid, deletion of one or more nucleotides of a target nucleic acid, insertion of one or more nucleotides into a target nucleic acid, substitution of one or more nucleotides of a target nucleic acid with an alternative nucleotide, more than one of the foregoing, or any combination thereof. In some embodiments, an ability of an effector protein to edit a target nucleic acid may depend upon the effector protein being complexed with a guide nucleic acid, the guide nucleic acid being hybridized to a target sequence of the target nucleic acid, the distance between the target sequence and a PAM sequence, or combinations thereof. A target nucleic acid comprises a target strand and a non-target strand. Accordingly, in some embodiments, the effector protein may edit a target strand and/or a non-target strand of a target nucleic acid.

[0092] Effector proteins may modify a nucleic acid by cis cleavage or trans cleavage. The modification of the target nucleic acid generated by an effector protein, as a non-limiting example, may result in modulation of the expression of the nucleic acid (e.g., increasing or decreasing expression of the nucleic acid) or modulation of the activity of a translation product of the target nucleic acid (e.g, inactivation of a protein binding to an RNA molecule or hybridization). In some embodiments, the modification of the target nucleic acid generated by an effector protein may, as a non-limiting example, result in modulation of the expression of the target nucleic acid (e.g., increasing or decreasing expression of the nucleic acid) or modulation of the activity of a translation product of the target nucleic acid (e.g. , inactivation of a protein binding to an RNA molecule or hybridization). Accordingly, in some embodiments, provided herein are methods of editing a target nucleic acid using an effector protein of the present disclosure, or compositions or systems thereof. Also provided herein are methods of modulating expression of a target nucleic acid using an effector protein of the present disclosure, or compositions or systems thereof. Further provided herein are methods of modulating the activity of a translation product of a target nucleic acid using an effector protein of the present disclosure, or compositions or systems thereof.

[0093] Effector proteins may function as a single protein, including a single protein that is capable of binding to a guide nucleic acid and modifying a target nucleic acid. Alternatively, effector proteins may function as part of a multiprotein complex, including, for example, a complex having two or more effector proteins, including two or more of the same effector proteins (e.g., dimer or multimer). A first effector protein, when functioning in a multiprotein complex, may have a first functional activity (e.g., binding to a guide nucleic acid), while a second effector protein present in the multiprotein complex is capable of a second functional activity (e.g., modifying a target nucleic acid). The first and second effector proteins may be the same. The first and second effector proteins may be different. The sequences of the first and second effector proteins may be 15% to 20% identical, 20% to 25% identical, 25% to 30% identical, 30% to 35% identical, 35% to 40% identical, 40% to 45% identical, 45% to 50% identical, 50% to 55% identical, 55% to 60% identical, 60% to 65% identical, 65% to 70% identical, 70% to 75% identical, 75% to 80% identical, 80% to 85% identical, 85% to 90% identical, 90% to 95% identical, 95% to 99.9% identical, or 100% identical.

[0094] TABLE 1 provides illustrative amino acid sequences of effector proteins.

[0095] In some embodiments, compositions, systems and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the amino acid sequence of the effector protein comprises at least about 200 contiguous amino acids or more of any one of the sequences recited in TABLE 1. In some embodiments, the amino acid sequence of an effector protein provided herein comprises at least about 200, at least about 220, at least about 240, at least about 260, at least about 280, at least about 300, at least about 320, at least about 340, at least about 360, at least about 380, at least about 400 contiguous amino acids, at least about 420 contiguous amino acids, at least about 440 contiguous amino acids, at least about 460 contiguous amino acids, at least about 480 contiguous amino acids, at least about 500 contiguous amino acids, at least about 520 contiguous amino acids, at least about 540 contiguous amino acids, at least about 560 contiguous amino acids, at least about 580 contiguous amino acids, at least about 600 contiguous amino acids, at least about 620 contiguous amino acids, at least about 640 contiguous amino acids, at least about 660 contiguous amino acids, at least about 680 contiguous amino acids, at least about 700 contiguous amino acids, or more of any one of the sequences of TABLE 1.

[0096] In some embodiments, compositions, systems and methods described herein comprise an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein comprises a portion of any one of the sequences recited in TABLE 1. In some embodiments, the effector protein comprises a portion of any one of the sequences recited in TABLE 1, wherein the portion does not comprise at least the first 10 amino acids, at least the first 20 amino acids, at least the first 40 amino acids, at least the first 60 amino acids, at least the first 80 amino acids, at least the first 100 amino acids, at least the first 120 amino acids, at least the first 140 amino acids, at least the first 160 amino acids, at least the first 180 amino acids, or at least the first 200 amino acids of any one of the sequences recited in TABLE 1 In some embodiments, the effector protein comprises a portion of any one of the sequences recited in TABLE 1, wherein the portion does not comprise the last 10 amino acids, the last 20 amino acids, the last 40 amino acids, the last 60 amino acids, the last 80 amino acids, the last 100 amino acids, the last 120 amino acids, the last 140 amino acids, the last 160 amino acids, the last 180 amino acids, or the last 200 amino acids of any one of the sequences recited in TABLE 1.

[0097] In some instances, the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of sequence of TABLE 1. In some instances, the amino acid sequence of the effector protein is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of sequences of TABLE 1.

[0098] In some instances, compositions, methods and systems described herein comprise an effector protein or a nucleic acid encoding the effector protein, wherein effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of sequences recited in TABLE 1. In some instances, the amino acid sequence is at least 80% identical to any one of sequences recited in TABLE 1. In some instances, the amino acid sequence is at least 85% identical to any one of sequences recited in TABLE 1. In some instances, the amino acid sequence is at least 90% identical to any one of sequences recited in TABLE 1. In some instances, the amino acid sequence is at least 95% identical to any one of sequences recited in TABLE 1. In some instances, the amino acid sequence is at least 97% identical to any one of sequences recited in TABLE 1. In some instances, the amino acid sequence is at least 99% identical to any one of sequences recited in TABLE 1. In some instances, the amino acid sequence is 100% identical to any one of sequences recited in TABLE 1.

[0099] In some embodiments, compositions, methods and systems described herein comprise an effector protein or a nucleic acid encoding the effector protein, wherein the amino acid sequence of the effector protein is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of sequences recited in TABLE 1. In some instances, the amino acid sequence of the effector protein is at least 80% identical to any one of sequences recited in TABLE 1. In some instances, the amino acid sequence of the effector protein is at least 85% identical to any one of sequences recited in TABLE 1. In some instances, the amino acid sequence of the effector protein is at least 90% identical to any one of sequences recited in TABLE 1. In some instances, the amino acid sequence of the effector protein is at least 95% identical to any one of sequences recited in TABLE 1. In some instances, the amino acid sequence of the effector protein is at least 97% identical to any one of sequences recited in TABLE 1. In some instances, the amino acid sequence of the effector protein is at least 99% identical to any one of sequences recited in TABLE 1. In some instances, the amino acid sequence of the effector protein is 100% identical to any one of sequences recited in TABLE 1.

[0100] In some instances, compositions, methods and systems described herein comprise an effector protein or a nucleic acid encoding the effector protein, wherein portion of the amino acid sequence of the effector protein is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to an equal length portion of any one of sequences of TABLE 1. In some instances, the length of the portion is selected from: 20 to 40 , 40 to 60 , 60 to 80, 80 to 100, 100 to 120, 120 to 140, 140 to 160, 160 to 180, 180 to 200, 200 to 220, 220 to 240, 240 to 260, 260 to 280, 280 to 300, 320 to 340, 340 to 360, 360 to 380, and 380 to 400 linked amino acids. In some instances, the length of the portion is selected from: 400 to 420, 420 to 440, 440 to 460, 460 to 480, 480 to 500, 520 to 540, 540 to 560, 560 to 580, 580 to 600, and 600 to 615 linked amino acids.

[0101] In some embodiments, compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% similar to any one of the sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 80% similar to any one of the sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 85% similar to any one of the sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 90% identical to any one of the sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 95% similar to any one of the sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 97% similar to any one of the sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 98% identical to any one of the sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 99% similar to any one of the sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is 100% similar to any one of the sequences as set forth in TABLE 1.

[0102] In some embodiments, when describing a certain percent (%) similarity in the context of an amino acid sequence, reference may be made to a value that is calculated by dividing a similarity score by the length of the alignment. In some embodiments, the similarity of two amino acid sequences can be calculated by using a BLOSUM62 similarity matrix (Henikoff and Henikoff, Proc. Natl. Acad. Set. USA., 89: 10915-10919 (1992)) that is transformed so that any value > 1 is replaced with +1 and any value < 0 is replaced with 0. For example, an He (I) to Leu (L) substitution is scored at +2.0 by the BLOSUM62 similarity matrix, which in the transformed matrix is scored at +1. This transformation allows the calculation of percent similarity, rather than a similarity score. Alternately, in some embodiments, when comparing two full protein sequences, the proteins can be aligned using pairwise MUSCLE alignment. Then, the % similarity can be scored at each residue and divided by the length of the alignment. For determining % similarity over a protein domain or motif, a multilevel consensus sequence (or PROSITE motif sequence) can be used to identify how strongly each domain or motif is conserved. In calculating the similarity of a domain or motif, the second and third levels of the multilevel sequence are treated as equivalent to the top level. Additionally, in some embodiments, if a substitution could be treated as conservative with any of the amino acids in that position of the multilevel consensus sequence, +1 point is assigned. For example, given the multilevel consensus sequence: RLG and YCK, the test sequence QIQ would receive three points. This is because in the transformed BLOSUM62 matrix, each combination is scored as: Q-R: +1; Q-Y: +0; I-L: +1; I-C: +0; Q-G: +0; Q-K: +1. For each position, the highest score is used when calculating similarity. In some embodiments, the % similarity can also be calculated using commercially available programs, such as the Geneious Prime software given the parameters matrix = BLOSUM62 and threshold > 1.

[0103] In some instances, effector proteins comprise a functional domain. The functional domain may comprise nucleic acid binding activity. The functional domain may comprise catalytic activity, also referred to as enzymatic activity. The catalytic activity may be nuclease activity. The nuclease activity may comprise cleaving a strand of a nucleic acid. The nuclease activity may comprise cleaving only one strand of a double stranded nucleic acid, also referred to as nicking. In some instances, the functional domain is an HNH domain. In some instances, the functional domain is a RuvC domain. In some instances, the RuvC domain comprises multiple subdomains. In some instances, the functional domain is a zinc finger binding domain. In some instances, the functional domain is a HEPN domain. In some instances, effector proteins lack a certain functional domain. In some instances, the effector protein lacks an HNH domain. In some embodiments, effector proteins lack a zinc finger binding domain.

[0104] In some instances, effector proteins disclosed herein may provide cleavage activity, such as cis cleavage activity, trans cleavage activity, nickase activity, nuclease activity, or a combination thereof. In some instances, effector proteins catalyze cleavage of a target nucleic acid in a cell or a sample. In some instances, the target nucleic acid is single stranded (ss). In some instances, effector proteins disclosed herein may cleave nucleic acids, including single stranded RNA (ssRNA), double stranded DNA (dsDNA), and single-stranded DNA (ssDNA). In some instances, the target nucleic acid is double stranded (ds). In some instances, the target nucleic acid is dsDNA. In some instances, the target nucleic acid is ssDNA. In some instances, the target nucleic acid is RNA. In some instances, effector proteins cleave the target nucleic acid within a target sequence of the target nucleic acid. In some instances, effector proteins cleave the target nucleic acid, as well as additional nucleic acids in the cell or the sample, which may be referred to as trans cleavage activity or simply trans cleavage. In some instances, trans cleavage may occur near, but not within or directly adjacent to, a target sequence of a target nucleic acid that is hybridized to a guide nucleic acid. Trans cleavage activity may be triggered by the hybridization of the guide nucleic acid to the target nucleic acid. In some embodiments, effector proteins described herein edit a target nucleic acid by cis cleavage activity on the target nucleic acid.

[0105] In some embodiments, cleaving a nucleic acid molecule includes hydrolysis of a phosphodiester bond of a nucleic acid molecule resulting in breakage of that bond. In some embodiments, the breakage is a nick (hydrolysis of a single phosphodiester bond on one side of a double-stranded molecule), single strand break (hydrolysis of a single phosphodiester bond on a single-stranded molecule) or double strand break (hydrolysis of two phosphodiester bonds on both sides of a double-stranded molecule) depending upon whether the nucleic acid molecule is single-stranded (e.g., ssDNA or ssRNA) or double-stranded (e.g., dsDNA) and the type of nuclease activity being catalyzed by the effector protein. [0106] In some instances, effector proteins catalyze cis cleavage activity. In some instances, effector proteins cleave both strands of dsDNA. In some instances, effector proteins described herein edit a target nucleic acid by cis cleavage activity on the target nucleic acid.

[0107] In some embodiments, compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises one or more amino acid alterations relative to any one of the sequences recited in TABLE 1. In some embodiments, the effector protein comprising one or more amino acid alterations is a variant of an effector protein described herein. It is understood that any reference to an effector protein herein also refers to an effector protein variant as described herein. In some embodiments, when describing a variant, reference may be made to a form or version of a protein that differs from the wild-type protein. A variant may have a different function or activity relative to the wild-type protein.

[0108] In some embodiments, the one or more amino acid alterations comprises conservative substitutions, non-conservative substitutions, conservative deletions, non-conservative deletions, or combinations thereof. In some embodiments, an effector protein or a nucleic acid encoding the effector protein comprises 1 amino acid alteration, 2 amino acid alterations, 3 amino acid alterations, 4 amino acid alterations, 5 amino acid alterations, 6 amino acid alterations, 7 amino acid alterations, 8 amino acid alterations, 9 amino acid alterations, 10 amino acid alterations or more relative to any one of the sequences recited in TABLE 1. In some embodiments, the one or more amino acid alterations may result in a change in activity of the effector protein relative to a naturally -occurring counterpart. For example, and as described in further detail below, the one or more amino acid alteration increases or decreases catalytic activity of the effector protein relative to a naturally-occurring counterpart. In some embodiments, the one or more amino acid alterations results in a catalytically inactive effector protein variant. [0109] Effector proteins may be a modified effector protein having reduced modification activity (e.g. , a catalytically defective effector protein). Effector proteins may be a modified effector protein having no modification activity (e.g., a catalytically inactive effector protein). In some instances, the effector protein may have a mutation in a nuclease domain. In some instances, the nuclease domain is a RuvC domain. By way of non-limiting example, the mutation may substitute an amino acid selected from glutamate and aspartate with an amino acid other than glutamate and aspartate. A RuvC domain may be characterized by a six-stranded beta sheet surrounded by four alpha helices, with three conserved subdomains contributing catalytic to the activity of the RuvC domain.

[0110] When describing a mutation that changes an amino acid residue or a nucleotide as described herein, such a change or changes can include, for example, deletions, insertions, and/or substitutions. The mutation can refer to a change in structure of an amino acid residue or nucleotide relative to the starting or reference residue or nucleotide. A mutation of an amino acid residue includes, for example, deletions, insertions and substituting one amino acid residue for a structurally different amino acid residue. Such substitutions can be a conservative substitution, a non-conservative substitution, a substitution to a specific sub-class of amino acids, or a combination thereof as described herein. A mutation of a nucleotide includes, for example, changing one naturally occurring base for a different naturally occurring base, such as changing an adenine to a thymine or a guanine to a cytosine or an adenine to a cytosine or a guanine to a thymine. A mutation of a nucleotide base may result in a structural and/or functional alteration of the encoding peptide, polypeptide or protein by changing the encoded amino acid residue of the peptide, polypeptide or protein. A mutation of a nucleotide base may not result in an alteration of the amino acid sequence or function of encoded peptide, polypeptide or protein, also known as a silent mutation.

[oni] It is understood that the terms “nucleotide” and “nucleoside” when used in the context of a nucleic acid molecule having multiple residues are used interchangeably and mean the sugar and base of the residue contained in the nucleic acid molecule. A skilled artisan could understand that linked nucleotides and/or linked nucleosides, as used in the context of a nucleic acid having multiple linked residues, are interchangeable and describe linked sugars and bases of residues contained in a nucleic acid molecule. Similarly, the term “nucleobase” when used in the context of a nucleic acid molecule can refer to the base of the residue contained in the nucleic acid molecule, for example, the base of a nucleotide or a nucleoside. Likewise, a linked nucleobase may refer to the base of linked nucleotides or linked nucleosides. A person of ordinary skill in the art when referring to nucleotides, nucleosides, and/or nucleobases would also understand the differences between RNA and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs, such as modified uridines, do not contribute to differences in identity or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5- methylcytosine, both of which have guanosine or modified guanosine as a complement). Thus, for example, the sequence 5'-AXG where X is any modified uridine, such as pseudouridine, Nl-methyl pseudouridine, or 5 -methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5' -CAU).

[0112] When a conservative substitution is described herein, such a substitution refers to the replacement of one amino acid for another such that the replacement takes place within a family of amino acids that are related in their side chains. Alternatively, a non-conservative substitution, when described herein, refers to the replacement of one amino acid residue for another such that the replaced residue is going from one family of amino acids to a different family of residues. Genetically encoded amino acids can be divided into four families: (1) acidic (negatively charged) = Asp (D), Glu (G); (2) basic (positively charged) = Lys (K), Arg (R), His (H); (3) non-polar (hydrophobic) = Cys (C), Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Met (M), Trp (W), Gly (G), Tyr (Y), with non-polar also being subdivided into: (i) strongly hydrophobic = Ala (A), Vai (V), Leu (L), He (I), Met (M), Phe (F); and (ii) moderately hydrophobic = Gly (G), Pro (P), Cys (C), Tyr (Y), Trp (W); and (4) uncharged polar = Asn (N), Gin (Q), Ser (S), Thr (T). In alternative fashion, the amino acid repertoire can be grouped as (1) acidic (negatively charged) = Asp (D), Glu (G); (2) basic (positively charged) = Lys (K), Arg

(R), His (H), and (3) aliphatic = Gly (G), Ala (A), Vai (V), Leu (L), He (I), Ser (S), Thr (T), with Ser

(S) and Thr (T) optionally being grouped separately as aliphatic-hydroxyl; (4) aromatic = Phe (F), Tyr (Y), Trp (W); (5) amide = Asn (N), Glu (Q); and (6) sulfur-containing = Cys (C) and Met (M) (see, for example, Biochemistry, 4th ed., Ed.

[0113] An effector protein may be brought into proximity of a target nucleic acid in the presence of a guide nucleic acid when the guide nucleic acid includes a nucleotide sequence that is complementary to or is a reverse complementary sequence to a target sequence in the target nucleic acid. The ability of an effector protein to modify a target nucleic acid may be dependent upon the effector protein being bound to a guide nucleic acid and the guide nucleic acid being hybridized to a target nucleic acid. An effector protein may recognize a protospacer adjacent motif (PAM) sequence present in the target nucleic acid, which may direct the modification activity of the effector protein. An effector protein may modify a nucleic acid by cis cleavage or trans cleavage. The modification of the target nucleic acid generated by an effector protein may, as a non-limiting example, result in modulation of the expression of the nucleic acid (e.g., increasing or decreasing expression of the nucleic acid) or modulation of the activity of a translation product of the target nucleic acid (e.g, inactivation of a protein binding to an RNA molecule or hybridization).

[0114] An effector protein may be a CRISPR-associated (“Cas”) protein. An effector protein may function as a single protein, including a single protein that is capable of binding to a guide nucleic acid and modifying a target nucleic acid. Alternatively, an effector protein may function as part of a multiprotein complex, including, for example, a complex having two or more effector proteins, including two or more of the same effector proteins (e.g., dimer or multimer). An effector protein, when functioning in a multiprotein complex, may have only one functional activity (e.g, binding to a guide nucleic acid), while other effector proteins present in the multiprotein complex are capable of the other functional activity (e.g. , modifying a target nucleic acid). An effector protein may be a modified effector protein having reduced modification activity (e.g., a catalytically defective effector protein) or no modification activity (e.g. , a catalytically inactive effector protein). Accordingly, an effector protein as used herein encompasses a modified or programmable nuclease that does not have nuclease activity.

Engineered Proteins

[0115] In some instances, effector proteins disclosed herein are engineered proteins. Engineered proteins are not identical to a naturally-occurring protein. In some embodiments, engineered proteins described herein have been modified. Such an engineered protein can include one or more mutations, including an insertion, deletion or substitution (e.g., conservative or non-conservative substitution). In some embodiments, modifications or mutations of effector proteins comprise the addition of one or more amino acids, deletion of one or more amino acids, substitution of one or more amino acids, or combinations thereof. In some embodiments, effector proteins disclosed herein are engineered proteins. Unless otherwise indicated, reference to effector proteins throughout the present disclosure include engineered proteins thereof.

[0116] In some embodiments, effector proteins described herein can be modified with the addition of one or more heterologous peptides or heterologous polypeptides (referred to collectively herein as a heterologous polypeptide). In some embodiments, an effector protein modified with the addition of one or more heterologous peptides or heterologous polypeptides may be referred to herein as a fusion protein. Such fusion proteins are described herein and throughout.

[0117] In some embodiments, a heterologous peptide or heterologous polypeptide comprises a subcellular localization signal. In some embodiments, a subcellular localization signal can be a nuclear localization signal (NLS). An effector protein disclosed herein or fusion effector protein may comprise a nuclear localization signal (NLS). In some embodiments, the NLS facilitates localization of a nucleic acid, protein, or small molecule to the nucleus, when present in a cell that contains a nuclear compartment. Exemplary NLS sequences are set forth in TABLE 2. The NLS may be located at a variety of locations, including, but not limited to 5 ’ of the effector protein, 5 ’ of the fusion partner, 3 ’ of the effector protein, 3 ’ of the fusion partner, between the effector protein and the fusion partner, within the fusion partner, within the effector protein.

[0118] In some embodiments, the subcellular localization signal is a nuclear export signal (NES), a sequence to keep an effector protein retained in the cytoplasm, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, an ER retention signal, and the like. In some embodiments, an effector protein described herein is not modified with a subcellular localization signal so that the polypeptide is not targeted to the nucleus, which can be advantageous depending on the circumstance (e.g., when the target nucleic acid is an RNA that is present in the cytosol). [0119] In some embodiments, a heterologous peptide or heterologous polypeptide comprises a chloroplast transit peptide (CTP), also referred to as a chloroplast localization signal or a plastid transit peptide, which targets the effector protein to a chloroplast. Chromosomal transgenes from bacterial sources may require a sequence encoding a CTP sequence fused to a sequence encoding an expressed protein (e.g. , the effector protein) if the expressed protein is to be compartmentalized in the plant plastid (e.g., chloroplast). The CTP may be removed in a processing step during translocation into the plastid. Accordingly, localization of an effector protein to a chloroplast is often accomplished by means of operably linking a polynucleotide sequence encoding a CTP sequence to the 5' region of a polynucleotide encoding the exogenous protein.

[0120] In some embodiments, the heterologous polypeptide is an endosomal escape peptide (EEP). An EEP is an agent that quickly disrupts the endosome in order to minimize the amount of time that a delivered molecule, such an effector protein, spends in the endosome-like environment, and to avoid getting trapped in the endosomal vesicles and degraded in the lysosomal compartment. An exemplary EEP is set forth in TABLE 2.

[0121] In some embodiments, the heterologous polypeptide is a cell penetrating peptide (CPP), also known as a Protein Transduction Domain (PTD). A CPP or PTD is a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane.

[0122] Further suitable heterologous polypeptides include, but are not limited to, proteins (or fragments/domains thereof) that are boundary elements (e.g., CTCF), proteins and fragments thereof that provide periphery recruitment (e.g., Lamin A, Lamin B, etc.), and protein docking elements (e.g., FKBP/FRB, Pill/Abyl, etc ).

[0123] In some embodiments, a heterologous peptide or heterologous polypeptide comprises a protein tag. In some embodiments, the protein tag is referred to as purification tag or a fluorescent protein. The protein tag may be detectable for use in detection of the effector protein and/or purification of the effector protein. Accordingly, in some embodiments, compositions, systems and methods comprise a protein tag or use thereof. Any suitable protein tag may be used depending on the purpose of its use. Non-limiting examples of protein tags include a fluorescent protein, a histidine tag, e.g., a 6XHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and maltose binding protein (MBP). In some embodiments, the protein tag is a portion of MBP that can be detected and/or purified. Non-limiting examples of fluorescent proteins include green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, and tdTomato.

[0124] A heterologous polypeptide may be located at or near the amino terminus (N-terminus) of the effector protein disclosed herein. A heterologous polypeptide may be located at or near the carboxy terminus (C-terminus) of the effector proteins disclosed herein. In some embodiments, a heterologous polypeptide is located internally in an effector protein described herein (i.e., is not at the N- or C- terminus of an effector protein described herein) at a suitable insertion site. [0125] In some embodiments, an effector protein described herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more heterologous polypeptides at or near the N-terminus, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more heterologous polypeptides at or near the C-terminus, or a combination of these (e.g., one or more heterologous polypeptides at the amino-terminus and one or more heterologous polypeptides at the carboxy terminus). When more than one heterologous polypeptide is present, each may be selected independently of the others, such that a single heterologous polypeptide may be present in more than one copy and/or in combination with one or more other heterologous polypeptides present in one or more copies. In some embodiments, a heterologous polypeptide is considered near the N- or C-terminus when the nearest amino acid of the heterologous polypeptide is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus.

[0126] In some embodiments, a heterologous polypeptide described herein comprises a heterologous polypeptide sequence recited in TABLE 2. In some embodiments, effector proteins described herein comprise an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any one of the sequences recited in TABLE 1 and further comprises one or more of the sequences set forth in TABLE 2. In some embodiments, a heterologous peptide described herein may be a fusion partner as described en supra.

[0127] An effector protein may be codon optimized for expression in a specific cell, for example, a bacterial cell, a plant cell, a eukaryotic cell, an animal cell, a mammalian cell, or a human cell. In some embodiments, the effector protein is codon optimized for a human cell. In some embodiments, effector proteins described herein are encoded by a codon optimized nucleic acid. In some embodiments, a nucleic acid sequence encoding an effector protein described herein is codon optimized. A nucleic acid encoding an effector protein may be codon optimized for expression in a specific cell, for example, a bacterial cell, a plant cell, a eukaryotic cell, an animal cell, a mammalian cell, or a human cell. This type of optimization can entail a mutation of an effector protein encoding nucleotide sequence to mimic the codon preferences of the intended host organism or cell while encoding the same polypeptide. Thus, the codons can be changed, but the encoded protein remains unchanged. For example, if the intended target cell was a human cell, a human codon-optimized effector protein-encoding nucleotide sequence could be used. Accordingly, in some embodiments, the nucleic acid encoding an effector protein is codon optimized for a human cell. As another non-limiting example, if the intended host cell were a mouse cell, then a mouse codon-optimized effector protein-encoding nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were a eukaryotic cell, then a eukaryote codon-optimized effector protein-encoding nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were a prokaryotic cell, then a prokaryote codon- optimized effector protein-encoding nucleotide sequence could be generated. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.or.jp/codon. [0128] It is understood that when describing coding sequences of polypeptides described herein, said coding sequences do not necessarily require a codon encoding a N-terminal Methionine (M) or a Valine (V) as described for the effector proteins described herein. One skilled in the art would understand that a start codon could be replaced or substituted with a start codon that encodes for an amino acid residue sufficient for initiating translation in a host cell.

[0129] An engineered protein, in some embodiments, includes at least one mutation relative to a reference protein (e.g., a naturally-occurring protein). In some embodiments, an engineered protein includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 mutations relative to a reference protein (e.g. , a naturally-occurring protein). Engineered proteins may not comprise an amino acid sequence that is identical to that of a naturally-occurring protein. In some instances, the amino acid sequence of an engineered protein is not identical to that of a naturally occurring protein. Engineered proteins may provide an increased activity relative to a naturally occurring protein. Engineered proteins may provide a reduced activity relative to a naturally occurring protein. The activity may be nuclease activity. The activity may be nickase activity. The activity may be nucleic acid binding activity.

[0130] Engineered proteins may provide an increased or reduced activity relative to a naturally occurring protein under a given condition of a cell or sample in which the activity occurs. The condition may be temperature. The temperature may be greater than 20°C, greater than 25°C, greater than 30°C, greater than 35°C, greater than 40°C, greater than 45°C, greater than 50°C, greater than 55°C, greater than 60°C, greater than 65°C, or greater than 70°C, but not greater than 80°C. The condition may be the presence of a salt. The salt may be a magnesium salt, a zinc salt, a potassium salt, a calcium salt, or a sodium salt. The condition may be the concentration of one or more salts.

[0131] In some instances, the amino acid sequence of an engineered protein comprises at least one residue that is different from that of a naturally occurring protein. In some instances, the amino acid sequence of an engineered protein comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 residues that are different from that of a naturally occurring protein. The residues in the engineered protein that differ from those at corresponding positions of the naturally occurring protein (when the engineered and naturally occurring proteins are aligned for maximal identity) may be referred to as substituted residues or amino acid substitutions. In some instances, the substituted residues are non-conserved residues relative to the residues at corresponding positions of the naturally occurring protein. A non-conserved residue has a different physicochemical property from the amino acid for which it substitutes. Physicochemical properties include aliphatic, cyclic, aromatic, basic, acidic and hydroxyl -containing. Glycine, alanine, valine, leucine, and isoleucine are aliphatic. Serine, Cysteine, threonine, and methionine are hydroxyl-containing. Proline is cyclic. Phenylalanine, tyrosine, tryptophan are basic. Aspartate, Glutamate, Asparagine, and glutamine are acidic.

[0132] In some embodiments, effector proteins may comprise one or more modifications that may provide altered activity as compared to a naturally-occurring counterpart (e.g., a naturally-occurring nuclease or nickase, activity which may be a naturally-occurring effector protein). In some embodiments, activity (e.g., nickase, nuclease, binding activity) of effector proteins described herein can be measured relative to a naturally-occurring effector protein or compositions containing the same in a cleavage assay. For example, effector proteins may comprise one or more modifications that may provide increased activity as compared to a naturally-occurring counterpart. As another example, effector proteins may provide increased catalytic activity (e.g., nickase, nuclease) or binding activity as compared to a naturally-occurring counterpart. Effector proteins may provide enhanced nucleic acid binding activity (e.g. , enhanced binding of a guide nucleic acid, and/or target nucleic acid) as compared to a naturally-occurring counterpart. An effector protein may have a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 200%, or more, increase of the activity of a naturally-occurring counterpart.

[0133] Alternatively, effector proteins may comprise one or more modifications that reduce the activity of the effector proteins relative to a naturally occurring nuclease, or nickase. An effector protein may have a 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less, decrease of the activity of a naturally occurring counterpart. Decreased activity may be decreased catalytic activity (e.g., nickase, nuclease) or binding activity as compared to a naturally-occurring counterpart.

[0134] In some instances, engineered proteins are designed to be catalytically inactive or to have reduced catalytic activity relative to a naturally occurring protein. In some embodiments, an effector protein that has decreased catalytic activity may be referred to as catalytically or enzymatically inactive, catalytically or enzymatically dead, as a dead protein or a dCas protein. In some embodiments, such a protein may comprise an enzymatically inactive domain (e.g. inactive nuclease domain). For example, a nuclease domain (e.g., RuvC domain) of an effector protein may be deleted or mutated relative to a wildtype counterpart so that it is no longer functional or comprises reduced nuclease activity.

[0135] A catalytically inactive effector protein may be generated by substituting an amino acid that confers a catalytic activity (also referred to as a “catalytic residue”) with a substituted residue that does not support the catalytic activity. In some instances, the substituted residue has an aliphatic side chain. In some instances, the substituted residue is glycine. In some instances, the substituted residue is valine. In some instances, the substituted residue is leucine. In some instances, the substituted residue is alanine. In some instances, the amino acid is aspartate, and it is substituted with asparagine. In some instances, the amino acid is glutamate, and it is substituted with glutamine.

[0136] An amino acid that confers catalytic activity may be identified by performing sequence alignment of an unmodified effector protein with a similar enzyme having at least one identified catalytic residue; selecting at least one putative catalytic residue in the unmodified effector protein within the portion of the unmodified effector protein that aligns with a portion of the similar enzyme that comprises the identified catalytic residue; substituting the at least one putative catalytic residue of the unmodified effector protein with the different amino acid; and comparing the catalytic activity of the unmodified effector protein to the modified effector protein.

[0137] A similar enzyme may be an enzyme that is at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identical to the unmodified effector protein. A similar enzyme may be an enzyme that is not greater than 99.9% identical to the unmodified effector protein. In some instances, the portion of the unmodified effector protein that aligns with a portion of the similar enzyme is at least 10 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, or at least 100 amino acids in length. In some instances, the portion of the unmodified effector protein that aligns with a portion of the similar enzyme is not greater than 200 amino acids. In some instances, the portion of the unmodified effector protein that aligns with a portion of the similar enzyme comprises a functional domain (e.g., HEPN, HNH, RuvC, zinc finger binding). In some instances, comparing the catalytic activity comprises performing a cleavage assay. An example of generating a catalytically inactive effector protein is provided in Example 8.

[0138] In some embodiments, a catalytically inactive effector protein may bind to a guide nucleic acid and/or a target nucleic acid but does not cleave the target nucleic acid. In some instances, a catalytically inactive effector protein may associate with a guide nucleic acid to activate or repress transcription of a target nucleic acid. In some instances, a catalytically inactive effector protein is fused to a fusion partner protein that confers an alternative activity to an effector protein activity. Such fusion proteins are described herein and throughout.

Fusion proteins

[0139] In some instances, compositions comprise a fusion effector protein, wherein the fusion effector protein comprises an effector protein described herein. In some instances, compositions comprise a nucleic acid encoding the fusion effector protein. In general, fusion effector proteins comprise an effector protein or a portion thereof, and a fusion partner protein. In some instances, compositions comprise a fusion effector protein and a guide nucleic acid, wherein at least a portion of the guide nucleic acid hybridizes to a target sequence of a target nucleic acid, and the fusion partner modulates the target nucleic acid or expression thereof. A fusion partner protein may also simply be referred to herein as a fusion partner. In general, the effector protein and the fusion partner protein are heterologous proteins.

[0140] In some cases, fusion effector proteins modify a target nucleic acid or the expression thereof. In some cases, the modifications are transient (e.g., transcription repression or activation). In some cases, the modifications are inheritable. For instance, epigenetic modifications made to a target nucleic acid, or to proteins associated with the target nucleic acid, e.g, nucleosomal histones, in a cell, are observed in cells produced by proliferation of the cell.

[0141] In some cases, fusion effector proteins modify a target nucleic acid or the expression thereof, wherein the target nucleic acid comprises a deoxyribonucleoside, a ribonucleoside or a combination thereof. The target nucleic acid may comprise or consist of a single stranded RNA (ssRNA), a doublestranded RNA (dsRNA), a single-stranded DNA (ssDNA), or a double stranded DNA (dsDNA). Nonlimiting examples of fusion partners for modifying ssRNA include, but are not limited to, splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation, elongation, and/or release factors; e.g., eIF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, e.g., adenosine deaminase acting on RNA (ADAR), including A to I and/or C to U editing enzymes); helicases; and RNA-binding proteins.

[0142] In some instances, the fusion partner protein is fused to the 5’ end of the effector protein. In some instances, the fusion partner protein is fused to the 3 ’ end of the effector protein. In some instances, the amino terminus of the fusion partner is linked/fused to the carboxy terminus of the effector protein. In some instances, the carboxy terminus of the fusion partner protein is linked/fused to the amino terminus of the effector protein by the linker. In some instances, the effector protein is located at an internal location of the fusion partner protein. In some instances, the fusion partner protein is located at an internal location of the Cas effector protein. For example, a base editing enzyme (e.g., a deaminase enzyme) is inserted at an internal location of a Cas effector protein. The effector protein may be fused directly or indirectly (e.g., via a linker) to the fusion partner protein. Exemplary linkers are described herein. Cas effector proteins may be fused to transcription activators or transcriptional repressors or deaminases or other nucleic acid modifying proteins. In some embodiments, Cas effector proteins need not be fused to a partner protein to accomplish the required protein (expression) modification. In some embodiments, the fusion partner protein is not fused to the effector protein.

[0143] In some embodiments, the fusion partner is not an effector protein as described herein. In some embodiments, the fusion partner comprises a second effector protein or a multimeric form thereof. Accordingly, in some embodiments, the fusion protein comprises more than one effector protein. In such embodiments, the fusion protein can comprise at least two effector proteins that are same. In some embodiments, the fusion protein comprises at least two effector proteins that are different. In some embodiments, the multimeric form is a homomeric form. In some embodiments, the multimeric form is a heteromeric form. Unless otherwise indicated, reference to effector proteins throughout the present disclosure include fusion proteins comprising the effector protein described herein and a fusion partner. [0144] In some embodiments, a fusion partner imparts some function or activity to a fusion protein that is not provided by an effector protein. Such activities may include but are not limited to nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, dimer forming activity (e.g., pyrimidine dimer forming activity), integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity, modification of a polypeptide associated with target nucleic acid (e.g., a histone), and/or signaling activity.

[0145] In some embodiments, a fusion partner may provide signaling activity. In some embodiments, a fusion partner may inhibit or promote the formation of multimeric complex of an effector protein. In an additional example, the fusion partner may directly or indirectly edit a target nucleic acid. Edits can be of a nucleobase, nucleotide, or nucleotide sequence of a target nucleic acid. In some embodiments, the fusion partner may interact with additional proteins, or functional fragments thereof, to make modifications to a target nucleic acid. In other embodiments, the fusion partner may modify proteins associated with a target nucleic acid. In some embodiments, a fusion partner may modulate transcription (e.g., inhibits transcription, increases transcription) of a target nucleic acid. In yet another example, a fusion partner may directly or indirectly inhibit, reduce, activate or increase expression of a target nucleic acid.

CRISPRi Fusions

[0146] In some instances, fusion partners inhibit or reduce expression of a target nucleic acid. Fusion proteins comprising such fusion partners and an effector protein may be referred to as CRISPRi fusions. In some instances, fusion partners reduce expression of the target nucleic acid relative to its expression in the absence of the fusion effector protein. Relative expression, including transcription and RNA levels, may be assessed, quantified, and compared, e.g. , by RT-qPCR. In some instances, fusion partners may comprise a transcriptional repressor. Transcriptional repressors may inhibit transcription via: recruitment of other transcription factor proteins; modification of target DNA such as methylation; recruitment of a DNA modifier; modulation of histones associated with target DNA; recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones; or a combination thereof.

[0147] Non-limiting examples of fusion partners that decrease or inhibit transcription include, but are not limited to: transcriptional repressors such as the Kriippel associated box (KRAB or SKD); K0X1 repression domain; the ZIM3 KRAB domain, the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), the SRDX repression domain (e.g., for repression in plants); histone lysine methyltransferases such as Pr-SET7/8, SUV4-20H1, RIZ1, and the like; histone lysine demethylases JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX, JARID1D/SMCY; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11; and DNA methylases such as Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), DNA methyltransferase 3 like (DNMT3L), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants); and periphery recruitment elements such as Lamin A, and Lamin B; and functional domains thereof.

CRISPRa Fusions

[0148] In some instances, fusion partners activate or increase expression of a target nucleic acid. Fusion proteins comprising such fusion partners and an effector protein may be referred to as CRISPRa fusions. In some instances, fusion partners increase expression of the target nucleic acid relative to its expression in the absence of the fusion effector protein. Relative expression, including transcription and RNA levels, may be assessed, quantified, and compared, e.g., by RT-qPCR. In some instances, fusion partners comprise a transcriptional activator. Transcriptional activators may promote transcription via: recruitment of other transcription factor proteins; modification of target DNA such as demethylation; recruitment of a DNA modifier; modulation of histones associated with target DNA; recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones; or a combination thereof. Non-limiting examples of fusion partners that activate or increase transcription include, but are not limited to: transcriptional activators such as VPR, VP 16, VP64, VP48, VP 160, p65 subdomain (e.g., from NFkB), and activation domain of EDLL and/or TAL activation domain (e.g. , for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1; histone lysine demethylases such as JHDM2a/b, UTX, JMJD3; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, SRC1, ACTR, P160, CLOCK; and DNA demethylases such as Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, TET2, DME, DML1, DML2, and ROS1; and functional domains thereof.

Modifying proteins

[0149] In some cases, a fusion partner provides enzymatic activity that modifies a protein (e.g., a histone) associated with a target nucleic acid. Such enzymatic activities include, but are not limited to, methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, de- ribosylation activity, myristoylation activity, and demyristoylation activity.

[0150] In some cases, the fusion partner has enzymatic activity that modifies a protein associated with a target nucleic acid. The protein may be a histone, an RNA binding protein, or a DNA binding protein. Examples of such protein modification activities include methyltransferase activity such as that provided by a histone methyltransferase (HMT) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A), euchromatic histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET/SETDB1, SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, DOT1L, Pr-SET7/8, SUV4-20H1, EZH2, RIZ1); demethylase activity such as that provided by a histone demethylase (e.g, Lysine Demethylase 1A (KDM1A also known as LSD1), JHDM2a/b, JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX, JARID1D/SMCY, UTX, JMJD3); acetyltransferase activity such as that provided by a histone acetylase transferase (e.g., catalytic core/fragment of the human acetyltransferase p300, GCN5, PCAF, CBP, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, HBO1/MYST2, HMOF/MYST1, SRC1, ACTR, P160, CLOCK); deacetylase activity such as that provided by ahistone deacetylase (e.g., HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11); kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, and demyristoylation activity.

Additional Fusion Partners

[0151] In some cases, the fusion partner is a chloroplast transit peptide (CTP), also referred to as a plastid transit peptide. In some embodiments, this targets the fusion protein to a chloroplast. Chromosomal transgenes from bacterial sources must have a sequence encoding a CTP sequence fused to a sequence encoding an expressed protein if the expressed protein is to be compartmentalized in the plant plastid (e.g. chloroplast). The CTP is removed in a processing step during translocation into the plastid. Accordingly, localization of an exogenous protein to a chloroplast is often accomplished by means of operably linking a polynucleotide sequence encoding a CTP sequence to the 5' region of a polynucleotide encoding the exogenous protein. In some cases, the CTP is located at the N-terminus of the fusion protein. Processing efficiency may, however, be affected by the amino acid sequence of the CTP and nearby sequences at the amino terminus (NH2 terminus) of the peptide.

[0152] In some cases, the fusion partner is an endosomal escape peptide (EEP). Exemplary EEPs are set forth in TABLE 2.

[0153] Further suitable fusion partners include, but are not limited to, proteins (or fragments/domains thereof) that are boundary elements (e.g. , CTCF), proteins and fragments thereof that provide periphery recruitment (e.g., Lamin A, Lamin B, etc.), protein docking elements (e.g., FKBP/FRB, Pill/Abyl, etc.). Other fusion partners are described throughout herein.

Base editors

[0154] In some instances, fusion partners modify a nucleobase of a target nucleic acid. Fusion proteins comprising such fusion partners and a catalytically inactive Cas effector protein may be referred to as base editors. In some instances, base editors modify a sequence of a target nucleic acid. In some embodiments, base editors provide a nucleobase change in a DNA molecule. In some instances, the nucleobase change in the DNA molecule is selected from: an adenine (A) to guanine (G); cytosine (C) to thymine (T); and cytosine (C) to guanine (G). In some embodiments, base editors provide a nucleobase change in an RNA molecule. In some instances, the nucleobase change in the RNA molecule is selected from: adenine (A) to guanine (G); uracil (U) to cytosine (C); cytosine (C) to guanine (G); and guanine (G) to adenine (A). In some instances, the fusion partner is a deaminase, e.g., ADAR1/2. Some base editors modify a nucleobase of on a single strand of DNA. In some embodiments, base editors modify a nucleobase on both strands of dsDNA. In some embodiments, upon binding to its target locus in DNA, base pairing between the guide RNA and target DNA strand leads to displacement of a small segment of single-stranded DNA in an “R-loop”. In some embodiments, DNA bases within the R-loop are modified by the deaminase enzyme. In some embodiments, DNA base editors for improved efficiency in eukaryotic cells comprise a catalytically inactive effector protein that may generate a nick in the non-edited DNA strand, inducing repair of the non-edited strand using the edited strand as a template.

[0155] In some embodiments, a catalytically inactive effector protein can comprise an effector protein that is modified relative to a naturally-occurring nuclease to have a reduced or eliminated catalytic activity relative to that of the naturally-occurring nuclease, but retains its ability to interact with a guide nucleic acid. The catalytic activity that is reduced or eliminated is often a nuclease activity. The naturally-occurring nuclease may be a wildtype protein. In some embodiments, the catalytically inactive effector protein is referred to as a catalytically inactive variant of a nuclease, e.g., a Cas nuclease.

[0156] Some base editors modify a nucleobase of an RNA. In some instances, RNA base editors comprise an adenosine deaminase. In some instances, ADAR proteins bind to RNAs and alter their sequence by changing an adenosine into an inosine. In some instances, RNA base editors comprise a Cas effector protein that is activated by or binds RNA. Non-limiting examples of Cas effector proteins that are activated by or bind RNA are Cas 13 proteins.

[0157] In some instances, base editors are used to treat a subject having or a subject suspected of having a disease related to a gene of interest. In some embodiments, base editors are useful for treating a disease or a disorder caused by a point mutation in a gene of interest. In some embodiments, compositions comprise a base editor and a guide nucleic acid, wherein the guide nucleic acid directs the base editor to a sequence in a target gene. The target gene may be associated with a disease. In some instances, the guide nucleic acid directs that base editor to or near a mutation in the sequence of a target gene. The mutation may be the deletion of one more nucleotides. The mutation may be the addition of one or more nucleotides. The mutation may be the substitution of one or more nucleotides. The mutation may be the insertion, deletion, or substitution of a single nucleotide, also referred to as a point mutation. The point mutation may be a SNP. The point mutation may be a chromosomal mutation, a copy number mutation, or any combination thereof. A point mutation optionally comprises a substitution, insertion, or deletion. In some embodiments, a mutation comprises a chromosomal mutation. A chromosomal mutation can comprise an inversion, a deletion, a duplication, or a translocation. In some embodiments, a mutation comprises a copy number variation. A copy number variation can comprise a gene amplification or an expanding trinucleotide repeat. The mutation may be associated with a disease. In some embodiments, the guide nucleic acid directs the base editor to bind a target sequence within the target nucleic acid that is within 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides ofthe mutation. In some embodiments, the guide nucleic acid comprises a sequence that is identical, complementary, or reverse complementary to a target sequence of a target nucleic acid that comprises the mutation. In some embodiments, the guide nucleic acid comprises a sequence that is identical, complementary, or reverse complementary to a target sequence of a target nucleic acid that is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides of the mutation.

[0158] In some embodiments, base editors are used to treat a subject having or a subject suspected of having a disease related to a gene of interest. In some embodiments, base editors are useful for treating a disease or a disorder caused by a point mutation in a gene of interest. In some embodiments, compositions comprise a base editor and a guide nucleic acid, wherein the guide nucleic acid directs the base editor to a sequence in a target gene.

[0159] In some embodiments, fusion partners comprise a base editing enzyme. In some embodiments, the base editing enzyme modifies the nucleotide of a deoxyribonucleotide. In some embodiments, the base editing enzyme modifies the nucleotide of a ribonucleotide. A base editing enzyme that converts a cytosine to a guanine or thymine may be referred to as a cytosine base editing enzyme. A base editing enzyme that converts an adenine to a to a guanine may be referred to as an adenine base editing enzyme. In some embodiments, the base editing enzyme comprises a deaminase enzyme. In some embodiments, the deaminase functions as a monomer. In some embodiments, the deaminase functions as heterodimer with an additional protein. In some embodiments, base editors comprise a DNA glycosylase inhibitor. In some embodiments, base editors comprise a uracil glycosylase inhibitor (UGI) or uracil N- glycosylase (UNG). In some embodiments, base editors do not comprise a UGI. In some embodiments, base editors do not comprise a UNG. In some embodiments, base editors do not comprise a functional fragment of a UGI. A functional fragment of a UGI is a fragment of a UGI that is capable of excising a uracil residue from DNA by cleaving an N-glycosydic bond. In some cases, a functional fragment comprises a fragment of a protein that retains some function relative to the entire protein. Non-limiting examples of functions are nucleic acid binding, protein binding, nuclease activity, nickase activity, deaminase activity, demethylase activity, or acetylation activity.

[0160] In some embodiments, a base editing enzyme can comprise a protein, polypeptide or fragment thereof that is capable of catalyzing the chemical modification of a nucleotide of a deoxyribonucleotide or a ribonucleotide. Such a base editing enzyme, for example, is capable of catalyzing a reaction that modifies a nucleotide that is present in a nucleic acid molecule, such as DNA or RNA (single stranded or double stranded). Non-limiting examples of the type of modification that a base editing enzyme is capable of catalyzing includes converting an existing nucleotide to a different nucleotide, such as converting a cytosine to a guanine or thymine or converting an adenine to a guanine, hydrolytic deamination of an adenine or adenosine, or methylation of cytosine (e.g., CpG, CpA, CpT or CpC). A base editing enzyme itself may or may not bind to the nucleic acid molecule containing the nucleotide. In some cases, a base editor can be a fusion protein comprising a base editing enzyme fused to an effector protein. The base editor is functional when the effector protein is coupled to a guide nucleic acid. The guide nucleic acid imparts sequence specific activity to the base editor. By way of nonlimiting example, the effector protein may comprise a catalytically inactive effector protein. Also, by way of non-limiting example, the base editing enzyme may comprise deaminase activity. Additional base editors are described herein.

[0161] In some embodiments, the base editor is a cytidine deaminase base editor generated by ancestral sequence reconstruction as described in WO2019226953, which is hereby incorporated by reference in its entirety.

[0162] Exemplary deaminase domains are described WO 2018027078 and W02017070632, and each are hereby incorporated in its entirety by reference. Also, additional exemplary deaminase domains are described in Komor et al., Nature, 533, 420-424 (2016); Gaudelli et al., Nature, 551, 464-471 (2017); Komor et al., Science Advances, 3:eaao4774 (2017), and Rees et al., Nat Rev Genet. 2018 Dec;19(12):770-788. doi: 10. 1038/s41576-018-0059-l, which are hereby incorporated by reference in their entirety.

[0163] In some embodiments, the base editor is a cytosine base editor (CBE). In general, a CBE comprises a cytosine base editing enzyme and a catalytically inactive effector protein. In some embodiments, the catalytically inactive effector protein is a catalytically inactive variant of an effector protein described herein. The CBE may convert a cytosine to a thymine. In some embodiments, the base editor is an adenine base editor (ABE). In general, an ABE comprises an adenine base editing enzyme and a catalytically inactive effector protein. In some embodiments, the catalytically inactive effector protein is a catalytically inactive variant of an effector protein described herein. The ABE generally converts an adenine to a guanine. In some embodiments, the base editor is a cytosine to guanine base editor (CGBE). In general, a CGBE converts a cytosine to a guanine.

[0164] In some embodiments, the base editor is a CBE. In some embodiments, the cytosine base editing enzyme is a cytidine deaminase. In some embodiments, the cytosine deaminase is an APOBEC1 cytosine deaminase, which accept ssDNA as a substrate but is incapable of cleaving dsDNA, fused to a catalytically inactive effector protein. In some embodiments, when bound to its cognate DNA, the catalytically inactive effector protein performs local denaturation of the DNA duplex to generate an R- loop in which the DNA strand not paired with the guide RNA exists as a disordered single-stranded bubble. In some embodiments, the catalytically inactive effector protein generated ssDNA R-loop enables the CBE to perform efficient and localized cytosine deamination in vitro. In some examples, deamination activity is exhibited in a window of about 4 to about 10 base pairs. In some embodiments, fusion to the catalytically inactive effector protein presents the target site to APOBEC 1 in high effective molarity, enabling the CBE to deaminate cytosines located in a variety of different sequence motifs, with differing efficacies. In some embodiments, the CBE is capable of mediating RNA-programmed deamination of target cytosines in vitro. In some embodiments, the CBE is capable of mediating RNA- programmed deamination of target cytosines in vivo. In some embodiments, the cytosine base editing enzyme is a cytosine base editing enzyme described by Koblan et al. (2018) Nature Biotechnology 36:848-846; Komor et al. (2016) Nature 533:420-424; Koblan et al. (2021) “Efficient OG-to-G’C base editors developed using CRISPRi screens, target-library analysis, and machine learning,” Nature Biotechnology, Kurt et al. (2021) Nature Biotechnology 39:41-46; Zhao et al. (2021) Nature Biotechnology 39:35-40; and Chen et al. (2021) Nature Communications 12: 1384, all incorporated herein by reference.

[0165] In some embodiments, CBEs comprise a uracil glycosylase inhibitor (UGI) or uracil N- glycosylase (UNG). In some embodiments, base excision repair (BER) of U*G in DNA is initiated by a UNG, which recognizes the U*G mismatch and cleaves the glyosidic bond between uracil and the deoxyribose backbone of DNA. In some embodiments, BER results in the reversion of the U*G intermediate created by the first CBE back to a C*G base pair. In some embodiments, UNG may be inhibited by fusion of uracil DNA glycosylase inhibitor (UGI), in some embodiments, a small protein from bacteriophage PBS, to the C-terminus of the CBE. In some embodiments, UGI is a DNA mimic that potently inhibits both human and bacterial UNG. In some embodiments, a UGI inhibitor is any protein or polypeptide that inhibits UNG. In some embodiments, the CBE mediates efficient base editing in bacterial cells and moderately efficient editing in mammalian cells, enabling conversion of a C*G base pair to a T«A base pair through a U*G intermediate. In some embodiments, the CBE is modified to increase base editing efficiency while editing more than one strand of DNA.

[0166] In some embodiments, the CBE nicks the non-edited DNA strand. In some embodiments, the non-edited DNA strand nicked by the CBE biases cellular repair of the U*G mismatch to favor a U*A outcome, elevating base editing efficiency. In some embodiments, the APOBEC1- nickase-UGI fusion efficiently edits in mammalian cells, while minimizing frequency of non-target indels.

[0167] In some embodiments, the cytidine deaminase is selected from APOBEC1, APOBEC2, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, APOBEC3A, BE1 (APOBECl-XTEN-dCas9), BE2 (APOBECl-XTEN-dCas9-UGI), BE3 (APOBEC1-XTEN- dCas9(A840H)-UGI), BE3-Gam, saBE3, saBE4-Gam, BE4, BE4-Gam, saBE4, or saBE4-Gam as described in WO2021163587, WO202108746, WO2021062227, and WO2020123887, which are incorporated herein by reference in their entirety.

[0168] In some embodiments, the fusion protein further comprises a non-protein uracil-DNA glcosylase inhibitor (npUGI). In some embodiments, the npUGI is selected from a group of small molecule inhibitors of uracil-DNA glycosylase (UDG), or a nucleic acid inhibitor of UDG. In some embodiments, the non-protein uracil-DNA glcosylase inhibitor (npUGI) is a small molecule derived from uracil. Examples of small molecule non-protein uracil-DNA glcosylase inhibitors, fusion proteins, and Cas-CRISPR systems comprising base editing activity are described in WO202108746, which is incorporated by reference in its entirety.

[0169] In some embodiments, the fusion partner is a deaminase, e.g., ADAR1/2, ADAR-2, or AID. In some embodiments, the base editor is an ABE. In some embodiments, the adenine base editing enzyme of the ABE is an adenosine deaminase. In some embodiments, the adenine base editing enzyme is selected from ABE8e, ABE8.20m, APOBEC3A, Anc APOBEC (a.k.a. AncBE4Max), and BtAPOBEC2. In some embodiments, the ABE base editor is an ABE7 base editor. In some embodiments, the deaminase or enzyme with deaminase activity is selected from ABE8. Im, ABE8.2m, ABE8.3m, ABE8.4m, ABE8.5m, ABE8.6m, ABE8.7m, ABE8.8m, ABE8.9m, ABE8. 10m, ABE8. 1 Im, ABE8.12m, ABE8.13m, ABE8.14m, ABE8.15m, ABE8.16m, ABE8.17m, ABE8.18m, ABE8.19m, ABE8.20m, ABE8.21m, ABE8.22m, ABE8.23m, ABE8.24m, ABE8.1d, ABE8.2d, ABE8.3d, ABE8.4d, ABE8.5d, ABE8.6d, ABE8.7d, ABE8.8d, ABE8.9d, ABE8.10d, ABE8.11d, ABE8.12d, ABE8.13d, ABE8.14d, ABE8.15d, ABE8.16d, ABE8.17d, ABE8.18d, ABE8.19d, ABE8.20d, ABE8.21d, ABE8.22d, ABE8.23d, or ABE8.24d. In some embodiments, the adenine base editing enzyme is ABE8. Id. In some embodiments, the adenosine base editor is ABE9. Exemplary deaminases are described in US20210198330, WO2021041945, W02021050571A1, and WO2020123887, all of which are incorporated herein by reference in their entirety. In some embodiments, the adenine base editing enzyme is an adenine base editing enzyme described in Chu et al., (2021) The CRISP R Journal 4:2: 169-177, incorporated herein by reference. In some embodiments, the adenine deaminase is an adenine deaminase described by Koblan et al. (2018) Nature Biotechnology 36:848-846, incorporated herein by reference. In some embodiments, the adenine base editing enzyme is an adenine base editing enzyme described by Tran et al. (2020) Nature Communications 11:4871. Additional examples of deaminase domains are also described in W02018027078 and W02017070632, which are hereby incorporated by reference in their entirety.

[0170] In some embodiments, an ABE converts an A«T base pair to a G*C base pair. In some embodiments, the ABE converts a target A«T base pair to G*C in vivo. In some embodiments, the ABE converts a target A«T base pair to G*C in vitro. In some embodiments, ABEs provided herein reverse spontaneous cytosine deamination, which has been linked to pathogenic point mutations. In some embodiments, ABEs provided herein enable correction of pathogenic SNPs (-47% of disease- associated point mutations). In some embodiments, the adenine comprises exocyclic amine that has been deaminated (e.g., resulting in altering its base pairing preferences). In some embodiments, deamination of adenosine yields inosine. In some embodiments, inosine exhibits the base-pairing preference of guanine in the context of a polymerase active site, although inosine in the third position of a tRNA anticodon is capable of pairing with A, U, or C in mRNA during translation. In some embodiments, an ABE comprises an engineered adenosine deaminase enzyme capable of acting on ssDNA.

[0171] In some embodiments, a base editor comprises an adenosine deaminase variant that differs from a naturally occurring deaminase. Relative to the naturally occurring deaminase, the adenosine deaminase variant may comprise a V82S alteration, a T166R alteration, or a combination thereof. In some embodiments, the adenosine deaminase variant comprises at least one of the following alterations relative to a naturally occurring adenosine deaminase: Y147T, Y147R, Q154S, Y123H, and Q154R., which are incorporated herein by reference in their entirety.

[0172] In some embodiments, a base editor comprises a deaminase dimer. In some embodiments, a base editor is a deaminase dimer further comprising a base editing enzyme and an adenine deaminase (e.g., TadA).

[0173] TadA comprises or consists of a sequence: SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIM ALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVL HHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 168).

[0174] In some embodiments, the adenosine deaminase is a TadA monomer (e.g., Tad*7.10, TadA*8 or TadA* 9). In some embodiments, the adenosine deaminase is a TadA* 8 variant. Such a TadA* 8 variant includes TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24 as described in WO2021163587 and W02021050571, which are each hereby incorporated by reference in its entirety.

[0175] In some embodiments, a base editor is a deaminase dimer comprising a base editing enzyme fused to TadA via a linker. In some embodiments the linker comprises or consists of a sequence: SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 169). In some embodiments, the amino acid sequence of the linker is 70%, 75%, 80%, 85%, 90%, or 95% identical to SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 169).

[0176] In some embodiments, the amino terminus of the fusion partner protein is linked to the carboxy terminus of the effector protein via the linker. In some embodiments, the carboxy terminus of the fusion partner protein is linked to the amino terminus of the effector protein via the linker.

[0177] In some embodiments, the base editing enzyme is fused to TadA at the N-terminus. In some embodiments, the base editing enzyme is fused to TadA at the C-terminus. In some embodiments, the base editing enzyme is a deaminase dimer comprising an ABE. In some embodiments, the deaminase dimer comprises an adenosine deaminase. In some embodiments, the deaminase dimer comprises TadA fused to an adenine base editing enzyme selected from ABE8e, ABE8.20m, APOBEC3A, Anc APOBEC (a.k.a. AncBE4Max), and BtAPOBEC2. In some embodiments TadA is fused to ABE8e or a variant thereof. In some embodiments TadA is fused to ABE8e or a variant thereof at the aminoterminus (ABE8e-TadA). In some embodiments, TadA is fused to ABE8e or a variant thereof at the carboxy terminus (ABE8e-TadA).

Prime Editing

[0178] In some embodiments, a fusion protein and/or a fusion partner can comprise a prime editing enzyme. In some embodiments, a prime editing enzyme is a protein, a polypeptide or a fragment thereof that is capable of catalyzing the modification (insertion, deletion, or base-to-base conversion) of a target nucleotide or nucleotide sequence in a nucleic acid. A prime editing enzyme capable of catalyzing such a reaction includes a reverse transcriptase. A prime editing enzyme may require a prime editing guide RNA (pegRNA) to catalyze the modification. Such a pegRNA can be capable of identifying the nucleotide or nucleotide sequence in the target nucleic acid to be edited and encoding the new genetic information that replaces the targeted nucleotide or nucleotide sequence in the nucleic acid. A prime editing enzyme may require a prime editing guide RNA (pegRNA) and a single guide RNA to catalyze the modification. In some embodiments, such a prime editing enzyme is an M-MLV RT enzyme or a mutant thereof. In some embodiments, the M-MLV RT enzyme comprises at least one mutation selected from D200N, L603W, T330P, T306K, and W313F relative to wildtype M-MLV RT enzyme.

Recombinases

[0179] In some embodiments, the fusion partners comprise a recombinase domain. In some embodiments, the recombinase is a site-specific recombinase. Examples of site-specific recombinases include a tyrosine recombinase, a serine recombinase, an integrase, or mutants or variants thereof. In some embodiments, the site-specific recombinase is a tyrosine recombinase. Non-limiting examples of a tyrosine recombinase is Cre, Flp or lambda integrase. In some embodiments, the recombinase is a serine recombinase. Non-limiting examples of serine recombinases include, but are not limited to, gamma-delta resolvase, Tn3 resolvase, Sin resolvase, Gin invertase, Hin invertase, Tn5044 resolvase, IS607 transposase, and IS607 integrase. In some embodiments, the site-specific recombinase is an integrase. Non-limiting examples of integrases include, but are not limited to: Bxbl, wBeta, BL3, phiR4, AI I8, TGI, MR11, phi370, SPBc, TP90I-I, phiRV, FC1, K38, phiBTl, and phiC31. In some embodiments, the recombinase is a Tn5 transposase, SB100X, phage encoded serine integrases/ recombinase 2, phage encoded serine integrase/ recombinase 13, or Human WT Exonuclease la. Further discussion and examples of suitable recombinase fusion partners are described in US 10,975,392, which is incorporated herein by reference in its entirety.

[0180] In some embodiments, the fusion protein comprises a linker that links the recombinase domain to the Cas-CRISPR domain of the effector protein. In some embodiments, the linker is The-Ser.

Modifying target nucleic acids

[0181] In some cases, fusion partners provide enzymatic activity that modifies a target nucleic acid. Such enzymatic activities include, but are not limited to, nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity. In some cases, nuclease activity comprises the enzymatic activity of an enzyme which allows the enzyme to cleave the phosphodiester bonds between the nucleotide subunits of nucleic acids. In some case, an enzyme with nuclease activity can comprise a nuclease.

[0182] Disclosed herein are compositions and methods for modifying a target nucleic acid. The target nucleic acid may be a gene or a portion thereof. Methods and compositions may modify a coding portion of a gene, a non-coding portion of a gene, or a combination thereof. Modifying at least one gene using the compositions and methods described herein may reduce or increase expression of one or more genes. In some embodiments, compositions and methods reduce expression of one or more genes by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, compositions and methods remove all expression of a gene, also referred to as genetic knock out. In some embodiments, compositions and methods increase expression of one or more genes by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%.

[0183] In some embodiments, compositions and methods use effector proteins that are fused to a heterologous protein. Heterologous proteins include, but are not limited to, transcriptional activators, transcriptional repressors, deaminases, methyltransferases, acetyltransferases, and other nucleic acid modifying proteins. In some cases, effector proteins need not be fused to a partner protein to accomplish the required protein (expression) modification.

[0184] In somecases, fusion partners have enzymatic activity that modifies the target nucleic acid. The target nucleic acid may comprise or consist of a ssRNA, dsRNA, ssDNA, or a dsDNA. Examples of enzymatic activity that modifies the target nucleic acid include, but are not limited to: nuclease activity such as that provided by a restriction enzyme (e.g., FokI nuclease); methyltransferase activity such as that provided by a methyltransferase (e.g., Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants)); demethylase activity such as that provided by a demethylase (e.g., Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1); DNA repair activity; DNA damage (e.g., oxygenation) activity; deamination activity such as that provided by a deaminase (e.g., a cytosine deaminase enzyme such as rat APOBEC1); dismutase activity; alkylation activity; depurination activity; oxidation activity; pyrimidine dimer forming activity; integrase activity such as that provided by an integrase and/or resolvase (e.g., Gin invertase such as the hyperactive mutant of the Gin invertase, GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase); transposase activity, recombinase activity such as that provided by a recombinase (e.g., catalytic domain of Gin recombinase); as well as polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.

[0185] Non-limiting examples of fusion partners for targeting ssRNA include, but are not limited to, splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation, elongation, and/or release factors; e.g., eIF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, e.g., adenosine deaminase acting on RNA (ADAR), including A to I and/or C to U editing enzymes); helicases; and RNA-binding proteins. It is understood that a fusion protein may include the entire protein or in some cases may include a fragment of the protein (e.g., a functional domain). In some embodiments, the functional domain interacts with or binds ssRNA, including intramolecular and/or intermolecular secondary structures thereof, e.g., hairpins, stem-loops, etc.). The functional domain may interact transiently or irreversibly, directly or indirectly. In some cases, a functional domain comprises a region of one or more amino acids in a protein that is required for an activity of the protein, or the full extent of that activity, as measured in an in vitro assay. Activities include, but are not limited to nucleic acid binding, nucleic acid modification, nucleic acid cleavage, protein binding. The absence of the functional domain, including mutations of the functional domain, would abolish or reduce activity. Fusion proteins may comprise a protein or domain thereof selected from: endonucleases (e.g., RNase III, the CRR22 DYW domain, Dicer, and PIN (PilT N-terminus); SMG5 and SMG6; domains responsible for stimulating RNA cleavage (e.g., CPSF, CstF, CFIm and CFIIm); exonucleases such as XRN-1 or Exonuclease T; deadenylases such as HNT3; protein domains responsible for nonsense mediated RNA decay (e.g., UPF1, UPF2, UPF3, UPF3b, RNP SI, Y14, DEK, REF2, and SRml60); protein domains responsible for stabilizing RNA (e.g., PABP); proteins and protein domains responsible for repressing translation (e.g., Ago2 and Ago4); proteins and protein domains responsible for stimulating translation (e.g., Staufen); proteins and protein domains responsible for (e.g., capable of) modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains responsible for polyadenylation of RNA (e.g., PAP1, GLD-2, and Star- PAP); proteins and protein domains responsible for polyuridinylation of RNA (e.g., CI DI and terminal uridylate transferase); proteins and protein domains responsible for RNA localization (e.g., from IMP1, ZBP1, She2p, She3p, and Bicaudal-D); proteins and protein domains responsible for nuclear retention of RNA (e.g., Rrp6); proteins and protein domains responsible for nuclear export of RNA (e.g., TAP, NXF1, THO, TREX, REF, and Aly); proteins and protein domains responsible for repression of RNA splicing (e.g., PTB, Sam68, and hnRNP Al); proteins and protein domains responsible for stimulation of RNA splicing (e.g., Serine/Arginine-rich (SR) domains); proteins and protein domains responsible for reducing the efficiency of transcription (e.g., FUS (TLS)); and proteins and protein domains responsible for stimulating transcription (e.g., CDK7 and HIV Tat). Alternatively, the effector domain may be a domain of a protein selected from the group comprising endonucleases; proteins and protein domains capable of stimulating RNA cleavage; exonucleases; deadenylases; proteins and protein domains having nonsense mediated RNA decay activity; proteins and protein domains capable of stabilizing RNA; proteins and protein domains capable of repressing translation; proteins and protein domains capable of stimulating translation; proteins and protein domains capable of modulating translation (e.g. , translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains capable of polyadenylation of RNA; proteins and protein domains capable of polyuridinylation of RNA; proteins and protein domains having RNA localization activity; proteins and protein domains capable of nuclear retention of RNA; proteins and protein domains having RNA nuclear export activity; proteins and protein domains capable of repression of RNA splicing; proteins and protein domains capable of stimulation of RNA splicing; proteins and protein domains capable of reducing the efficiency of transcription; and proteins and protein domains capable of stimulating transcription. Another suitable fusion partner is a PUF RNA- binding domain, which is described in more detail in WO2012068627, which is hereby incorporated by reference in its entirety.

[0186] In some instances, fusion partners comprise an RNA splicing factor. The RNA splicing factor may be used (in whole or as fragments thereof) for modular organization, with separate sequencespecific RNA binding modules and splicing effector domains. Non-limiting examples of RNA splicing factors include members of the Serine/ Arginine-rich (SR) protein family contain N-terminal RNA recognition motifs (RRMs) that bind to exonic splicing enhancers (ESEs) in pre-mRNAs and C-terminal RS domains that promote exon inclusion. As another example, the hnRNP protein hnRNP Al binds to exonic splicing silencers (ESSs) through its RRM domains and inhibits exon inclusion through a C- terminal Glycine-rich domain. Some splicing factors may regulate alternative use of splice site (ss) by binding to regulatory sequences between the two alternative sites. For example, ASF/SF2 may recognize ESEs and promote the use of intron proximal sites, whereas hnRNP Al may bind to ESSs and shift splicing towards the use of intron distal sites. One application for such factors is to generate ESFs that modulate alternative splicing of endogenous genes, particularly disease associated genes. For example, Bcl-x pre-mRNA produces two splicing isoforms with two alternative 5' splice sites to encode proteins of opposite functions. The long splicing isoform Bcl-xL is a potent apoptosis inhibitor expressed in long-lived postmitotic cells and is up-regulated in many cancer cells, protecting cells against apoptotic signals. The short isoform Bcl-xS is a pro-apoptotic isoform and expressed at high levels in cells with a high turnover rate (e.g., developing lymphocytes). The ratio of the two Bcl-x splicing isoforms is regulated by multiple ccb-clcmcnts that are located in either the core exon region or the exon extension region (i.e., between the two alternative 5' splice sites). For more examples, see W02010075303, which is hereby incorporated by reference in its entirety.

Linkers

[0187] In general, effector proteins and fusion partners of a fusion effector protein are connected via a linker. The linker may comprise or consist of a covalent bond. The linker may comprise or consist of a chemical group. In some instances, the linker comprises an amino acid. In some embodiments, a linker comprises a bond or molecule that links a first polypeptide to a second polypeptide. In some instances, a peptide linker comprises at least two amino acids linked by an amide bond. In general, the linker connects a terminus of the effector protein to a terminus of the fusion partner. In some instances, the carboxy terminus of the effector protein is linked to the amino terminus of the fusion partner. In some instances, the carboxy terminus of the fusion partner is linked to the amino terminus of the effector protein. In some embodiments, the effector protein and the fusion partner are directly linked by a covalent bond.

[0188] In some cases, fusion effector proteins disclosed herein comprise a linker, wherein the linker comprises or consists of a peptide. The peptide may have any of a variety of amino acid sequences. The peptide may comprise a region of rigidity (e.g., beta sheet, alpha helix), a region of flexibility, or any combination thereof. In some embodiments, the linker comprises small amino acids, such as glycine and alanine, that impart linker flexibility. In some embodiments, the linker comprises amino acids that impart linker rigidity, such as valine and isoleucine. In some embodiments, the linker comprises small amino acids, such as glycine and alanine, that impart high degrees of flexibility. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any desired element may include linkers that are all or partially flexible, such that the linker may include a flexible linker as well as one or more portions that confer less flexible structure. Suitable linkers include proteins of 4 linked amino acids to 40 linked amino acids in length, or between 4 linked amino acids and 25 linked amino acids in length. In some embodiments, the linker is from 1 to 100 amino acids in length. In some embodiments, the linker is more 100 amino acids in length. In some embodiments, the linker is from 10 to 27 amino acids in length.

[0189] In some cases, a linked amino acid comprises at least two amino acids linked by an amide bond. These linkers may be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins, or may be encoded by a nucleic acid sequence encoding a fusion protein comprising an effector protein (e.g, an effector protein coupled to a fusion partner). Linkers may comprise glycine(s), serine(s), and combinations thereof. Nonlimiting examples of linker includes glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, GSGGSn (SEQ ID NO: 204), GGSGGSn (SEQ ID NO: 205), and GGGSn (SEQ ID NO: 206), where n is an integer of at least one), glycinealanine polymers, and alanine-serine polymers. Exemplary linkers may comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 207), GGSGG (SEQ ID NO: 208), GSGSG (SEQ ID NO: 209), GSGGG (SEQ ID NO: 210), GGGSG (SEQ ID NO: 211), and GSSSG (SEQ ID NO: 212). In some embodiments, the linker comprises one or more repeats a tri -peptide GGS. In some embodiments, the linker is an XTEN linker. In some embodiments, the XTEN linker is an XTEN80 linker. In some embodiments, the XTEN linker is an XTEN20 linker. In some embodiments, the XTEN20 linker has an amino acid sequence of GSGGSPAGSPTSTEEGTSESATPGSG (SEQ ID NO: 204).

[0190] In some embodiments, linkers comprise or consist of 4 to 60, 6 to 55, 8 to 50, 10 to 45, 12 to 40, 14 to 35, 16 to 30, 18 to 25 linked amino acids. In some embodiments, linkers comprise or consist of 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, or 50 to 60 linked amino acids. In some embodiments, linkers comprise or consist of 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 55 or 55 to 60 linked amino acids. In some embodiments, linkers comprise or consist of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55 or about 60 amino acids.

[0191] In some instances, linkers comprise or consist of a non-peptide linker. Non-limiting examples of non-peptide linkers are linkers comprising polyethylene glycol (PEG), polypropylene glycol (PPG), co-poly(ethylene/propylene) glycol, polyoxyethylene (POE), polyurethane, polyphosphazene, polysaccharides, dextran, polyvinyl alcohol, polyvinylpyrrolidones, polyvinyl ethyl ether, polyacrylamide, polyacrylate, polycyanoacrylates, a nucleotide, a polynucleotide, a lipid, a polymer, lipid polymers, chitins, hyaluronic acid, heparin, an alkyl linker, or a combination thereof.

[0192] In some instances, linkers comprise or consist of a nucleic acid. In some instances, the nucleic acid comprises DNA. In some instances, the nucleic acid comprises RNA. In some instances, the effector protein and the fusion partner each interact with the nucleic acid, the nucleic acid thereby linking the effector protein and the fusion partner. In some instances, the nucleic acid serves as a scaffold for both the effector protein and the fusion partner to interact with, thereby linking the effector protein and the fusion partner. Such nucleic acids include those described by Tadakuma et al., (2016), Progress in Molecular Biology and Translational Science, Volume 139, Pages 121-163, incorporated herein by reference.

[0193] In some instances, the fusion effector protein or the guide nucleic acid comprises a chemical modification that allows for direct crosslinking between the guide nucleic acid or the effector protein and the fusion partner. By way of non-limiting example, the chemical modification may comprise any one of a SNAP -tag, CLIP -tag, ACP-tag, Halo-tag, and an MCP-tag. In some instances, modifications are introduced with a Click Reaction, also known as Click Chemistry. The Click reaction may be copper dependent or copper independent.

[0194] In some instances, guide nucleic acids comprise an aptamer. The aptamer may serve as a linker between the effector protein and the fusion partner by interacting non-covalently with both. In some instances, the aptamer binds a fusion partner, wherein the fusion partner is a transcriptional activator. In some instances, the aptamer binds a fusion partner, wherein the fusion partner is a transcriptional inhibitor. In some instances, the aptamer binds a fusion partner, wherein the fusion partner comprises a base editor. In some instances, the aptamer binds the fusion partner directly. In some instances, the aptamer binds the fusion partner indirectly. Aptamers may bind the fusion partner indirectly through an aptamer binding protein. By way of non-limiting example, the aptamer binding protein may be MS2 and the aptamer sequence may be ACATGAGGATCACCCATGT (SEQ ID NO: 170); the aptamer binding protein may be PP7 and the aptamer sequence may be GGAGCAGACGATATGGCGTCGCTCC (SEQ ID NO: 171); or the aptamer binding protein may be BoxB and the aptamer sequence may be GCCCTGAAGAAGGGC (SEQ ID NO: 172).

[0195] In some instances, the fusion partner is located within effector protein. For example, the fusion partner may be a domain of a fusion partner protein that is internally integrated into the effector protein. In other words, the fusion partner may be located between the 5 ’ and 3 ’ ends of the effector protein without disrupting the ability of the fusion effector protein to recognize/bind a target nucleic acid. In some instances, the fusion partner replaces a portion of the effector protein. In some instances, the fusion partner replaces a domain of the effector protein. In some instances, the fusion partner does not replace a portion of the effector protein.

[0196] A person of ordinary skill in the art would understand that any suitable linker described herein for fusion partners may be used to link any two peptides or polypeptides described herein.

Multimeric Complexes

[0197] Compositions, systems, and methods of the present disclosure may comprise a multimeric complex or uses thereof, wherein the multimeric complex comprises one or more effector proteins that non-covalently interact with one another. A multimeric complex may comprise enhanced activity relative to the activity of any one of its effector proteins alone. For example, a multimeric complex comprising two effector proteins (e.g., in dimeric form) may comprise greater nucleic acid binding affinity and/or nuclease activity than that of either of the effector proteins provided in monomeric form. In another example, a multimeric complex comprising an effector protein and an effector partner may comprise greater nucleic acid binding affinity and/or nuclease activity than that of either of the effector protein or effector partner provided in monomeric form. A multimeric complex may have an affinity for a target sequence of a target nucleic acid and is capable of catalytic activity (e.g, cleaving, nicking, inserting or otherwise editing the nucleic acid) at or near the target sequence. A multimeric complex may have an affinity for a donor nucleic acid and is capable of catalytic activity (e.g., cleaving, nicking, editing or otherwise modifying the nucleic acid by creating cuts) at or near one or more ends of the donor nucleic acid. Multimeric complexes may be activated when complexed with a guide nucleic acid. Multimeric complexes may be activated when complexed with a target nucleic acid. Multimeric complexes may be activated when complexed with a guide nucleic acid, a target nucleic acid, and/or a donor nucleic acid. In some embodiments, the multimeric complex cleaves the target nucleic acid. In some embodiments, the multimeric complex nicks the target nucleic acid.

[0198] Various aspects of the present disclosure include compositions and methods comprising multiple effector proteins, and uses thereof, respectively. An effector protein comprising at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to any one of the sequences of TABLE 1 may be provided with a second effector protein. Two effector proteins may target different nucleic acid sequences. Two effector proteins may target different types of nucleic acids (e.g. , a first effector protein may target double- and single-stranded nucleic acids, and a second effector protein may only target single-stranded nucleic acids). It is understood that when discussing the use of more than one effector protein in compositions, systems, and methods provided herein, the multimeric complex form is also described.

[0199] In some embodiments, multimeric complexes comprise at least one effector protein comprising an amino acid sequence with at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identity to any one of the sequences of TABLE 1. In some embodiments, the multimeric complex is a dimer comprising two effector proteins of identical amino acid sequences. In some embodiments, the multimeric complex comprises a first effector protein and a second effector protein, wherein the amino acid sequence of the first effector protein is at least 90%, at least 92%, at least 94%, at least 96%, at least 98% identical, or at least 99% identical to the amino acid sequence of the second effector protein.

[0200] In some embodiments, the multimeric complex is a heterodimeric complex comprising at least two effector proteins of different amino acid sequences. In some embodiments, the multimeric complex is a heterodimeric complex comprising a first effector protein and a second effector protein, wherein the amino acid sequence of the first effector protein is less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% identical to the amino acid sequence of the second effector protein.

[0201] In some embodiments, a multimeric complex comprises at least two effector proteins. In some embodiments, a multimeric complex comprises more than two effector proteins. In some embodiments, a multimeric complex comprises two, three or four effector proteins. In some embodiments, at least one effector protein of the multimeric complex comprises an amino acid sequence with at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identity to any one of the sequences of TABLE 1. In some embodiments, each effector protein of the multimeric complex independently comprises an amino acid sequence with at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identity to any one of the sequences of TABLE 1.

Synthesis, Isolation and Assaying

[0202] Effector proteins of the present disclosure may be synthesized, using any suitable method. Effector proteins of the present disclosure may be produced in vitro or by eukaryotic cells or by prokaryotic cells. Effector proteins can be further processed by unfolding, e.g., heat denaturation, dithiothreitol reduction, etc. and may be further refolded, using any suitable method. Effector proteins of the present disclosure of the present disclosure may be synthesized, using any suitable method.

[0203] Any suitable method of generating and assaying the effector proteins described herein may be used. Such methods include, but are not limited to, site-directed mutagenesis, random mutagenesis, combinatorial libraries, and other mutagenesis methods described herein (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); Gillman et al., Directed Evolution Library Creation: Methods and Protocols (Methods in Molecular Biology) Springer, 2nd ed (2014)). One non-limiting example of a method for preparing an effector protein is to express recombinant nucleic acids encoding the effector protein in a suitable microbial organism, such as a bacterial cell, a yeast cell, or other suitable cell, using methods well known in the art. Exemplary methods are also described in the Examples provided herein.

[0204] In some embodiments, effector proteins described herein can be isolated and purified for use in compositions, systems, and/or methods described herein. Methods described here can include the step of isolating the effector protein described herein. Compositions and/or systems described herein can further comprise a purification tag that can be attached to an effector protein, or a nucleic acid encoding for a purification tag that can be attached to a nucleic acid encoding for an effector protein as described herein. A purification tag, as used herein, can be an amino acid sequence which can attach or bind with high affinity to a separation substrate and assist in isolating the protein of interest from its environment, which can be its biological source, such as a cell lysate. Attachment of the purification tag can be at the N or C terminus of the effector protein. Furthermore, an amino acid sequence recognized by a protease or a nucleic acid encoding for an amino acid sequence recognized by a protease, such as TEV protease or the HRV3C protease can be inserted between the purification tag and the effector protein, such that biochemical cleavage of the sequence with the protease after initial purification liberates the purification tag. Purification and/or isolation can be through high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. In some embodiments, purification tags can be a fluorescent protein, e.g., green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, tdTomato, and the like; a histidine tag, e.g., a 6XHis tag (SEQ ID NO: 213); a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like.

[0205] For example, in some embodiments, effector proteins described herein are isolated from cell lysate. In some embodiments, the compositions described herein can comprise 20% or more by weight, 75% or more by weight, 95% or more by weight, or 99.5% or more by weight of an effector protein, related to the method of preparation of compositions described herein and its purification thereof, wherein percentages can be upon total protein content in relation to contaminants. Thus, in some cases, an effector protein described herein is at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure (e.g., free of contaminants, non-engineered polypeptide proteins or other macromolecules, etc.).

Protospacer Adjacent Motif (PAM)

[0206] In some instances, effector proteins cleave or nick a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid. In some instances, cleavage occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of a 5’ or 3’ terminus of a PAM sequence. In some instances, cleavage occurs within 20, 30 or 40 nucleotides of a 5’ or 3’ terminus of a PAM sequence. In some embodiments, the target nucleic acid is a double stranded DNA (dsDNA) molecule. In some embodiments, the target nucleic acid is a double stranded nucleic acid comprising a target strand and a non-target strand. In some embodiments, the PAM is immediately adjacent to the 5’ end of the target sequence on the sense strand of the dsDNA molecule. In some embodiments, the PAM is immediately adjacent to the 5’ end of the target sequence on the antisense strand of the dsDNA molecule. A target nucleic acid may comprise a PAM sequence adjacent to a sequence that is complementary to a guide nucleic acid spacer sequence. In some embodiments, effector proteins described herein recognize a PAM sequence. In some embodiments, recognizing a PAM sequence comprises interacting with a sequence adjacent to the PAM. In some embodiments, a target nucleic acid comprises a target sequence that is adjacent to a PAM sequence. In some instances, effector proteins do not require a PAM sequence to bind to a target nucleic acid. In some instances, effector proteins do not require a PAM sequence to cleave or a nick a target nucleic acid.

[0207] In some embodiments, a target nucleic acid is a single stranded target nucleic acid comprising a target sequence. Accordingly, in some embodiments, the single stranded target nucleic acid comprises a PAM sequence described herein that is adjacent (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides) or directly adjacent to the target sequence. In some embodiments, an RNP cleaves the single stranded target nucleic acid. In some embodiments, an RNP describes a complex of one or more nucleic acids and one or more polypeptides described herein. It is understood that the one or more nucleic acid may comprise deoxyribonucleotides (DNA), ribonucleotides (RNA), a combination thereof (e.g., RNA with a thymine base), biochemically or chemically modified nucleobases (e.g., one or more engineered modifications described herein), or combinations thereof.

[0208] In some embodiments, a target nucleic acid is a double stranded nucleic acid comprising a target strand and a non-target strand, wherein the target strand comprises a target sequence. In some embodiments, the PAM sequence is located on the target strand. In some embodiments, the PAM sequence is located on the non-target strand. In some embodiments, the PAM sequence described herein is adjacent (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides) to the target sequence on the target strand or the non-target strand. In some embodiments, such a PAM described herein is directly adjacent to the target sequence on the target strand or the non-target strand. In some embodiments, an RNP cleaves the target strand or the non-target strand. In some embodiments, the RNP cleaves both, the target strand and the non-target strand. In some embodiments, an RNP recognizes the PAM sequence, and hybridizes to a target sequence of the target nucleic acid. In some embodiments, the RNP cleaves the target nucleic acid, wherein the RNP has recognized the PAM sequence and is hybridized to the target sequence.

[0209] In some embodiments, an effector protein described herein, or a multimeric complex thereof, recognizes a PAM on a target nucleic acid. In some embodiments, multiple effector proteins of the multimeric complex recognize a PAM on a target nucleic acid. In some embodiments, at least two of the multiple effector proteins recognize the same PAM sequence. In some embodiments, at least two of the multiple effector proteins recognize different PAM sequences. In some embodiments, only one effector protein of the multimeric complex recognizes a PAM on a target nucleic acid. [0210] An effector protein of the present disclosure, or a multimeric complex thereof, may cleave or nick a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid. In some embodiments, cleavage occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of a 5’ or 3’ terminus of a PAM sequence.

[0211] In some embodiments, the sequence of the PAM is an exemplary PAM sequence provided in TABLE 3. PAMs used in compositions, systems, and methods herein are further described throughout the application.

[0212] In some embodiments, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of sequences recited in TABLE 1, and wherein the target nucleic acid comprises a corresponding PAM sequence as identified in TABLE 3. In some instances, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1, and wherein the target nucleic acid comprises a PAM represented by SEQ ID NO: 10. In some instances, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 2, and wherein the target nucleic acid comprises a PAM represented by SEQ ID NO: 11. In some instances, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 3, and wherein the target nucleic acid comprises a PAM represented by SEQ ID NO: 12. In some instances, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 4, and wherein the target nucleic acid comprises a PAM represented by SEQ ID NO: 13. In some instances, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 5, and wherein the target nucleic acid comprises a PAM represented by SEQ ID NO: 14. In some instances, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 6, and wherein the target nucleic acid comprises a PAM represented by SEQ ID NO: 15. In some instances, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 7, and wherein the target nucleic acid comprises a PAM represented by SEQ ID NO: 16. In some instances, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 8, and wherein the target nucleic acid comprises a PAM represented by SEQ ID NO: 17. In some e instances, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 9, and wherein the target nucleic acid comprises a PAM represented by SEQ ID NO: 18.

[0213] In some embodiments, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 87, and wherein the target nucleic acid comprises a PAM represented by SEQ ID NO: 98. In some embodiments, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 88, and wherein the target nucleic acid comprises a PAM represented by any one of SEQ ID NO: 98-99. In some embodiments, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 89, and wherein the target nucleic acid comprises a PAM represented by any one of SEQ ID NO: 98-99. In some embodiments, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 90, and wherein the target nucleic acid comprises a PAM represented by SEQ ID NO: 98 or 100. In some embodiments, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 91, and wherein the target nucleic acid comprises a PAM represented by SEQ ID NO: 98 or 101. In some embodiments, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 92, and wherein the target nucleic acid comprises a PAM represented by SEQ ID NO: 98 or 102. In some embodiments, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 93, and wherein the target nucleic acid comprises a PAM represented by SEQ ID NO: 98 or 102. In some embodiments, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 94, and wherein the target nucleic acid comprises a PAM represented by SEQ ID NO: 103 or 104. In some embodiments, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 95, and wherein the target nucleic acid comprises a PAM represented by SEQ ID NO: 105 or 106. In some embodiments, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 96, and wherein the target nucleic acid comprises a PAM represented by SEQ ID NO: 98. In some embodiments, methods comprise contacting a target nucleic acid with an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 97, and wherein the target nucleic acid comprises a PAM represented by SEQ ID NO: 107 or 108.

III. Nucleic Acid Systems

Guide Nucleic Acids

[0214] The compositions, systems, and methods of the present disclosure may comprise a guide nucleic acid, or a nucleic acid, such as a DNA molecule, encoding the guide nucleic acid, or a use thereof. Also provided herein are compositions, systems and methods that comprise at least one of: one or more guide nucleic acids and DNA molecule(s) encoding the guide nucleic acids. A person of ordinary skill in the art understands that a DNA molecule that “encodes” a nucleic acid, such as a guide nucleic acid, refers to a DNA molecule having a nucleic acid that produces an RNA molecule (e.g., a guide nucleic acid) when transcribed. It is understood that when referring to a guide nucleic acid as described herein, a DNA molecule, such as an expression vector, encoding the guide nucleic acid is also described.

[0215] In general, the guide nucleic acid comprises a CRISPR RNA (crRNA), at least a portion of which is complementary to a target sequence of a target nucleic acid. In some instances, the crRNA comprises a sequence that is bound by an effector protein. In some instances, the crRNA comprises a repeat sequence that is bound by an effector protein. In some embodiments, the guide nucleic acid comprises a trans-activating CRISPR RNA (tracrRNA) that interacts with the effector protein. In some embodiments, the guide nucleic acid comprises an intermediary RNA. In some embodiments, the crRNA and the intermediary RNA are covalently linked (e.g., phosphodiester), also referred to herein as a single guide RNA (sgRNA). In some embodiments, the crRNA and the intermediary RNA are linked by one or more nucleotides. In some embodiments, a guide nucleic acid is an sgRNA. In some embodiments, the sgRNA comprises a handle sequence. In some embodiments, the handle sequence interacts with the effector protein. In some embodiments, the handle sequence comprises a repeat sequence, an intermediary RNA, a linker sequence, or a combination thereof. In some embodiments, the sgRNA does not comprise a nucleotide sequence that is transactivating. In some instances, the crRNA and the tracrRNA are covalently linked. In some embodiments, the crRNA and the tracrRNA are linked by a phosphodiester bond. In some instances, the crRNA and the tracrRNA are linked by one or more linked nucleotides. In some embodiments, a guide nucleic acid does not comprise a tracrRNA. Accordingly, in some embodiments, a composition does not comprise a tracrRNA. [0216] In some instances, a crRNA and a tracrRNA function as two separate, unlinked molecules, wherein the tracrRNA hybridizes with the crRNA and interacts with an effector protein. Accordingly, in this context the tracrRNA is transactivating.

[0217] Guide nucleic acids may comprise DNA, RNA, or a combination thereof (e.g., RNA with a thymine base). Guide nucleic acids may include a chemically modified nucleobase or phosphate backbone. In some embodiments, a guide nucleic acid of the present disclosure comprises one or more of the following: a) a single nucleic acid molecule; b) a DNA base; c) an RNA base; d) a modified base; e) a modified sugar; f) a modified backbone; and the like. Modifications are described herein and throughout the present disclosure (e.g, in the section entitled “Engineered Modifications”).

[0218] Guide nucleic acids may be referred to herein as a guide RNA (gRNA). However, a guide RNA is not limited to ribonucleotides, but may comprise deoxyribonucleotides and other chemically modified nucleotides. A guide nucleic acid, as well as any components thereof (e.g., spacer sequence, repeat sequence, linker nucleotide sequence, handle sequence, intermediary sequence etc.) may comprise one or more deoxyribonucleotides, ribonucleotides, biochemically or chemically modified nucleotides (e.g. , one or more engineered modifications as described herein), or any combinations thereof.

[0219] A guide nucleic acid may comprise a naturally occurring guide nucleic acid. A guide nucleic acid may comprise a non-naturally occurring guide nucleic acid, including a guide nucleic acid that is designed to contain a chemical or biochemical modification. In some embodiments, a guide nucleic acid may comprise a non-naturally occurring sequence, wherein the sequence of the guide nucleic acid, or any portion thereof, may be different from the sequence of a naturally occurring guide nucleic acid. The sequence of a guide nucleic acid may comprise two or more heterologous sequences. Guide RNAs may be chemically synthesized or recombinantly produced, including by any suitable method.

[0220] The guide nucleic acid may also form complexes as described through herein. For example, a guide nucleic acid may hybridize to another nucleic acid, such as target nucleic acid, or a portion thereof. In another example, a guide nucleic acid may complex with an effector protein. In such embodiments, a guide nucleic acid-effector protein complex may be described herein as an RNP. In some embodiments, when in a complex, at least a portion of the complex may bind, recognize, and/or hybridize to a target nucleic acid. For example, when a guide nucleic acid and an effector protein are complexed to form an RNP, at least a portion of the guide nucleic acid hybridizes to a target sequence in a target nucleic acid. Those skilled in the art in reading the below specific examples of guide nucleic acids as used in RNPs described herein, will understand that in some embodiments, a RNP may hybridize to one or more target sequences in a target nucleic acid, thereby allowing the RNP to modify and/or recognize a target nucleic acid or sequence contained therein (e.g., PAM) or to modify and/or recognize non-target sequences depending on the guide nucleic acid, and in some embodiments, the effector protein, used.

[0221] Guide nucleic acids, when complexed with an effector protein, may bring the effector protein into proximity of a target nucleic acid. Sufficient conditions for hybridization of a guide nucleic acid to a target nucleic acid and/or for binding of a guide nucleic acid to an effector protein include in vivo physiological conditions of a desired cell type or in vitro conditions sufficient for assaying catalytic activity of a protein, polypeptide or peptide described herein, such as the nuclease activity of an effector protein.

[0222] In some instances, fusion effector proteins are targeted by a guide nucleic acid (e.g., a guide RNA) to a specific location in the target nucleic acid where they exert locus-specific regulation. Nonlimiting examples of locus-specific regulation include blocking RNA polymerase binding to a promoter (which selectively inhibits transcription activator function), and/or modifying local chromatin (e.g, when a fusion sequence is used that modifies the target nucleic acid or modifies a protein associated with the target nucleic acid). The guide nucleic acid (e.g. , a guide RNA) may bind (hybridize) to a target nucleic acid (e.g., a single strand of a target nucleic acid) or a portion thereof, an amplicon thereof, or a portion thereof. The target nucleic acid, in some embodiments, comprises a mutation. In some embodiments, the mutation is located in a non-coding region of a gene . By way of non-limiting example, a guide nucleic acid may bind (hybridize) to a target nucleic acid, such as DNA or RNA, from a cancer gene or gene associated with a genetic disorder, or an amplicon thereof, as described herein.

[0223] In some cases, an effector protein cleaves a precursor RNA (“pre-crRNA”) to produce a guide RNA, also referred to as a “mature guide RNA.” An effector protein that cleaves pre-crRNA to produce a mature guide RNA is said to have pre-crRNA processing activity. In some cases, a repeat sequence of a guide RNA comprises mutations or truncations relative to respective regions in a corresponding pre-crRNA.

[0224] The guide nucleic acid may comprise a first region complementary to a target nucleic acid (FR1) and a second region that is not complementary to the target nucleic acid (FR2). In some cases, FR1 is located 5’ to FR2 (FR1-FR2). In somecases, FR2 is located 5’ to FR1 (FR2-FR1). In some embodiments, the FR2 comprises one or more repeat sequences or intermediary sequence. In some embodiments, an effector protein binds to at least a portion of the FR. In some embodiments, the FR1 comprises a spacer sequence, wherein the spacer sequence can interact in a sequence-specific manner with (e.g., has complementarity with, or can hybridize to a target sequence in) a target nucleic acid.

[0225] In some embodiments, a guide nucleic acid may comprise or form intramolecular secondary structure (e.g., hairpins, stem-loops, etc.). In some instances, a guide nucleic acid comprises a stemloop structure comprising a stem region and a loop region. In some embodiments, the stem region is 4 to 8 linked nucleotides in length. In some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length. In some embodiments, the guide nucleic acid comprises a pseudoknot (e.g., a secondary structure comprising a stem, at least partially, hybridized to a second stem or half-stem secondary structure). An effector protein may recognize a guide nucleic acid comprising multiple stem regions. In some embodiments, the nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, the guide nucleic acid comprises at least 2, at least 3, at least 4, or at least 5 stem regions.

[0226] In some embodiments, the compositions, systems, and methods of the present disclosure comprise two ormore guide nucleic acids (e.g., 2, 3, 4, 5, 6, 7, 9, 10 ormore guide nucleic acids), and/or uses thereof. Multiple guide nucleic acids may target an effector protein to different locations in the target nucleic acid by hybridizing to different target sequences. In some embodiments, a first guide nucleic acid may hybridize within a location of the target nucleic acid that is different from where a second guide nucleic acid may hybridize the target nucleic acid. In some embodiments, the first loci and the second loci of the target nucleic acid may be located at least 1, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 nucleotides apart. In some embodiments, the first loci and the second loci of the target nucleic acid may be located between 100 and 200, 200 and 300, 300 and 400, 400 and 500, 500 and 600, 600 and 700, 700 and 800, 800 and 900 or 900 and 1000 nucleotides apart.

[0227] In some embodiments, the first loci and/or the second loci of the target nucleic acid are located in an intron of a gene. In some embodiments, the first loci and/or the second loci of the target nucleic acid are located in an exon of a gene. In some embodiments, the first loci and/or the second loci of the target nucleic acid span an exon-intron junction of a gene. In some embodiments, the first portion and/or the second portion of the target nucleic acid are located on either side of an exon and cutting at both sites results in deletion of the exon. In some embodiments, composition, systems, and methods comprise a donor nucleic acid that may be inserted in replacement of a deleted or cleaved sequence of the target nucleic acid. In some embodiments, compositions, systems, and methods comprising multiple guide nucleic acids or uses thereof comprise multiple effector proteins, wherein the effector proteins may be identical, non-identical, or combinations thereof.

[0228] In some cases, the guide nucleic acid comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,

49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 linked nucleotides. In general, a guide nucleic acid comprises at least linked nucleotides. In some instances, a guide nucleic acid comprises at least 25 linked nucleotides. A guide nucleic acid may comprise 10 to 50 linked nucleotides. In some cases, the guide nucleic acid comprises or consists essentially of about 12 to about 80 linked nucleotides, about 12 to about 50, about 12 to about 45, about 12 to about 40, about 12 to about 35, about 12 to about 30, about 12 to about 25, from about 12 to about 20, about 12 to about 19 , about 19 to about 20, about 19 to about 25, about 19 to about 30, about 19 to about 35, about 19 to about 40, about 19 to about 45, about 19 to about 50, about 19 to about 60, about 20 to about 25, about 20 to about 30, about 20 to about 35, about 20 to about 40, about 20 to about 45, about 20 to about 50, or about 20 to about 60 linked nucleotides. In some cases, the guide nucleic acid has about 10 to about 60, about 20 to about

50, or about 30 to about 40 linked nucleotides. [0229] In some embodiments, aguide nucleic acid comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that are complementary to a eukaryotic sequence. Such a eukaryotic sequence is a nucleotide sequence that is present in a host eukaryotic cell. Such a nucleotide sequence is distinguished from nucleotide sequences present in other host cells, such as prokaryotic cells, or viruses. Said sequences present in a eukaryotic cell can be located in a gene, an exon, an intron, a non-coding (e.g., promoter or enhancer) region, a selectable marker, tag, signal, and the like. In some embodiments, a target sequence is a eukaryotic sequence.

[0230] [221] In some embodiments, guide nucleic acids comprise additional elements that contribute additional functionality (e.g., stability, heat resistance, etc.) to the guide nucleic acid. Such elements may be one or more nucleotide alterations, nucleotide sequences, intermolecular secondary structures, or intramolecular secondary structures (e.g., one or more hair pin regions, one or more bulges, etc.).

[0231] [222] In some embodiments, guide nucleic acids comprise one or more linkers connecting different nucleotide sequences as described herein. A linker may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides. A linker may be any suitable linker, examples of which are described herein.

[0232] In some embodiments, the guide nucleic acid comprises a nucleotide sequence as described herein (e.g., TABLE 4, TABLE 5, TABLE 6, or TABLE 7). Such nucleotide sequences described herein (e.g. , TABLE 4, TABLE 5, TABLE 6, or TABLE 7) may be described as a nucleotide sequence of either DNA or RNA, however, no matter the form the sequence is described, it is readily understood that such nucleotide sequences can be revised to be RNA or DNA, as needed, for describing a sequence within a guide nucleic acid itself or the sequence that encodes a guide nucleic acid, such as a nucleic acid described herein for a viral vector. Similarly, disclosure of the nucleotide sequences described herein (e.g., TABLE 4, TABLE 5, TABLE 6, or TABLE 7) also discloses the complementary nucleotide sequence, the reverse nucleotide sequence, and the reverse complement nucleotide sequence, any one of which can be a nucleotide sequence for use in a guide nucleic acid as described herein. In some embodiments, guide nucleic acid sequence(s) comprises one or more nucleotide alterations at one or more positions in any one of the sequences described herein. Alternative nucleotides may be any one or more of A, C, G, T or U, or a deletion, or an insertion.

[0233] In some embodiments, compositions, systems and methods described herein comprise a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one ofthe sequences recited in TABLE 4, TABLE 5, TABLE 6, or TABLE 7

Repeat Sequence

[0234] In some embodiments, a guide nucleic acids comprise a repeat sequence that interacts with the effector protein. The repeat sequence may be capable of interacting with an effector protein. Typically, the repeat sequence is adjacent to the spacer region. In some embodiments, a repeat sequence is adjacent to a spacer region, wherein the spacer region comprises a spacer sequence. In some embodiments, the repeat sequence is followed by 5’ of the spacer sequence. In some embodiments, the spacer sequence is followed by 5’ of the repeat sequence. In some embodiments, the repeat sequence is between 10 and 50, 12 and 48, 14 and 46, 16 and 44, and 18 and 42 nucleotides in length. In some embodiments, the repeat sequence is between 19 and 37 nucleotides in length. In some embodiments, the repeat sequence non-covalently binds to an effector protein as described herein.

[0235] In some embodiments, a repeat sequence is connected to another sequence of a guide nucleic acid, such as an intermediary sequence, that is capable of non-covalently interacting with an effector protein. In some embodiments, a repeat sequence is adjacent to an intermediary sequence. In some embodiments, a repeat sequence is 3’ to an intermediary sequence. In some embodiments, an intermediary sequence is followed by a repeat sequence, which is followed by a spacer sequence in the 5’ to 3’ direction. In some embodiments, a repeat sequence is linked to a spacer sequence and/or an intermediary sequence. In some embodiments, a guide nucleic acid comprises a repeat sequence linked to a spacer sequence and/or to an intermediary sequence, which may be a direct link or by any suitable linker, examples of which are described herein.

[0236] In some embodiments, guide nucleic acids comprise more than one repeat sequence. In some embodiments, guide nucleic acids comprise two or more, three or more, or four or more repeat sequences. In some embodiments, a guide nucleic acid comprises more than one repeat sequence separated by another sequence of the guide nucleic acid. For example, in some embodiments, a guide nucleic acid comprises two repeat sequences, wherein the first repeat sequence is followed by a spacer sequence, and the spacer sequence is followed by a second repeat sequence in the 5 ’ to 3 ’ direction. In some embodiments, the more than one repeat sequences are identical. In some embodiments, the more than one repeat sequences are not identical.

[0237] In some embodiments, a repeat sequence includes a nucleotide sequence that is capable of forming a guide nucleic acid-effector protein complex (e.g., a RNP complex). In some embodiments, the repeat sequence comprises two sequences that are complementary to each other and hybridize to form a double stranded RNA duplex (dsRNA duplex). In some embodiments, the two sequences are not directly linked and hybridize to form a stem loop structure. In some embodiments, the dsRNA duplex comprises 5, 10, 15, 20 or 25 base pairs (bp). In some embodiments, not all nucleotides of the dsRNA duplex are paired, and therefore the duplex forming region can include a bulge. In some embodiments, the repeat sequence comprises a hairpin or stem -loop structure, optionally at the 5’ portion of the repeat sequence. In some embodiments, a strand of the stem portion comprises a sequence and the other strand of the stem portion comprises a sequence that is, at least partially, complementary. In some embodiments, such sequences may have 65% to 100% complementarity (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementarity). In some embodiments, a guide nucleic acid comprises nucleotide sequence that when involved in hybridization events may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a bulge, a loop structure or hairpin structure, etc.).

[0238] In some embodiments, a repeat sequence for use with compositions, systems and methods described herein comprises a sequence that has at least 65%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99%, or 100% sequence identity to any one of the sequences set forth in TABLE 4.

[0239] In some embodiments, the repeat sequence comprises one or more nucleotide alterations at one or more positions in any one of the sequences recited in TABLE 4. Alternative nucleotides can be any one or more of A, C, G, T or U, or a deletion, or an insertion.

[0240] In some embodiments, the repeat sequence comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 at least 25, or at least 30 contiguous nucleotides. TABLE 4 provides exemplary repeat sequences. In some embodiments, the repeat sequence comprises any one of the nucleotide sequences set for the in TABLE 4. In some embodiments, the repeat sequence comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 at least 25, or at least 30 contiguous nucleotides of any one of the sequences recited in TABLE 4. In some embodiments, the repeat sequence comprises a nucleotide sequence that has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences recited in TABLE 4. In some embodiments, the repeat sequence is bound by the corresponding effector protein as identified in TABLE 4. In some embodiments, the effector protein as identified in TABLE 4 comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of the sequences recited in TABLE 1.

[0241] In some embodiments, a nucleotide sequence that is bound by an effector protein comprises at least a portion of or all of any one of the sequences recited in TABLE 4. In some embodiments, the portion refers to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 at least 25, at least 30, or at least 35 contiguous nucleotides of any one of the sequences recited in TABLE 4. In some embodiments, a nucleotide sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of the sequences recited in TABLE 4, wherein the nucleotide sequence is bound by the corresponding effector protein as identified in TABLE 4. In some embodiments, the effector protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of the sequences recited in TABLE 1.

Spacer Sequence

[0242] In some embodiments, guide nucleic acids comprise a spacer sequence that hybridizes with a target sequence of a target nucleic acid. The spacer sequence may have complementarity with (e.g., hybridize to) a target sequence of a target nucleic acid. The spaceer sequence may be a reverse complementary sequence of the target sequence. In some embodiments, a spacer sequence comprises a nucleotide sequence that is, at least partially, hybridizable to an equal length of a sequence (e.g. , a target sequence) of a target nucleic acid. Exemplary hybridization conditions are described herein. The spacer sequence may function to direct an effector protein complexed to the guide nucleic acid to the target nucleic acid for detection and/or modification. In some embodiments, a repeat sequence includes a nucleotide sequence that is capable of forming a guide nucleic acid-effector protein complex (e.g., a RNP complex).

[0243] In some embodiments, when describing hybridizing and grammatical equivalents thereof, reference may be made to a nucleotide sequence that is able to noncovalently interact, i. e. form Watson- Crick base pairs and/or G/U base pairs, or anneal, to another nucleotide sequence in a sequence-specific, antiparallel, manner (i. e. , a nucleotide sequence specifically interacts to a complementary nucleotide sequence) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) for both DNA and RNA. In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule (e.g., when a DNA target nucleic acid base pairs with a guide RNA, etc.): guanine (G) can also base pair with uracil (U). For example, G/U base-pairing is at least partially responsible for the degeneracy (i. e. , redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. Thus, a guanine (G) can be considered complementary to both an uracil (U) and to an adenine (A). Accordingly, when a G/U base-pair can be made at a given nucleotide position, the position is not considered to be non-complementary, but is instead considered to be complementary. While hybridization typically occurs between two nucleotide sequences that are complementary, mismatches between bases are possible. It is understood that two nucleotide sequences need not be 100% complementary to be specifically hybridizable, hybridizable, partially hybridizable, or for hybridization to occur. Moreover, a nucleotide sequence may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g. , a bulge, a loop structure or hairpin structure, etc.). The conditions appropriate for hybridization between two nucleotide sequences depend on the length of the sequence and the degree of complementarity, variables which are well known in the art. For hybridizations between nucleic acids with short stretches of complementarity (e.g. complementarity over 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides) the position of mismatches may become important (see Sambrook et al., supra, 11.7-11.8). Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more). Any suitable in vitro assay may be utilized to assess whether two sequences “hybridize”. One such assay is a melting point analysis where the greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Temperature, wash solution salt concentration, and other conditions may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001).

[0244] In some embodiments, a spacer sequence comprises at least 5 to about 50 contiguous nucleotides that are complementary to a target sequence in a target nucleic acid. In some embodiments, a spacer sequence comprises at least 5 to about 50 linked nucleotides. In some embodiments, a spacer sequence comprises at least 5 to about 50, at least 5 to about 25, at least about 10 to at least about 25, or at least about 15 to about 25 linked nucleotides.

[0245] In some cases, the spacer sequence is 15-28 linked nucleotides in length. In some embodiments, the spacer sequence is 15-26, 15-24, 15-22, 15-20, 15-18, 16-28, 16-26, 16-24, 16-22, 16-20, 16-18, 17-26, 17-24, 17-22, 17-20, 17-18, 18-26, 18-24, or 18-22 linked nucleotides in length. In some cases, the spacer sequence is 18-24 linked nucleotides in length. In some cases, the spacer sequence is at least 15 linked nucleotides in length. In some cases, the spacer sequence is at least 16, 18, 20, or 22 linked nucleotides in length. In some cases, the spacer sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some cases, the spacer sequence is at least 17 linked nucleotides in length. In some cases, the spacer sequence is at least 18 linked nucleotides in length. In some cases, the spacer sequence is at least 20 linked nucleotides in length.

[0246] In some cases, the spacer sequence is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence of the target nucleic acid. In some cases, the spacer sequence is 100% complementary to the target sequence of the target nucleic acid. In some cases, the spacer sequence comprises at least 15 contiguous nucleobases that are complementary to the target nucleic acid.

[0247] Typically, the repeat sequence is adjacent to the spacer sequence. For example, a guide RNA that interacts with an effector protein comprises a repeat sequence that is 5 ’ of the spacer sequence or 3’ of the spacer sequence. In some embodiments, a spacer sequence is adjacent to a repeat sequence. In some embodiments, a spacer sequence follows a repeat sequence in a 5’ to 3’ direction. In some embodiments, a spacer sequence precedes a repeat sequence in a 5’ to 3’ direction. In some embodiments, the spacer sequence(s) and the repeat sequence(s) of the guide nucleic acid are present within the same molecule. In some embodiments, the spacer(s) and repeat sequence(s) are linked directly to one another. In some embodiments, a linker is present between the spacer(s) and repeat sequences. Linkers may be any suitable linker. In some embodiments, the spacer sequence(s) and the repeat sequence(s) of the guide nucleic acid are present in separate molecules, which are joined to one another by base pairing interactions.

[0248] In some embodiments, a spacer sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence of a target nucleic acid. A spacer sequence is capable of hybridizing to an equal length portion of a target nucleic acid (e.g., a target sequence). In some embodiments, a target nucleic acid, such as DNA or RNA, may be a cancer gene or gene associated with a genetic disorder, or an amplicon thereof, as described herein. In some embodiments, a target nucleic acid is a gene selected from TABLE 8. In some embodiments, a spacer sequence comprises a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence of a target nucleic acid selected from TABLE 8. In some embodiments, a target nucleic acid is a nucleic acid associated with a disease or syndrome set forth in TABLE 9. In some embodiments, a spacer sequence comprises a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence of a target nucleic acid associated with a disease or syndrome set forth in TABLE 9. In some embodiments, the spacer sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that are capable of hybridizing to the target sequence. In some embodiments, the spacer sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that are complementary to the target sequence. [0249] It is understood that the sequence of a spacer sequence need not be 100% complementary to that of a target sequence of a target nucleic acid to hybridize or hybridize specifically to the target sequence. The guide nucleic acid may comprise at least one uracil between nucleic acid residues 5 to 20 of the spacer sequence that is not complementary to the corresponding nucleoside of the target sequence. The guide nucleic acid may comprise at least one uracil between nucleic acid residues 5 to 9, 10 to 14, or 15 to 20 of the spacer sequence that is not complementary to the corresponding nucleoside of the target sequence. In some cases, the region of the target nucleic acid that is complementary to the spacer sequence comprises an epigenetic modification or a post-transcriptional modification. In some cases, the epigenetic modification comprises acetylation, methylation, or thiol modification. In another embodiment, the spacer sequence may comprise at least one alteration, such as a substituted or modified nucleotide, that is not complementary to the corresponding nucleotide of the target sequence. Spacer sequences are further described throughout herein.

Linkers for Nucleic Acids

[0250] In some embodiments, a guide nucleic acid for use with compositions, systems, and methods described herein comprises one or more linkers, or a nucleic acid encoding one or more linkers. In some embodiments, the guide nucleic acid comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten linkers. In some embodiments, the guide nucleic acid comprises one, two, three, four, five, six, seven, eight, nine, or ten linkers. In some embodiments, the guide nucleic acid comprises more than one linker. In some embodiments, at least two of the more than one linker are the same. In some embodiments, at least two of the more than one linker are not same.

[0251] In some embodiments, a linker comprises one to ten, one to seven, one to five, one to three, two to ten, two to eight, two to six, two to four, three to ten, three to seven, three to five, four to ten, four to eight, four to six, five to ten, five to seven, six to ten, six to eight, seven to ten, or eight to ten linked nucleotides. In some embodiments, the linker comprises one, two, three, four, five, six, seven, eight, nine, or ten linked nucleotides. In some embodiments, a linker comprises a nucleotide sequence of 5’- GAAA-3’.

[0252] In some embodiments, a guide nucleic acid comprises one or more linkers connecting one or more repeat sequences. In some embodiments, the guide nucleic acid comprises one or more linkers connecting one or more repeat sequences and one or more spacer sequences. In some embodiments, the guide nucleic acid comprises at least two repeat sequences connected by a linker.

Intermediary nucleotide sequence

[0253] In some instances, guide nucleic acids comprise an intermediary nucleotide sequence. In general, an intermediary nucleotide sequence is not transactivated or transactivating. The intermediary nucleotide sequence may also be referred to as an intermediary RNA, although it may comprise deoxyribonucleotides in addition to ribonucleotides. In general, the intermediary nucleotide sequence interacts an effector protein. In some instances, the intermediary nucleotide sequence forms a secondary structure in a cell, and an effector protein binds the secondary structure.

[0254] In general, an intermediary RNA functions in a single nucleic acid system. The single nucleic acid system refers to a system wherein a guide nucleic acid comprises a first sequence that hybridizes to a target nucleic acid and a second sequence that intereacts with an effector protein. The first sequence and the second sequence are present in a single, linked molecule. The first sequence may be 5’ or 3’ of the second sequence. In some embodiments, guide nucleic acids comprises a repeat sequence, a linker, an intermediary RNA, a spacer sequence, or a combination thereof. In some embodiments, the repeat sequence, the intermediary RNA, or a combination thereof interacts with the effector protein to form an RNP complex. In some embodiments, the intermediary RNA forms the RNP complex along with any one of the effector proteins described herein. In some embodiments, the RNP complex recognizes a PAM sequence within a target nucleic acid.

[0255] In some embodiments, compositions and methods comprise an RNP complex or uses thereof, wherein the RNP complex recognizes a PAM of a target nucleic acid and interacts with the target nucleic acid in a single nucleic acid system. In some instances, the single nucleic acid system, wherein the RNP complex comprises a guide nucleic acid, and any one of the effector proteins described herein, wherein the guide nucleic acid comprises an intermediary RNA, a repeat sequence, a spacer sequence, and optionally a linker sequence in a single, linked molecule but not necessarily in that order. In some instances, the order from 5’ to 3’ is intermediary RNA, linker, sequence, repeat sequence, spacer sequence. In some e instances, the order from 5’ to 3’ is intermediary RNA, sequence, repeat sequence, linker sequence, spacer sequence. In some instances, the order from 5’ to 3’ is intermediary RNA, first linker sequence, repeat sequence, second linker sequence, spacer sequence.

[0256] In some embodiments, an RNP complex provides a trans cleavage of a target nucleic acid or a non-target nucleic acid. In some embodiments, the RNP complex provides a cis cleavage of a target nucleic acid. In some embodiments, the RNP complex cleaves a target strand of the target nucleic acid. In some embodiments, the RNP complex cleaves a non-target strand of the target nucleic acid. In some embodiments, the effector protein of the RNP complex comprises nuclease activity for cleaving the target nucleic acid. In some embodiments, the effector protein of the RNP complex does not comprise nuclease activity for cleaving the target nucleic acid. In some embodiments, an effector protein is covalently linked to a fusion partner protein that provides an activity on the target nucleic acid.

[0257] In some embodiments, the length of an intermediary RNAs is not greater than 50, 56, 68, 71, 73, 95, or 105 linked nucleotides. In some embodiments, the length of an intermediary RNA is about 30 to about 120 linked nucleotides. In some embodiments, the length of an intermediary RNA is about 50 to about 105, about 50 to about 95, about 50 to about 73, about 50 to about 71, about 50 to about 68, or about 50 to about 56 linked nucleotides. In some embodiments, the length of an intermediary RNA is 56 to 105 linked nucleotides, from 56 to 105 linked nucleotides, 68 to 105 linked nucleotides, 71 to 105 linked nucleotides, 73 to 105 linked nucleotides, or 95 to 105 linked nucleotides. In some embodiments, the length of an intermediary RNA is 40 to 60 nucleotides. In some embodiments, the length of the intermediary RNA is 50, 56, 68, 71, 73, 95, or 105 linked nucleotides. In some embodiments, the length of the intermediary RNA is 50 nucleotides.

[0258] In some embodiments, an intermediary sequence may also comprise or form a secondary structure (e.g., one or more hairpin loops) that facilitates the binding of an effector protein to a guide nucleic acid and/or modification activity of an effector protein on a target nucleic acid (e.g., a hairpin region). In some embodiments, the hairpin region may comprise a first sequence, a second sequence that is reverse complementary to the first sequence, and a stem-loop linking the first sequence and the second sequence. In some embodiments, an exemplary intermediary RNA may comprise a stem -loop structure comprising a stem region and a loop region. In some embodiments, the stem region is 4 to 8 linked nucleotides in length. In some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length. In some embodiments, the intermediary RNA comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure). In some embodiments, an effector protein may recognize an intermediary RNA comprising a single stem region. An effector protein may recognize an intermediary RNA comprising multiple stem regions. In some embodiments, the amino acid sequences of the multiple stem regions are identical to one another. In some embodiments, the amino acid sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, the intermediary RNA comprises at least 2, at least 3, at least 4, or at least 5 stem regions.

[0259] In some embodiments, an exemplary intermediary RNA may comprise, from 5’ to 3’, a 5’ region, a hairpin region, and a 3 ’ region. In some embodiments, the 5 ’ region may hybridize to the 3 ’ region. In some embodiments, the 5’ region does not hybridize to the 3’ region. In some embodiments, the 3’ region is covalently linked to the guide nucleic acid (e.g., through a phosphodiester bond). In some embodiments, one or more of the 3’ region and the 5’ region may have a length of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, or about 20 linked nucleotides. In some embodiments, the length of one or more of the 3’ region and the 5’ region is 0 to 20 linked nucleotides.

[0260] In some embodiments, the intermediary nucleotide sequence comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 220, at least 240, at least 260, at least 280, at least 300, at least 320, at least 340, or at least 360 contiguous nucleotides.

Handle Sequence

[0261] In some embodiments, guide nucleic acids described herein may comprise one or more handle sequences. In some embodiments, the handle sequence comprises an intermediary sequence. In such instances, at least a portion of an intermediary sequence non-covalently bonds with an effector protein. In some embodiments, the intermediary sequence is at the 3 ’-end of the handle sequence. In some embodiments, the intermediary sequence is at the 5’- end of the handle sequence. Additionally, or alternatively, in some embodiments, the handle sequence further comprises one or more of linkers and repeat sequences. In such instances, at least a portion of an intermediary sequence, or both of at least a portion of the intermediary sequence and at least a portion of repeat sequence, non-covalently interacts with an effector protein. In some embodiments, an intermediary sequence and repeat sequence are directly linked (e.g., covalently linked, such as through a phosphodiester bond). In some embodiments, the intermediary sequence and repeat sequence are linked by a suitable linker, examples of which are provided herein. In some embodiments, the linker comprises a sequence of 5’-GAAA-3’. In some embodiments, the intermediary sequence is 5’ to the repeat sequence. In some embodiments, the intermediary sequence is 5 ’ to the linker. In some embodiments, the intermediary sequence is 3 ’ to the repeat sequence. In some embodiments, the intermediary sequence is 3’ to the linker. In some embodiments, the repeat sequence is 3 ’ to the linker. In some embodiments, the repeat sequence is 5 ’ to the linker. In general, a single guide nucleic acid, also referred to as a single guide RNA (sgRNA), comprises a handle sequence comprising an intermediary sequence, and optionally one or more of a repeat sequence and a linker.

[0262] A handle sequence may comprise or form a secondary structure (e.g. , one or more hairpin loops) that facilitates the binding of an effector protein to a guide nucleic acid and/or modification activity of an effector protein on a target nucleic acid (e.g., a hairpin region). In some embodiments, handle sequences comprise a stem-loop structure comprising a stem region and a loop region. In some embodiments, the stem region is 4 to 8 linked nucleotides in length. In some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length. In some embodiments, the handle sequence comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure). An effector protein may recognize a handle sequence comprising multiple stem regions. In some embodiments, the nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, the handle sequence comprises at least 2, at least 3, at least 4, or at least 5 stem regions.

[0263] In some embodiments, a length of the handle sequence is at least 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides. In some embodiments, a length of the handle sequence is not greater than 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides. In some embodiments, the length of the handle sequence is about 30 to about 210, about 60 to about 210, about 90 to about 210, about 120 to about 210, about 150 to about 210, about 180 to about 210, about 30 to about 180, about 60 to about 180, about 90 to about 180, about 120 to about 180, or about 150 to about 180 linked nucleotides. [0264] In some embodiments, the handle sequence comprises one or more nucleotide alterations at one or more positions in any one of the sequences recited in TABLE 5. Alternative nucleotides can be any one or more of A, C, G, T or U, or a deletion, or an insertion.

[0265] In some embodiments, the length of a handle sequence is not greater than about 240, about 200, about 150, about 100, about 50, or about 20 linked nucleotides. In some embodiments, the length of a handle sequence is about 20 to about 240 linked nucleotides. In some embodiments, the length of a handle sequence is about 20 to about 200, about 20 to about 150, about 20 to about 100, about 20 to about 50, about 50 to about 240, about 50 to about 200, about 50 to about 150, about 50 to about 100, about 100 to about 240, about 100 to about 200, about 100 to about 150, about 150 to about 240, about 150 to about 200, or about 200 to about 240 linked nucleotides. In some embodiments, the length of a handle sequence is greater than about 20, about 50, about 100, about 150, about 200, or about 240 linked nucleotides. TABLE 5 provides exemplary handle sequences.

[0266] In some embodiments, a handle sequence comprises a first nucleotide sequence and a second nucleotide sequence, wherein in the first is connected to the second sequence directly or via a repeat sequence or via a linker. In some embodiments, the first nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NOS: 48, 52-53, 58, 60, 62-63, 65-66, 70, 72, 74, and 127-141. In some embodiments, the second nucleotide sequence is a spacer seqeunce as described herein. In some embodiments, the repeat sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of the sequences recited in TABLE 4. In some embodiments, the handle sequence comprising the repeat sequence interacts with any one of the effector proteins described herein as identified in TABLE 4. In some embodiments, the linker comprises a nucleotide sequence of 5’- GAAA-3’.

A Single Nucleic Acid System

[0267] In some embodiments, compositions, systems and methods described herein comprise a single nucleic acid system comprising a guide nucleic acid or a nucleotide sequence encoding the guide nucleic acid, and one or more effector proteins or a nucleotide sequence encoding the one or more effector proteins. Such a single nucleic acid system includes a system that uses a guide nucleic acid complexed with one or more polypeptides described herein, wherein the complex is capable of interacting with a target nucleic acid in a sequence specific manner, and wherein the guide nucleic acid is capable of non- covalently interacting with the one or more polypeptides described herein, and wherein the guide nucleic acid is capable of hybridizing with a target sequence of the target nucleic acid. In some embodiments, a single nucleic acid system lacks a duplex of a guide nucleic acid as hybridized to a second nucleic acid, wherein in such a duplex the second nucleic acid, and not the guide nucleic acid, is capable of interacting with the effector protein. In some embodiments, in a single nucleic system, the guide nucleic acid is not transactivating or transactivated. In some embodiments, in a single nucleic acid system, the guide nucleic acid-polypeptide complex (e.g., an RNP complex) is not transactivated or transactivating.

[0268] In some embodiments, FR2 of the guide nucleic acid non-covalently interacts with the one or more polypeptides described herein. In some embodiments, a FR1 of the guide nucleic acid hybridizes with a target sequence of the target nucleic acid. In the single nucleic acid system having a complex of the guide nucleic acid and the effector protein, the effector protein is not transactivated by the guide nucleic acid. In other words, activity of effector protein does not require binding to a second non-target nucleic acid molecule. An exemplary guide nucleic acid for a single nucleic acid system is a crRNA or a sgRNA. crRNA

[0269] In some embodiments, a guide nucleic acid comprises a crRNA. In some embodiments, the guide nucleic acid is the crRNA. Guide nucleic acids and portions thereof may be found in or identified from a CRISPR array present in the genome of a host organism. A crRNA may be the product of processing of a longer precursor CRISPR RNA (pre-crRNA) transcribed from the CRISPR array by cleavage of the pre-crRNA within each direct repeat sequence to afford shorter, mature crRNAs. A crRNA may be generated by a variety of mechanisms, including the use of dedicated endonucleases (e.g., Cas6 or Cas5d in Type I and III systems), coupling of a host endonuclease (e.g., RNase III) with tracrRNA (Type II systems), or a ribonuclease activity endogenous to the effector protein itself (e.g, Cpfl, from Type V systems). A crRNA may also be specifically generated outside of processing of a pre-crRNA and individually contacted to an effector protein in vivo or in vitro.

[0270] In general, a crRNA comprises a first sequence, often referred to herein as a spacer sequence, that hybridizes to a target sequence of a target nucleic acid, and a second sequence that is capable of being connected to an effector protein by either being non-covalently bound by an effector protein or by hybridization to an intermediary RNA. In some embodiments, the second sequence is often referred to herein as a repeat sequence that interacts with an effector protein directly. Accordingly, in some instances, the crRNA of the guide nucleic acid comprises a repeat sequence and a spacer sequence, wherein the repeat sequence binds to the effector protein and the spacer sequence hybridizes to a target sequence of the target nucleic acid. In some embodiments, the repeat sequence and the spacer sequences are directly connected to each other (e.g. , covalent bond (phosphodiester bond)). In some embodiments, the repeat sequence and the spacer sequence are connected by a linker. The repeat sequence of the crRNA may interact with an effector protein, allowing for the guide nucleic acid and the effector protein to form a complex. In some embodiments, the crRNA interacts with an effector protein indirectly via hybridizing to a tracrRNA, wherein the tracrRNA is transactivating.

[0271] In some embodiments, a crRNA may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof. In some embodiments, a crRNA comprises about: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 linked nucleotides. In some embodiments, a crRNA comprises at least: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 linked nucleotides. In some embodiments, the length of the crRNA is not greater than about 40, about 45, about 50, about 55, about 60, about 65, about 70 or about 75 linked nucleotides. In some embodiments, the length of the crRNA is about 30 to about 120 linked nucleotides. In some embodiments, the length of a crRNA is about 20 to about 120, about 20 to about 100, about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 40 to about 50, about 50 to about 90, about 50 to about 80, about 50 to about 70, or about 50 to about 60 linked nucleotides. In some embodiments, the length of a crRNA is about 40, about 45, about 50, about 55, about 60, about 65, about 70 or about 75 linked nucleotides. TABLE 6 provides exemplary crRNA sequences.

[0272] In some embodiments, a crRNA comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one ofthe crRNA sequences in TABLE 6. In some embodiments, a crRNA sequence comprises a repeat sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in TABLE 4, and a spacer sequence. In some embodiments, a crRNA comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 contiguous nucleotides of any one of the crRNA sequences recited in TABLE 6. In some embodiments, a crRNA sequence comprises at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides of any one of the repeat sequences recited in TABLE 4 and a spacer sequence. sgRNA

[0273] In some embodiments, a guide nucleic acid comprises a single guide RNA (sgRNA). In some embodiments, the guide nucleic acid is a sgRNA. In some embodiments, a single guide nucleic acid, single guide RNA or sgRNA as used interchangeable herein, in the context of a single nucleic acid system, may describe a guide nucleic acid, wherein the guide nucleic acid is a single polynucleotide chain having all the required sequence for a functional complex with an effector protein (e.g., being bound by an effector protein, including in some instances activating the effector protein, and hybridizing to a target nucleic acid, without the need for a second nucleic acid molecule). For example, an sgRNA can have two or more linked guide nucleic acid components (e.g., an intermediary sequence, a repeat sequence, a spacer sequence and optionally a linker, or a handle sequence and a spacer sequence). The combination of a spacer sequence (e.g., a nucleotide sequence that hybridizes to a target sequence in a target nucleic acid) with a handle sequence may be referred to herein as a single guide RNA (sgRNA), wherein the spacer sequence and the handle sequence are covalently linked. In some embodiments, the spacer sequence and handle sequence are directly connected to each other by a covalent bond. In some embodiments, the spacer sequence and handle sequence are linked by a phosphodiester bond. In some embodiments, the spacer sequence and handle sequence are linked by one or more linked nucleotides. In some embodiments, the spacer sequence and handle sequence are linked by a linker.

[0274] In general, a sgRNA comprises a first sequence, sometimes referred to herein as a handle sequence, that interacts with an effector protein; and a second sequence, sometimes referred to herein as a spacer sequence that hybridizes to a target sequence of a target nucleic acid. In some embodiments, the handle sequence comprises a repeat sequence. In some embodiments, the handle sequence comprises an intermediary nucleotide sequence. In some embodiments, the handle sequence comprises a repeat sequece and an intermediary nucleotide sequence. In some embodiment, the handle sequence comprises a linker sequence. In some embodiments, the handle sequence is adjacent to the spacer sequence. For example, a guide RNA that interacts with an effector protein may comprise a handle sequence that is 5’ of the spacer sequence or 3’ of the spacer sequence. In some embodiments, the handle sequence is connected to the spacer sequence via a linker sequence. In some embodiments, the handle sequence may be linked to the spacer sequence in an sgRNA by any suitable, examples of which are provided herein. In general, the handle sequence interacts with the effector protein to form the RNP complex.

[0275] In some embodiments, a sgRNA comprises an intermediary sequence and an crRNA. In some embodiments, an intermediary sequence is 5 ’ to a crRNA in an sgRNA. In some embodiments, a sgRNA comprises a linked intermediary sequence and crRNA. In some embodiments, an intermediary sequence and a crRNA are linked in an sgRNA directly (e.g. , covalently linked, such as through a phosphodiester bond) In some embodiments, an intermediary sequence and a crRNA are linked in an sgRNA by any suitable linker, examples of which are provided herein.

[0276] In some embodiments, the length of the sgRNA is not greater than about 240, about 200, about 150, about 100, or about 50 linked nucleotides. In some embodiments, the length of the sgRNA is about 20 to about 240 linked nucleotides. In some embodiments, the length of a sgRNA is about 20 to about 200, about 20 to about 150, about 20 to about 100, about 20 to about 50, about 50 to about 240, about 50 to about 200, about 50 to about 150, about 50 to about 100, about 100 to about 240, about 100 to about 200, about 100 to about 150, about 150 to about 240, about 150 to about 200, or about 200 to about 240 linked nucleotides. In some embodiments, the length of a sgRNA is greater than about 20, about 50, about 100, about 150, about 200, or about 240 linked nucleotides. TABLE 7 provides exemplary sgRNAs.

[0277] In some embodiments, compositions, methods and system described herein comprise an effector protein or a nucleic acid encoding the effector protein and a guide nucleic acid or a nucleic acid encoding the guide nucleic acid. In some embodiments, the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of sequences recited in TABLE 1. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of the sequences recited in TABLE 4, TABLE 5, and TABLE 7, and, wherein the guide nucleic acid corresponds to the effector protein as identified in TABLE 4, TABLE 5, and TABLE 7. In some embodiments, the guide nucleic acid is a sgRNA. In some embodiments, the sgRNA comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of the sequences recited in TABLE 4, TABLE 5, and TABLE 7, wherein the sgRNA corresponds to the effector protein as identified in TABLE 4, TABLE 5, and TABLE 7. In some embodiments, the sgRNA comprises a handle sequence and a spacer sequence. In some embodiments, the handle sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of sequences recited in TABLE 4, and TABLE 5, wherein the handle sequence corresponds to the effector protein as identified in TABLE 4, and TABLE 5. In some embodiments, the handle sequence comprises one or more of a linker, a repeat sequence, and an intermediary RNA. In some embodiments, the repeat sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of sequences recited in TABLE 4, wherein the repeat sequence corresponds to the effector protein as identified in TABLE 4. In some embodiments, the effector protein contacts a target nucleic acid, wherein the target nucleic acid comprises a PAM sequence corresponding to the effector protein as identified in TABLE 3.

[0278] In some embodiments, the sgRNA comprises one or more nucleotide alterations at one or more positions in any one of the sequences recited in TABLE 7. Alternative nucleotides can be any one or more of A, C, G, T or U, or a deletion, or an insertion.

[0279] In some embodiments, a composition, a method, or a system described herein comprises an effector protein or a nucleic acid encoding the effector protein, and a sgRNA or a nucleic acid encoding the sgRNA. In some embodiments, the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 1, and the sgRNA comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 49 or SEQ ID NO: 50. In some embodiments, the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 2, and the sgRNA comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NOs: 54-56. In some embodiments, the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 6, and the sgRNA comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 67 or SEQ ID NO: 68.

[0280] In some embodiments, a composition, a method, or a system described herein comprises an effector protein or a nucleic acid encoding the effector protein, and a sgRNA or a nucleic acid encoding the sgRNA, wherein the sgRNA comprises a sgRNA sequence (e.g., handle sequence, intermediary nucleotide sequence and a linker, or intermediary nucleotide sequence) described herein. In some embodiments, the handle sequence of an sgRNA comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of sequences recited in TABLE 5

[0281] In some embodiments, a sgRNA is bound by an effector protein comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 1, wherein a handle sequence of the sgRNA comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 176 or SEQ ID NO: 177. In some embodiments, a sgRNA is bound by an effector protein comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 2, wherein a handle sequence of the sgRNA comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NOs: 178-180. In some embodiments, a sgRNA is bound by an effector protein comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 6, wherein a handle sequence of the sgRNA comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 181 or SEQ ID NO: 182

[0282] In some embodiments, the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of the sequences recited in TABLE 1, and the sgRNA comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of the sequences recited in TABLE 4, TABLE 5, and TABLE 7. In some embodiments, the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of the sequences recited in TABLE 1, and the sgRNA comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of the sequences recited in TABLE 4, TABLE 5, and TABLE 7, wherein the effector protein recognizes any one of corresponding PAM sequences recited in TABLE 3.

[0283] In some embodiments, the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 1, and the sgRNA comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 176, and SEQ ID NO: 177. In some embodiments, the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 2, and the sgRNA sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NOs: 54- 56, and SEQ ID NOs: 178-180. In some embodiments, the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 6, and the sgRNA sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 181, and SEQ ID NO: 182

A Dual Nucleic Acid System

[0284] In some embodiments, compositions, systems and methods described herein comprise a dual nucleic acid system comprising a crRNA or a nucleotide sequence encoding the crRNA, a tracrRNA or a nucleotide sequence encoding the tracrRNA, and one or more effector protein or a nucleotide sequence encoding the one or more effector protein, wherein the crRNA and the tracrRNA are separate, unlinked molecules, wherein a repeat hybridization region of the tracrRNA is capable of hybridizing with an equal length portion of the crRNA to form a tracrRNA-crRNA duplex, wherein the equal length portion of the crRNA does not include a spacer sequence of the crRNA, and wherein the spacer sequence is capable of hybridizing to a target sequence of the target nucleic acid. In the dual nucleic acid system having a complex of the guide nucleic acid, tracrRNA, and the effector protein, the effector protein is transactivated by the tracrRNA. In other words, activity of effector protein requires binding to a tracrRNA molecule.

[0285] In some embodiments, a repeat hybridization sequence is at the 3 ’ end of a tracrRNA. In some embodiments, a repeat hybridization sequence may have a length of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, or about 20 linked nucleotides. In some embodiments, the length of the repeat hybridization sequence is 1 to 20 linked nucleotides.

[0286] A tracrRNA and/or tracrRNA-crRNA duplex may form a secondary structure that facilitates the binding of an effector protein to a tracrRNA or a tracrRNA-crRNA. In some embodiments, the secondary structure modifies activity of the effector protein on a target nucleic acid. In some embodiments, the secondary structure comprises a stem-loop structure comprising a stem region and a loop region. In some embodiments, the stem region is 4 to 8 linked nucleotides in length. In some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length. In some embodiments, the secondary structure comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure). An effector protein may recognize a secondary structure comprising multiple stem regions. In some embodiments, nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, the secondary structure comprises at least two, at least three, at least four, or at least five stem regions. In some embodiments, the secondary structure comprises one or more loops. In some embodiments, the secondary structure comprises at least one, at least two, at least three, at least four, or at least five loops. [0287] In some embodiments, a guide nucleic acid forms an RNP complex comprising an effector protein, a crRNA and a tracrRNA. In general, the crRNA and the tracrRNA function as separate, unlinked molecules. The crRNA and the tracrRNA may hybridize to each other. A tracrRNA may include a repeat hybridization sequence and a hairpin region. The repeat hybridization sequence may hybridize to all or part of the repeat sequence of a crRNA. The repeat hybridization sequence may be positioned 3’ of the hairpin region. The repeat hybridization sequence may be positioned 5’ of the hairpin region. A tracrRNA may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof.

[0288] In general, a tracrRNA comprises a nucleotide sequence that is bound by an effector protein. In some embodiments, a tracrRNA may comprise at least one secondary structure (e.g., hairpin loop) that facilitates the binding of an effector protein. The hairpin region may include a first sequence, a second sequence that is reverse complementary to the first sequence, and a stem-loop linking the first sequence and the second sequence.

[0289] In some instances, tracrRNAs comprise a stem-loop structure comprising a stem region and a loop region. In some cases, the stem region is 4 to 8 linked nucleotides in length. In some cases, the stem region is 5 to 6 linked nucleotides in length. In some cases, the stem region is 4 to 5 linked nucleotides in length. In some cases, the tracrRNA comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure). An effector protein may recognize a tracrRNA comprising multiple stem regions. In some instances, the amino acid sequences of the multiple stem regions are identical to one another. In some instances, the amino acid sequences of at least one of the multiple stem regions is not identical to those of the others. In some cases, the tracrRNA comprises at least 2, at least 3, at least 4, or at least 5 stem regions.

[0290] In some instances, the length of a tracrRNA is not greater than 50, 56, 68, 71, 73, 95, or 105 linked nucleotides. In some embodiments, the length of a tracrRNA is about 30 to about 120 linked nucleotides. In some embodiments, the length of atracrRNA is about 50 to about 105, about 50 to about 95, about 50 to about 73, about 50 to about 71, about 50 to about 68, or about 50 to about 56 linked nucleotides. In some embodiments, the length of a tracrRNA is 56 to 105 linked nucleotides, from 56 to 105 linked nucleotides, 68 to 105 linked nucleotides, 71 to 105 linked nucleotides, 73 to 105 linked nucleotides, or 95 to 105 linked nucleotides. In some embodiments, the length of a tracrRNA is 40 to 60 nucleotides. In some embodiments, the length of atracrRNA is 50, 56, 68, 71, 73, 95, or 105 linked nucleotides. In some embodiments, the length of a tracrRNA is 50 nucleotides.

[0291] An exemplary tracrRNA may comprise, from 5’ to 3’, a 5’ region, a hairpin region, a repeat hybridization sequence, and a 3’ region. In some cases, the 5’ region may hybridize to the 3’ region. In some embodiments, the 5 ’ region does not hybridize to the 3 ’ region. In some embodiments, a tracrRNA may comprise an unhybridized region at the 3’ end of the tracrRNA. The unhybridized region may have a length of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, or about 20 linked nucleotides. In some embodiments, the length of the un-hybridized region is 0 to 20 linked nucleotides.

[0292] In some instances, compositions do not comprise a tracrRNA. In some instances, methods do not comprise the use of atracrRNA. In some instancves, the guide RNA does not comprise atracrRNA. In some cases, an effector protein does not require a tracrRNA to locate and/or cleave a target nucleic acid.

IV. Engineered Modifications

[0293] Polypeptides (e.g., effector proteins) and nucleic acids (e.g., guide nucleic acids) described herein can be further modified as described throughout and as further described herein. In some embodiments, when describing an engineered modification, reference may be made to a structural change of one or more nucleic acid residues of a nucleotide sequence or one or more amino acid residue of an amino acid sequence, such as chemical modification of one or more nucleobases; or a chemical change to the phosphate backbone, a nucleotide, a nucleobase, or a nucleoside. Such modifications can be made to an effector protein amino acid sequence or guide nucleic acid nucleotide sequence, or any sequence disclosed herein (e.g. , a nucleic acid encoding an effector protein or a nucleic acid that encodes a guide nucleic acid). Methods of modifying a nucleic acid or amino acid sequence are known. One of ordinary skill in the art will appreciate that the engineered modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid, protein, composition or system is not substantially decreased. Nucleic acids provided herein can be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vz/ro-transcription, cloning, enzymatic, or chemical cleavage, etc. In some instances, the nucleic acids provided herein are not uniformly modified along the entire length of the molecule. Different nucleotide modifications and/or backbone structures can exist at various positions within the nucleic acid.

[0294] Engineered modifications described herein do not alter the primary sequence of the polypeptides or nucleic acids provided herein. Examples are modifications of interest that do not alter primary sequence, including chemical derivatization of polypeptides, e.g., acylation, acetylation, carboxylation, amidation, etc. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

[0295] Modifications disclosed herein can also include modification of described polypeptides and/or guide nucleic acids through any suitable method, such as molecular biological techniques and/or synthetic chemistry, to improve their resistance to proteolytic degradation, to change the target sequence specificity, to optimize solubility properties, to alter protein activity (e.g., transcription modulatory activity, enzymatic activity, etc.) or to render them more suitable. In some embodiments, modification of described polypeptides and/or guide nucleic acids render them more suitable for their intended purpose (e.g., in vivo administration, in vitro methods, or ex vivo applications). Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D- amino acids or non-naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues. Modifications can also include modifications with non-naturally occurring unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.

[0296] Modifications can further include the introduction of various groups to polypeptides and/or guide nucleic acids described herein. For example, groups can be introduced during synthesis or during expression of a polypeptide (e.g., an effector protein), which allow for linking to other molecules or to a surface. Thus, e.g., cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like. [0297] Modifications can further include modification of nucleic acids described herein (e.g., guide nucleic acids) to provide the nucleic acid with a new or enhanced feature, such as improved stability. Such modifications of a nucleic acid include a base editing, base modification, a backbone modification, a sugar modification, or combinations thereof, of one or more nucleotides, nucleosides, or nucleobases in a nucleic acid.

[0298] In some embodiments, nucleic acids (e.g., engineered guide nucleic acids or nucleic acids encoding the same and/or nucleic acids encoding effector proteins, ) described herein comprise one or more modifications comprising: 2’0-methyl modified nucleotides, 2’ Fluoro modified nucleotides; locked nucleic acid (LNA) modified nucleotides; peptide nucleic acid (PNA) modified nucleotides; nucleotides with phosphorothioate linkages; a 5’ cap (e.g., a 7-methylguanylate cap (m7G)), phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3 '-alkylene phosphonates, 5 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphor amidates, thionoalkylphosphonates , thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3 '-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more intemucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage; phosphorothioate and/or heteroatom intemucleoside linkages, such as -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2- (known as a methylene (methylimino) or MMI backbone), -CH2-O-N(CH3)-CH2-, -CH2-N(CH3)- N(CH3)-CH2- and -O-N(CH3)-CH2-CH2- (wherein the native phosphodiester intemucleotide linkage is represented as -O- P(=O)(OH)-O-CH2-); morpholino linkages (formed in part from the sugar portion of a nucleoside); morpholino backbones; phosphorodiamidate or other non-phosphodiester intemucleoside linkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; other backbone modifications having mixed N, O, S and CH2 component parts; and combinations thereof.

V. Vectors and Multiplexed Expression Vectors

[0299] In some embodiments, compositions, methods and systems provided herein comprise a vector system, wherein the vector system comprises one or more vectors. When a vector is described herein, such a vector can be used as a vehicle to introduce one or more molecules of interest into a host cell. A molecule of interest can comprise a polypeptide (e.g., an effector protein), an engineered guide or a component thereof (e.g., crRNA, intermediary RNA, tracrRNA, or sgRNA), a donor nucleic acid, a nucleic acid encoding a polypeptide, a nucleic acid encoding an engineered guide or a component thereof. For example, vector systems described herein can comprise one or more vectors comprising a polypeptide (e.g., an effector protein), an engineered guide nucleic acid (e.g., crRNA, intermediary RNA, tracrRNA, or sgRNA), or a nucleic acid encoding for the same (e.g., a DNA molecule), or a nucleic acid or nucleic acids encoding a polypeptide, engineered guide nucleic acid, a donor nucleic acid, or any combination thereof. [0300] In some embodiments, compositions and systems provided herein comprise a vector system comprising a polypeptide (e.g., an effector protein) described herein. In some embodiments, compositions and systems provided herein comprise a vector system comprising a guide nucleic acid (e.g., crRNA, intermediary RNA, tracrRNA, or sgRNA) described herein. In some embodiments, compositions and systems provided herein comprise a vector system comprising a donor nucleic acid described herein.

[0301] In some embodiments, compositions and systems provided herein comprise a vector system encoding a polypeptide (e.g., an effector protein) described herein. In some embodiments, compositions and systems provided herein comprise a vector system encoding a guide nucleic acid (e.g., crRNA, intermediary RNA, tracrRNA, or sgRNA) described herein. In some embodiments, compositions and systems provided herein comprise a multi-vector system encoding an effector protein and a guide nucleic acid described herein, wherein the guide nucleic acid and the effector protein are encoded by the same or different vectors. In some embodiments, the guide nucleic acid and the effector protein are encoded by different vectors of the system. In some embodiments, a vector system comprises a library of vectors each encoding one or more component of a composition or system described herein. In some embodiments, a nucleic acid encoding a polypeptide (e.g., an effector protein) comprises an expression vector. In some embodiments, a nucleic acid encoding a polypeptide is a messenger RNA. In some embodiments, an expression vector comprises or encodes an engineered guide nucleic acid. In some cases, the expression vector encodes the crRNA or sgRNA.

[0302] In some embodiments, a vector may encode one or more effector proteins. In some embodiments, a vector comprises a nucleotide sequence encoding one or more effector proteins as described herein. In some embodiments, the one or more effector proteins comprise at least two effector proteins. In some embodiments, the at least two effector protein are the same. In some embodiments, the at least two effector proteins are different from each other. In some embodiments, the nucleotide sequence is operably linked to a promoter that is operable in a target cell, such as a eukaryotic cell. In some embodiments, a vector may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or more effector proteins. In some embodiments, a vector can encode one or more effector proteins comprising any one of amino acid sequences recited in TABLE 1.

[0303] In some embodiments, a vector may encode one or more guide nucleic acids. In some embodiments, a vector comprises one or more guide nucleic acids, or a nucleotide sequence encoding the one or more guide nucleic acids as described herein. In some embodiments, the one or more guide nucleic acids comprise at least two guide nucleic acids. In some embodiments, the at least two guide nucleic acids are the same. In some embodiments, the at least two guide nucleic acids are different from each other. In some embodiments, the guide nucleic acid or the nucleotide sequence encoding the guide nucleic acid is operably linked to a promoter that is operable in a target cell, such as a eukaryotic cell. In some embodiments, a vector may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or more different guide nucleic acids as described herein.

[0304] In some embodiments, a vector comprises one or more donor nucleic acids as described herein. In some embodiments, the one or more donor nucleic acids comprise at least two donor nucleic acids. In some embodiments, the at least two donor nucleic acids are the same. In some embodiments, the at least two donor nucleic acids are different from each other. In some embodiments, the vector comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more donor nucleic acids. [0305] In some embodiments, a vector can comprise or encode one or more regulatory elements. Regulatory elements can refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence or a coding sequence and/or regulate translation of an encoded polypeptide. In some embodiments, a vector can comprise or encode for one or more additional elements, such as, for example, replication origins, antibiotic resistance (or a nucleic acid encoding the same), a tag (or a nucleic acid encoding the same), selectable markers, and the like. In some embodiments, a vector comprises or encodes for one or more elements, such as, for example, ribosome binding sites, and RNA splice sites.

[0306] Vectors described herein can encode a promoter - a regulatory region on a nucleic acid, such as a DNA sequence, capable of initiating transcription of a downstream (3' direction) coding or non-coding sequence. As used herein, a promoter can be bound or linked at its 3' terminus to a nucleic acid the expression or transcription of which is desired, and extends upstream (5' direction) to include bases or elements necessary to initiate transcription or induce expression, which could be measured at a detectable level. A promoter can comprise a nucleotide sequence, referred to herein as a “promoter sequence”. A promoter sequence can include a transcription initiation site, and one or more protein binding domains responsible for the binding of transcription machinery, such as RNA polymerase. When eukaryotic promoters are used, such promoters can contain “TATA” boxes and “CAT” boxes. Various promoters, including inducible promoters, may be used to drive expression, i. e. , transcriptional activation, of the nucleic acid of interest. Accordingly, in some embodiments, the nucleic acid of interest can be operably linked to a promoter.

[0307] Promotors can be any suitable type of promoter envisioned for the compositions, systems, and methods described herein. Examples include constitutively active promoters (e.g., CMV promoter), inducible promoters (e.g, heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.), spatially restricted and/or temporally restricted promoters (e.g., a tissue specific promoter, a cell type specific promoter, etc.), etc. Suitable promoters include, but are not limited to: SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6), an enhanced U6 promoter, and a human Hl promoter (Hl). By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by 2-fold, by 5 -fold, by 10-fold, by 50-fold, by 100-fold, by 500-fold, or by 1000-fold, or more. In addition, vectors used for providing a nucleic acid encoding an engineered guide nucleic acid and/or an effector protein to a cell may include nucleic acid sequences that encode for selectable markers in the target cells, so as to identify cells that have taken up the engineered guide nucleic acid and/or an effector protein.

[0308] In general, vectors provided herein comprise at least one promotor or a combination of promoters driving expression or transcription of one or more genome editing tools described herein. In some embodiments, the vector comprises a nucleotide sequence of a promoter. In some embodiments, the vector comprises two promoters. In some embodiments, the vector comprises three promoters. In some embodiments, a length of the promoter is less than about 500, less than about 400, less than about 300, or less than about 200 linked nucleotides. In some embodiments, a length of the promoter is at least 100, at least 200, at least 300, at least 400, or at least 500 linked nucleotides. Non-limiting examples of promoters include CMV, 7SK, EFla, RPBSA, hPGK, EFS, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, GALI-10, Hl, TEF1, GDS, ADH1, CaMV35S, HSV TK, Ubi, U6, MNDU3, MSCV, MND and CAG.

[0309] In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter only drives expression of its corresponding coding sequence (e.g., polypeptide or guide nucleic acid) when a signal is present, e.g, a hormone, a small molecule, a peptide. Non-limiting examples of inducible promoters are the T7 RNA polymerase promoter, the T3 RNA polymerase promoter, the Isopropyl-beta-D- thiogalactopyranoside (IPTG)-regulated promoter, a lactose induced promoter, a heat shock promoter, a tetracycline-regulated promoter (tetracycline-inducible or tetracycline-repressible), a steroid regulated promoter, a metal-regulated promoter, and an estrogen receptor-regulated promoter. In some embodiments, the promoter is an activation-inducible promoter, such as a CD69 promoter. In some embodiments, the promoter for expressing effector protein is a ubiquitous promoter. In some embodiments, the ubiquitous promoter comprises MND or CAG promoter sequence.

[0310] In some embodiments, the promoters are prokaryotic promoters (e.g., drive expression of a gene in a prokaryotic cell). In some embodiments, the promoters are eukaryotic promoters, (e.g., drive expression of a gene in a eukaryotic cell). In some embodiments, the promoter is EFla. In some embodiments, the promoter is ubiquitin. In some embodiments, vectors are bicistronic or polycistronic vector (e.g., having or involving two or more loci responsible for generating a protein) having an internal ribosome entry site (IRES) is for translation initiation in a cap-independent manner.

[0311] In some embodiments, a vector described herein is a nucleic acid expression vector. In some embodiments, a vector described herein is a recombinant expression vector. In some embodiments, a vector described herein is a messenger RNA. [0312] In some embodiments, a vector described herein is a delivery vector. In some embodiments, the delivery vector is a eukaryotic vector, a prokaryotic vector (e.g., a bacterial vector) a viral vector, or any combination thereof. In some embodiments, the delivery vehicle is a non-viral vector. In some embodiments, the delivery vector is a plasmid. In some embodiments, the plasmid comprises DNA. In some embodiments, the plasmid comprises RNA. In some embodiments, the plasmid comprises circular double-stranded DNA. In some embodiments, the plasmid is linear. In some embodiments, the plasmid comprises one or more coding sequences of interest and one or more regulatory elements. In some embodiments, the plasmid comprises a bacterial backbone containing an origin of replication and an antibiotic resistance gene or other selectable marker for plasmid amplification in bacteria. In some embodiments, the plasmid is a minicircle plasmid. In some embodiments, the plasmid contains one or more genes that provide a selective marker to induce a target cell to retain the plasmid. In some examples, the plasmids are engineered through synthetic or other suitable means known in the art. For example, in some cases, the genetic elements are assembled by restriction digest of the desired genetic sequence from a donor plasmid or organism to produce ends of the DNA which is then be readily ligated to another genetic sequence.

[0313] In some embodiments, vectors comprise an enhancer. Enhancers are nucleotide sequences that have the effect of enhancing promoter activity. In some embodiments, enhancers augment transcription regardless of the orientation of their sequence. In some embodiments, enhancers activate transcription from a distance of several kilo basepairs. Furthermore, enhancers are located optionally upstream or downstream of a gene region to be transcribed, and/or located within the gene, to activate the transcription. Exemplary enhancers include, but are not limited to, WPRE; CMV enhancers; the R-U5' segment in LTR of HTLV-I.

Administration of a non-viral vector

[0314] In some embodiments, an administration of a non-viral vector comprises contacting a cell, such as a host cell, with the non-viral vector. In some embodiments, a physical method or a chemical method is employed for delivering the vector into the cell. Exemplary physical methods include electroporation, gene gun, sonoporation, magnetofection, or hydrodynamic delivery. Exemplary chemical methods include delivery of the recombinant polynucleotide by liposomes such as, cationic lipids or neutral lipids; lipofection; dendrimers; lipid nanoparticle (LNP); or cell-penetrating peptides.

[0315] In some embodiments, an effector protein (or a nucleic acid encoding same) and/or an engineered guide nucleic acid (or a nucleic acid encoding same) are coadministered with a donor nucleic acid. Coadministration can be contact with a target nucleic acid, administered to a cell, such as a host cell, or administered as method of nucleic acid detection, editing, and/or treatment as described herein, in a single vehicle, such as a single expression vector. In certain embodiments, an effector protein (or a nucleic acid encoding same) and/or an engineered guide nucleic acid (or a nucleic acid encoding same) are not co-administered with donor nucleic acid in a single vehicle. In some embodiments, at least two of the three components, a nucleic acid encoding one or more effector proteins, one or more donor nucleic acids, and one or more guide nucleic acids or a nucleic acid encoding the one or more guide nucleic acid, are provided in the single expression vector. In certain embodiments, an effector protein (or a nucleic acid encoding same) and/or an engineered guide nucleic acid (or a nucleic acid that, when transcribed, produces same) are not co-administered with donor nucleic acid in a single vehicle. In some embodiments, an effector protein (or a nucleic acid encoding same), an engineered guide nucleic acid (or a nucleic acid encoding same), and/or donor nucleic acid are administered in one or more or two or more vehicles, such as one or more, or two or more expression vectors.

Lipid Particles and Non-viral Vectors

[0316] In some embodiments, compositions and systems provided herein comprise a lipid particle. In some embodiments, a lipid particle is a lipid nanoparticle (LNP). In some embodiments, a lipid or a lipid nanoparticle can encapsulate an expression vector. In some embodiments, the expression vector incorporates the effector protein, the guide nucleic acid, the nucleic acid encoding the effector protein and/or the DNA molecule encoding the guide nucleic acid. LNPs are a non-viral delivery system for gene therapy. LNPs are also a non-viral delivery system for composition and/or system components described herein. LNPs are effective for delivery of nucleic acids. Beneficial properties of LNP include ease of manufacture, low cytotoxicity and immunogenicity, high efficiency of nucleic acid encapsulation and cell transfection, multi-dosing capabilities and flexibility of design (Kulkami et al., (2018) Nucleic Acid Therapeutics, 28(3): 146-157). In some embodiments, a method can comprise contacting a cell with an expression vector. In some embodiments, contacting can comprise electroporation, lipofection, or lipid nanoparticle (LNP) delivery of an expression vector. In some embodiments, a nucleic acid expression vector is a non-viral vector. In some embodiments, compositions and methods comprise a lipid, polymer, nanoparticle, or a combination thereof, or use thereof, to introduce an effector protein (e.g., a Cas protein), guide nucleic acid, donor nucleic acid (e.g., a donor template) or any combination thereof to a cell. Non-limiting examples of lipids and polymers are cationic polymers, cationic lipids, ionizable lipids, or bio-responsive polymers. In some embodiments, the ionizable lipids exploits chemical-physical properties of the endosomal environment (e.g., pH) offering improved delivery of nucleic acids. In some embodiments, the ionizable lipids are neutral at physiological pH. In some embodiments, the ionizable lipids are protonated under acidic pH. In some embodiments, the bio-responsive polymer exploits chemical-physical properties of the endosomal environment (e.g., pH) to preferentially release the genetic material in the intracellular space.

[0317] In some embodiments, a LNP comprises an outer shell and an inner core. In some embodiments, the outer shell comprises lipids. In some embodiments, the lipids comprise modified lipids. In some embodiments, the modified lipids comprise pegylated lipids. In some embodiments, the lipids comprise one or more of cationic lipids, anionic lipids, ionizable lipids, and non-ionic lipids. In some embodiments, the LNP comprises one or more of N¹,N³,N⁵-tris(3-(didodecylamino)propyl)benzene- 1,3, 5 -tricarboxamide (TT3), 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l-palmitoyl-2- oleoylsn-glycero-3 -phosphoethanolamine (POPE), l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), cholesterol (Choi), 1,2-dimyristoyl-sn-glycerol, and methoxypolyethylene glycol (DMG- PEChooo), derivatives, analogs, or variants thereof. In some embodiments, the LNP has a negative net overall charge prior to complexation with one or more of a guide RNA, a nucleic acid encoding the one or more guide RNA, and a nucleic acid encoding one or more effector protein. In some embodiments, the inner core is a hydrophobic core. In some embodiments, the guide RNA or the nucleic acid encoding the guide RNA forms a complex with one or more of the cationic lipids and the ionizable lipids. In some embodiments, the nucleic acid encoding the effector protein or the nucleic acid encoding the guide RNA is self-replicating.

[0318] In some embodiments, a LNP comprises one or more of a cationic lipid, an ionizable lipid and a modified version thereof. In some embodiments, the ionizable lipid comprises TT3 or a derivative thereof. Accordingly, in some embodiments, the LNP comprises one or more of TT3 and pegylated TT3. The publication WO2016187531 is hereby incorporated by reference in its entirety, which describes representative LNP formulations in Table 2 and Table 3, and a representative methods of delivering LNP formulations in Example 7.

[0319] In some embodiments, a LNP comprises a lipid composition targeting to a specific organ. In some embodiments, the lipid composition comprises lipids having a specific alkyl chain length that controls accumulation of the LNP in the specific organ (e.g., liver or spleen). In some embodiments, the lipid composition comprises a biomimetic lipid that controls accumulation of the LNP in the specific organ (e.g., brain). In some embodiments, the lipid composition comprises lipid derivatives (e.g., cholesterol derivatives) that controls accumulation of the LNP in a specific cell (e.g., liver endothelial cells, Kupffer cells, hepatocytes).

Delivery of Viral Vectors

[0320] An expression vector can be a viral vector. In some embodiments, a viral vector comprises a nucleic acid to be delivered into a host cell via a recombinantly produced virus or viral particle. The nucleic acid may be single-stranded or double stranded, linear or circular, segmented or non-segmented. The nucleic acid may comprise DNA, RNA, or a combination thereof. In some embodiments, the expression vector is an adeno-associated viral vector. There are a variety of viral vectors that are associated with various types of viruses, including but not limited to retroviruses (e.g., lentiviruses and y-retroviruses), adenoviruses, arenaviruses, alphaviruses, adeno-associated viruses (AAVs), baculoviruses, vaccinia viruses, herpes simplex viruses and poxviruses. In some embodiments, the viral vector is a replication-defective viral vector, comprising an insertion of a therapeutic gene inserted in genes essential to the lytic cycle, preventing the virus from replicating and exerting cytotoxic effects. A viral vector provided herein can be derived from or based on any such virus. Often the viral vectors provided herein are an adeno-associated viral vector (AAV vector).

[0321] In some embodiments, the vector is an adeno-associated viral (AAV) vector. In some embodiments, the viral vector is a recombinant viral vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the retroviral vector comprises gamma-retroviral vector. A viral vector provided herein may be derived from or based on any such virus. For example, in some embodiments, the gamma- retroviral vector is derived from a Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV) or a Murine Stem cell Virus (MSCV) genome. In some embodiments, the lentiviral vector is derived from the human immunodeficiency virus (HIV) genome. In some embodiments, the viral vector is a chimeric viral vector. In some embodiments, the chimeric viral vector comprises viral portions from two or more viruses. In some embodiments, the viral vector corresponds to a virus of a specific serotype. [0322] In some embodiments, a viral vector is an adeno-associated viral vector (AAV vector). In some embodiments, a viral particle that delivers a viral vector described herein is an AAV. In some embodiments, the AAV comprises any AAV known in the art. In some embodiments, the viral vector corresponds to a virus of a specific AAV serotype. In some embodiments, the AAV serotype is selected from an AAV1 serotype, an AAV2 serotype, AAV3 serotype, an AAV4 serotype, AAV5 serotype, an AAV6 serotype, AAV7 serotype, an AAV8 serotype, an AAV9 serotype, an AAV10 serotype, an AAV 11 serotype, an AAV 12 serotype, an AAV-rhlO serotype, and any combination, derivative, or variant thereof. In some embodiments, the AAV vector is a recombinant vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementary AAV (scAAV) vector, a single-stranded AAV, or any combination thereof. scAAV genomes are generally known in the art and contain both DNA strands which can anneal together to form double-stranded DNA.

[0323] In some embodiments, an AAV vector described herein is a chimeric AAV vector. In some embodiments, the chimeric AAV vector comprises an exogenous amino acid or an amino acid substitution, or capsid proteins from two or more serotypes. In some examples, a chimeric AAV vector may be genetically engineered to increase transduction efficiency, selectivity, or a combination thereof. [0324] Generally, an AAV vector has two inverted terminal repeats (ITRs). According, in some embodiments, the viral vector provided herein comprises two inverted terminal repeats of AAV. The DNA sequence in between the ITRs of an AAV vector provided herein may be referred to herein as the sequence encoding the genome editing tools. These genome editing tools can include, but are not limited to, an effector protein, effector protein modifications, fusion proteins (e.g., nuclear localization signal (NLS), polyA tail), guide nucleic acid(s), respective promoter(s), and a donor nucleic acid, nucleic acids encoding the same, or combinations thereof.

[0325] In general, viral vectors provided herein comprise at least one promotor or a combination of promoters driving expression or transcription of one or more genome editing tools described herein. In some embodiments, the length of the promoter is less than about 500, less than about 400, or less than about 300 linked nucleotides. In some embodiments, the length of the promoter is at least 100 linked nucleotides. Non-limiting examples of promoters include ApoE, TBG, CMV, 7SK, EFla, RPBSA, hPGK, EFS, SV40, PGK1, Ubc, human beta actin promoter, CAG, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, GALI, Hl, TEF1, GDS, ADH1, CaMV35S, Ubi, U6, MNDU3, Ck8e, SPC5-12, Desmin, MND and MSCV. In some embodiments, the promoter is an inducible promoter that only drives expression of its corresponding gene when a signal is present, e.g, a hormone, a small molecule, a peptide. Non-limiting examples of inducible promoters are the T7 RNA polymerase promoter, the T3 RNA polymerase promoter, the Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, a lactose induced promoter, a heat shock promoter, a tetracycline-regulated promoter (tetracyclineinducible or tetracycline-repressible), a steroid regulated promoter, a metal -regulated promoter, and an estrogen receptor-regulated promoter. In some embodiments, the promoter is an activation-inducible promoter, such as a CD69 promoter, as described further in Kulemzin et al., (2019), BMC Med Genomics, 12:44.

[0326] In some embodiments, the coding region of the AAV vector forms an intramolecular doublestranded DNA template thereby generating an AAV vector that is a self-complementary AAV (scAAV) vector. In general, the sequence encoding the genome editing tools of an scAAV vector has a length of about 2 kb to about 3 kb. The scAAV vector can comprise nucleotide sequences encoding an effector protein, providing guide nucleic acids described herein, and a donor nucleic acid described herein. In some embodiments, the AAV vector provided herein is a self-inactivating AAV vector.

[0327] In some embodiments, an AAV vector provided herein comprises a modification, such as an insertion, deletion, chemical alteration, or synthetic modification, relative to a wild-type AAV vector. [0328] In some embodiments, the viral particle that delivers the viral vector described herein is an AAV. AAVs are characterized by their serotype. Non-limiting examples of AAV serotypes are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, scAAV, AAV-rhlO, chimeric or hybrid AAV, or any combination, derivative, or variant thereof.

Producing AAV Delivery Vectors

[0329] In some embodiments, methods of producing AAV delivery vectors herein comprise packaging a nucleic acid encoding an effector protein and a guide nucleic acid, or a combination thereof, into an AAV vector. In some embodiments, methods of producing the delivery vector comprises, (a) contacting a cell with at least one nucleic acid encoding: (i) a guide nucleic acid; (ii) a Replication (Rep) gene; and (iii) a Capsid (Cap) gene that encodes an AAV capsid protein; (b) expressing the AAV capsid protein in the cell; (c) assembling an AAV particle; and (d) packaging an effector encoding nucleic acid into the AAV particle, thereby generating an AAV delivery vector. In some embodiments, promoters, staffer sequences, and any combination thereof may be packaged in the AAV vector. In some examples, the AAV vector may package 1, 2, 3, 4, or 5 guide nucleic acids or copies thereof. In some embodiments, the AAV vector comprises inverted terminal repeats, e.g. , a 5 ’ inverted terminal repeat and a 3 ’ inverted terminal repeat. In some embodiments, the AAV vector comprises a mutated inverted terminal repeat that lacks a terminal resolution site.

[0330] In some embodiments, a hybrid AAV vector is produced by transcapsidation, e.g., packaging an inverted terminal repeat (ITR) from a first serotype into a capsid of a second serotype, wherein the first and second serotypes may be not the same. In some examples, the Rep gene and ITR from a first AAV serotype (e.g., AAV2) may be used in a capsid from a second AAV serotype (e.g., AAV9), wherein the first and second AAV serotypes may be not the same. As a non-limiting example, a hybrid AAV serotype comprising the AAV2 ITRs and AAV9 capsid protein may be indicated AAV2/9. In some examples, the hybrid AAV delivery vector comprises an AAV2/1, AAV2/2, AAV 2/4, AAV2/5, AAV2/8, or AAV2/9 vector.

Producing AAV Particles

[0331] The AAV particles described herein can be referred to as recombinant AAV (rAAV). Often, rAAV particles are generated by transfecting AAV producing cells with an AAV-containing plasmid carrying the sequence encoding the genome editing tools, a plasmid that carries viral encoding regions, i.e., Rep and Cap gene regions; and a plasmid that provides the helper genes such as El A, E1B, E2A, E4ORF6 and VA. In some embodiments, the AAV producing cells are mammalian cells. In some embodiments, host cells for rAAV viral particle production are mammalian cells. In some embodiments, a mammalian cell for rAAV viral particle production is a COS cell, a HEK293T cell, a HeLa cell, a KB cell, a derivative thereof, a variant thereof, or a combination thereof. In some embodiments, rAAV virus particles can be produced in the mammalian cell culture system by providing the rAAV plasmid to the mammalian cell. In some embodiments, producing rAAV virus particles in a mammalian cell can comprise transfecting vectors that express the rep protein, the capsid protein, and the gene-of-interest expression construct flanked by the ITR sequence on the 5’ and 3’ ends. Methods of such processes are provided in, for example, Naso et al., BioDrugs, 2017 Aug;31(4):317-334 and Benskey et al., (2019), Methods Mol Biol., 1937:3-26, each of which is incorporated by reference in their entireties.

[0332] In some embodiments, rAAV is produced in a non-mammalian cell. In some embodiments, rAAV is produced in an insect cell. In some embodiments, an insect cell for producing rAAV viral particles comprises a Sf9 cell. In some embodiments, production of rAAV virus particles in insect cells can comprise baculovirus. In some embodiments, production of rAAV virus particles in insect cells can comprise infecting the insect cells with three recombinant baculoviruses, one carrying the cap gene, one carrying the rep gene, and one carrying the gene-of-interest expression construct enclosed by an ITR on both the 5 ’ and 3 ’ end. In some embodiments, rAAV virus particles are produced by the One Bac system. In some embodiments, rAAV virus particles can be produced by the Two Bac system. In some embodiments, in the Two Bac system, the rep gene and the cap gene of the AAV is integrated into one baculovirus virus genome, and the ITR sequence and the gene-of-interest expression construct is integrated into another baculovirus virus genome. In some embodiments, in the One Bac system, an insect cell line that expresses both the rep protein and the capsid protein is established and infected with a baculovirus virus integrated with the ITR sequence and the gene-of-interest expression construct. Details of such processes are provided in, for example, Smith et. al., (1983), Mol. Cell. Biol., 3(12):2156-65; Urabe et al., (2002), Hum. Gene. Then, 1; 13(16): 1935-43; and Benskey et al., (2019), Methods Mol Biol., 1937:3-26, each of which is incorporated by reference in its entirety.

VI. Target Nucleic Acids

[0333] Disclosed herein are compositions, systems, and methods for modifying and detecting target nucleic acids. In some embodiments, the target nucleic acid is a double stranded nucleic acid. In some instances, the target nucleic acid is a single stranded nucleic acid. In some instances, the target nucleic acid is a double stranded nucleic acid that is prepared into single stranded nucleic acids before or upon contacting an RNP, a reagent or a sample. In some embodiments, the target nucleic acid comprises DNA. In some instances, the target nucleic acid comprises RNA. The target nucleic acids include but are not limited to mRNA, rRNA, tRNA, non-coding RNA, long non-coding RNA, single stranded RNA (ssRNA), and microRNA (miRNA). In some instances, the target nucleic acid is complementary DNA (cDNA) synthesized from a single-stranded RNA template in a reaction catalyzed by a reverse transcriptase. In some cases, the target nucleic acid is single-stranded RNA (ssRNA) or mRNA. In some embodiments, the target nucleic acid is from a virus, a parasite, or a bacterium described herein.

[0334] In some embodiments, the target nucleic acid is a double stranded nucleic acid comprising a target strand and a non-target strand, wherein the target strand comprises a target sequence. In some embodiments, where a target strand comprises a target sequence, at least a portion of the engineered guide nucleic acid is complementary to the target sequence on the target strand. In some embodiments, where the target nucleic acid is a double stranded nucleic acid comprising a target strand and a nontarget strand, and wherein the target strand comprises a target sequence, at least a portion of the engineered guide nucleic acid is complementary to the target sequence on the target strand. In some embodiments, a target nucleic acid comprises a PAM as described herein that is located on the nontarget strand. Such a PAM described herein, in certain embodiments, is adjacent (e.g., within 1, 2, 3, 4 or 5 nucleotides) to the 3 ’ end of the target sequence on the non-target strand of the double stranded DNA molecule. In some embodiments, such a PAM described herein is directly adjacent to the 3’ end of a target sequence on the non-target strand of the double stranded DNA molecule.

[0335] In some embodiments, a target nucleic acid comprising a target sequence comprises a PAM sequence. In some embodiments, the PAM sequence is adjacent to the target sequence. In some embodiments, the PAM sequence is 3’ to the target sequence. In some embodiments, the PAM sequence is directly 3’ to the target sequence. In some embodiments, the PAM sequence 5’ to the target sequence. In some embodiments, the PAM sequence is directly 5’ to the target sequence. In some embodiments, the target nucleic acid as described in the methods herein does not initially comprise a PAM sequence. However, any target nucleic acid of interest may be generated using the methods described herein to comprise a PAM sequence, and thus be a PAM target nucleic acid. A PAM target nucleic acid, as used herein, refers to a target nucleic acid that has been amplified to insert a PAM sequence that is recognized by an effector protein system.

[0336] In some embodiments, a target nucleic acid comprises 5 to 100, 5 to 90, 5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 linked nucleotides. In some embodiments, the target nucleic acid comprises 10 to 90, 20 to 80, 30 to 70, or 40 to 60 linked nucleotides. In some embodiments, the target nucleic acid comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, or 100 linked nucleotides. In some embodiments, the target nucleic acid comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 linked nucleotides.

[0337] In some embodiments, compositions, systems, and methods described herein comprise a target nucleic acid may be responsible for a disease, contain a mutation (e.g., single strand polymorphism, point mutation, insertion, or deletion), be contained in an amplicon, or be uniquely identifiable from the surrounding nucleic acids (e.g, contain a unique sequence of nucleotides). In some embodiments, the target nucleic acid has undergone a modification (e.g., an editing) after contacting with an RNP. In some embodiments, the editing is a change in the sequence of the target nucleic acid. In some embodiments, the change comprises an insertion, deletion, or substitution of one or more nucleotides compared to the target nucleic acid that has not undergone any modification.

[0338] In some embodiments, the target nucleic acid comprises a nucleic acid sequence from a pathogen responsible for a disease. Non-limiting examples of pathogens are bacteria, a virus and a fungus. The target nucleic acid, in some embodiments, is a portion of a nucleic acid from a sexually transmitted infection or a contagious disease. In some embodiments, the target nucleic acid is a portion of a nucleic acid from a genomic locus, or any DNA amplicon, such as a reverse transcribed mRNA or a cDNA from a gene locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus in at least one of: human immunodeficiency virus (HIV), human papillomavirus (HPV), chlamydia, gonorrhea, syphilis, trichomoniasis, sexually transmitted infection, malaria, Dengue fever, Ebola, chikungunya, and leishmaniasis. Pathogens include viruses, fungi, helminths, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites. Helminths include roundworms, heartworms, and phytophagous nematodes, flukes, Acanthocephala, and tapeworms. Protozoan infections include infections from Giardia spp., Trichomonas spp., African trypanosomiasis, amoebic dysentery, babesiosis, balantidial dysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis. Examples of pathogens such as parasitic/protozoan pathogens include, but are not limited to: Plasmodium falciparum, P. vivax, Trypanosoma cruzi and Toxoplasma gondii. Fungal pathogens include, but are not limited to Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans. Pathogenic viruses include but are not limited to coronavirus (e.g., SARS-CoV-2); immunodeficiency virus (e.g., HIV); influenza virus; dengue; West Nile virus; herpes virus; yellow fever virus; Hepatitis Virus C; Hepatitis Virus A; Hepatitis Virus B; papillomavirus; and the like. Pathogens include, e.g., HIV virus, Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Hemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II, human serum parvo-like virus, respiratory syncytial virus (RSV), M. genitalium, T. vaginalis, varicella-zoster virus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, blue tongue virus, Sendai virus, feline leukemia virus, Reovirus, polio virus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, West Nile virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria tenella, Onchocerca volvulus, Leishmania tropica, Mycobacterium tuberculosis, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium and M. pneumoniae. In some embodiments, the target sequence is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus of bacterium or other agents responsible for a disease in the sample comprising a mutation that confers resistance to a treatment, such as a single nucleotide mutation that confers resistance to antibiotic treatment.

[0339] In some embodiments, the target nucleic acid comprises a nucleic acid sequence of a virus, a bacterium, or other pathogen responsible for a disease in a plant (e.g., a crop). Methods and compositions of the disclosure may be used to treat or detect a disease in a plant. For example, the methods of the disclosure may be used to target a viral nucleic acid sequence in a plant. An effector protein of the disclosure may cleave the viral nucleic acid. In some embodiments, the target nucleic acid comprises a nucleic acid sequence of a virus or a bacterium or other agents (e.g., any pathogen) responsible for a disease in the plant (e.g., a crop). In some embodiments, the target nucleic acid comprises RNA. The target nucleic acid, in some embodiments, is a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease in the plant (e.g., a crop). In some embodiments, the target nucleic acid is a portion of a nucleic acid from a genomic locus, or any NA amplicon, such as a reverse transcribed mRNA or a cDNA from a gene locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus in at a virus or a bacterium or other agents (e.g., any pathogen) responsible for a disease in the plant (e.g., a crop). A virus infecting the plant may be an RNA virus. A virus infecting the plant may be a DNA virus. Non-limiting examples of viruses that may be targeted with the disclosure include Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Potato virus Y (PVY), Cauliflower mosaic virus (CaMV) (RT virus), Plum pox virus (PPV), Brome mosaic virus (BMV) and Potato virus X (PVX).

[0340] In some embodiments, a target nucleic acid comprises a portion or a specific region of a nucleic acid from a genomic locus, any DNA amplicon of, a reverse transcribed mRNA, or a cDNA from a gene described herein. In some embodiments, the target nucleic acid is an amplicon of at least a portion of a gene. Non-limiting examples of genes are recited in TABLE 9. Nucleic acid sequences of target nucleic acids and/or corresponding genes are readily available in public databases as known and used in the art. In some embodiments, the target nucleic acid is selected from TABLE 9. In some embodiments, the target nucleic acid comprises one or more target sequences. In some embodiments, the one or more target sequence is within any one of the target nucleic acids set forth in TABLE 9.

[0341] In some embodiments, the target nucleic acid is any one of: a naturally occurring eukaryotic sequence, a naturally occurring prokaryotic sequence, a naturally occurring viral sequence, a naturally occurring bacterial sequence, a naturally occurring fungal sequence, an engineered eukaryotic sequence, an engineered prokaryotic sequence, an engineered viral sequence, an engineered bacterial sequence, an engineered fungal sequence, a fragment of a naturally occurring sequence, a fragment of an engineered sequence, and combinations thereof.

[0342] In some embodiments, the target nucleic acid is isolated from any one of: a naturally occurring cell, a eukaryotic cell, a prokaryotic cell, a plant cell, a fungal cell, an animal cell, cell of an invertebrate, a fly cell, a cell of a vertebrate, a mammalian cell, a primate cell, a non-human primate cell, a human cell, a living cell, a non-living cell, a modified cell, a derived cell, and a non-naturally occurring cell.

[0343] Nucleic acids, such as DNA and pre-mRNA, described herein can contain at least one intron and at least one exon, wherein as read in the 5 ’ to the 3 ’ direction of a nucleic acid strand, the 3 ’ end of an intron can be adjacent to the 5’ end of an exon, and wherein said intron and exon correspond for transcription purposes. If a nucleic acid strand contains more than one intron and exon, the 5’ end of the second intron is adjacent to the 3’ end of the first exon, and 5’ end of the second exon is adjacent to the 3’ end of the second intron. The junction between an intron and an exon can be referred to herein as a splice junction, wherein a 5’ splice site (SS) can refer to the +1/+2 position at the 5’ end of intron and a 3’SS can refer to the last two positions at the 3’ end of an intron. Alternatively, a 5’ SS can refer to the 5’ end of an exon and a 3’SS can refer to the 3’ end of an exon. In some embodiments, nucleic acids can contain one or more elements that act as a signal during transcription, splicing, and/or translation. In some embodiments, signaling elements include a 5’SS, a 3’SS, a premature stop codon, U1 and/or U2 binding sequences, and cis acting elements such as branch site (BS), polypyridine tract (PYT), exonic and intronic splicing enhancers (ESEs and ISEs) or silencers (ESSs and ISSs). In some embodiments, nucleic acids may also comprise a untranslated region (UTR), such as a 5 ’ UTR or a 3 ’ UTR. In some embodiments, the start of an exon or intron is referred to interchangeably herein as the 5’ end of an exon or intron, respectively. Likewise, in some embodiments, the end of an exon or intron is referred to interchangeably herein as the 3’ end of an exon or intron, respectively. [0344] In some embodiments, at least a portion of at least one target sequence is within about 1, about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, about 50 or more, about 55 or more, about 60 or more, about 65 or more, about 70 or more, about 75 or more, about 80 or more, about 85 or more, about 90 or more, about 95 or more, about 100 or more, about 105 or more, about 110 or more, about 115 or more, about 120 or more, about 125 or more, about 130 or more, about 135 or more, about 140 or more, about 145 or more, or about 150 to about 300 nucleotides adjacent to: the 5’ end of an exon; the 3’ end of an exon; the 5 ’ end of an intron; the 3 ’ end of an intron; one or more signaling element comprising a 5’SS, a 3’SS, a premature stop codon, U1 binding sequence, U2 binding sequence, a BS, a PYT, ESE, an ISE, an ESS, an ISS; a 5’ UTR; a 3’ UTR; more than one of the foregoing, or any combination thereof. In some embodiments, the target nucleic acid comprises a target locus. In some embodiments, the target nucleic acid comprises more than one target loci. In some embodiments, the target nucleic acid comprises two target loci. Accordingly, in some embodiments, the target nucleic acid can comprise one or more target sequences.

[0345] In some embodiments, compositions, systems, and methods described herein comprise an edited target nucleic acid which can describe a target nucleic acid wherein the target nucleic acid has undergone a change, for example, after contact with an effector protein. In some embodiments, the editing is an alteration in the sequence of the target nucleic acid. In some embodiments, the edited target nucleic acid comprises an insertion, deletion, or replacement of one or more nucleotides compared to the unedited target nucleic acid. In some embodiments, the editing is a mutation.

Mutations

[0346] In some instances, target nucleic acids comprise a mutation. In some embodiments, a composition, system or method described herein can be used to modify a target nucleic acid comprising a mutation such that the mutation is modified to be a wild-type nucleotide or nucleotide sequence. In some embodiments, a composition, system or method described herein can be used to detect a target nucleic acid comprising a mutation. A mutation may result in the insertion of at least one amino acid in a protein encoded by the target nucleic acid. A mutation may result in the deletion of at least one amino acid in a protein encoded by the target nucleic acid. A mutation may result in the substitution of at least one amino acid in a protein encoded by the target nucleic acid. A mutation that results in the deletion, insertion, or substitution of one or more amino acids of a protein encoded by the target nucleic acid may result in misfolding of a protein encoded by the target nucleic acid. A mutation may result in a premature stop codon, thereby resulting in a truncation of the encoded protein.

[0347] Non-limiting examples of mutations are insertion-deletion (indel), a point mutation, single nucleotide polymorphism (SNP), a chromosomal mutation, a copy number mutation or variation, and frameshift mutations. In some embodiments, an indel mutation is an insertion or deletion of one or more nucleotides. In some embodiments, a point mutation comprises a substitution, insertion, or deletion. In some embodiments, a frameshift mutation occurs when the number of nucleotides in the insertion/deletion is not divisible by three, and it occurs in a protein coding region. In some embodiments, a chromosomal mutation can comprise an inversion, a deletion, a duplication, or a translocation of one or more nucleotides. In some embodiments, a copy number variation can comprise a gene amplification or an expanding trinucleotide repeat. In some embodiments, an SNP is associated with a phenotype of the sample or a phenotype of the organism from which the sample was taken. In some cases, an SNP is associated with altered phenotype from wild type phenotype. In some embodiments, the SNP is a synonymous substitution or a nonsynonymous substitution. In some embodiments, the nonsynonymous substitution is a missense substitution or a nonsense point mutation. In some embodiments, the synonymous substitution is a silent substitution.

[0348] In some embodiments, a target nucleic acid described herein comprises a mutation of one or more nucleotides. The mutation may be a mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. The mutation can be a deletion, insertion, and/or substitution of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 nucleotides. The mutation can be a deletion, insertion, and/or substitution of 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to 85, 85 to 90, 90 to 95, 95 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, 900 to 1000, 1 to 50, 1 to 100, 25 to 50, 25 to 100, 50 to 100, 100 to 500, 100 to 1000, or 500 to 1000 nucleotides.

[0349] The mutation may be located in a non-coding region or a coding region of a gene. In some embodiments, the target nucleic acid is a gene. The mutation can be in the open reading frame of a target nucleic acid that results in the insertion of at least one amino acid in a polypeptide encoded by the target nucleic acid. The mutation may be in the open reading frame of a target nucleic acid that results in the deletion of at least one amino acid in a polypeptide encoded by the target nucleic acid. The mutation can be in the open reading frame of a target nucleic acid that results in the substitution of at least one amino acid in a protein encoded by the target nucleic acid. A mutation that results in the deletion, insertion, or substitution of one or more amino acids of a polypeptide encoded by the target nucleic acid may result in misfolding of the polypeptide. The mutation may result in a premature stop codon. The mutation may result in a truncation of the protein.

[0350] In some instances at least a portion of a guide nucleic acid of a composition described herein hybridizes to a region of the target nucleic acid comprising the mutation. In some instances, at least a portion of a guide nucleic acid of a composition described herein hybridizes to a region of the target nucleic acid that is within 10 nucleotides, within 50 nucleotides, within 100 nucleotides, or within 200 nucleotides of the mutation. [0351] In some instances, the mutation is an autosomal dominant mutation. In some instances, the mutation is a dominant negative mutation. In some instances, the mutation is a loss of function mutation. In some instances, the mutation is a single nucleotide mutation or a single nucleotide polymorphism (SNP). In some instances, the single nucleotide mutation or the SNP is associated with a phenotype of the sample or a phenotype of the organism from which the sample was taken. In some embodiments, a single nucleotide mutation, SNP, or deletion described herein is associated with a disease, such as a genetic disease. The single nucleotide mutation or SNP, in some embodiments, is associated with altered phenotype from wild type phenotype. The SNP may be a synonymous substitution or a nonsynonymous substitution. The nonsynonymous substitution may be a missense substitution, or a nonsense point mutation. The synonymous substitution may be a silent substitution. The mutation may be a deletion of one or more nucleotides. Often, the single nucleotide mutation, SNP, or deletion is associated with a disease such as cancer or a genetic disorder. The mutation, such as a single nucleotide mutation, a SNP, or a deletion, may be encoded in the sequence of a target nucleic acid from the germline of an organism or may be encoded in a target nucleic acid from a diseased cell, such as a cancer cell.

[0352] In some embodiments, the mutation is associated with a disease, such as a genetic disorder. In some embodiments, the mutation may be encoded in the sequence of a target nucleic acid from the germline of an organism or may be encoded in a target nucleic acid from a diseased cell. In some instances, the target nucleic acid comprises a mutation associated with a disease. In some examples, a mutation associated with a disease refers to a mutation whose presence in a subject indicates that the subject is susceptible to, or suffers from, a disease, disorder, condition, pathological state, or syndrome. A “syndrome”, as used herein, refers to a group of symptoms which, taken together, characterize a condition. In some examples, a mutation associated with a disease, disorder, condition, or syndrome refers to a mutation which causes, contributes to the development of, or indicates the existence of the disease, disorder, condition, or syndrome. A mutation associated with a disease may also refer to any mutation which generates transcription or translation products at an abnormal level, or in an abnormal form, in cells affected by a disease relative to a control without the disease. In some examples, a mutation associated with a disease refers to a mutation whose presence in a subject indicates that the subject is susceptible to or suffers from, a disease, disorder, condition, or syndrome. In some examples, a mutation associated with a disease refers to a mutation which causes, contributes to the development of, or indicates the existence of the disease, disorder, condition, or syndrome. A mutation associated with a disease may also refer to any mutation which generates transcription or translation products at an abnormal level, or in an abnormal form, in cells affected by a disease relative to a control without the disease. In some examples, a mutation associated with a disease refers to a mutation whose presence in a subject indicates that the subject is susceptible to, or suffers from, a disease, disorder, or pathological state. In some embodiments, a mutation associated with a disease, comprises the cooccurrence of a mutation and the phenotype of a disease. The mutation may occur in a gene, wherein transcription or translation products from the gene occur at a significantly abnormal level or in an abnormal form in a cell or subject harboring the mutation as compared to a non-disease control subject not having the mutation. In some instances, the mutation causes the disease. In some embodiments, the target nucleic acid is from a gene with a mutation associated with a genetic disorder, from a gene whose overexpression is associated with a genetic disorder, from a gene associated with abnormal cellular growth resulting in a genetic disorder, or from a gene associated with abnormal cellular metabolism resulting in a genetic disorder. In some embodiments, a target nucleic acid described herein comprises a mutation associated with a disease, wherein the target nucleic acid is any one of the target nucleic acids set forth in TABLE 8. In some embodiments, a target nucleic acid described herein comprises a mutation associated with a disease, wherein the disease is any one of the diseases set forth in TABLE 9.

Detection and Identification of Target Nucleic Acid

[0353] In some embodiments, a target nucleic acid is in a cell. In some embodiments, the cell is a single-cell eukaryotic organism; a plant cell an algal cell; a fungal cell; an animal cell; a cell of an invertebrate animal; a cell of a vertebrate animal such as fish, amphibian, reptile, bird, and mammal; or a cell of a mammal such as a human, a non-human primate, an ungulate, a feline, a bovine, an ovine, and a caprine. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell, a human cell, or a plant cell. In some embodiments, the cell is a human cell. In some embodiments, the human cell is a: muscle cell, liver cell, lung cell, cardiac cell, visceral cell, cardiac muscle cell, smooth muscle cell, cardiomyocyte, nodal cardiac muscle cell, smooth muscle cell, visceral muscle cell, skeletal muscle cell, myocyte, red (or slow) skeletal muscle cell, white (fast) skeletal muscle cell, intermediate skeletal muscle, muscle satellite cell, muscle stem cell, myoblast, muscle progenitor cell, induced pluripotent stem cell (iPS), or a cell derived from an iPS cell, modified to have its gene edited and differentiated into myoblasts, muscle progenitor cells, muscle satellite cells, muscle stem cells, skeletal muscle cells, cardiac muscle cells or smooth muscle cells.

[0354] In some embodiments, an effector protein-guide nucleic acid complex may comprise high selectivity for a target sequence. In some embodiments, an RNP comprise a selectivity of at least 200: 1, 100: 1, 50: 1, 20: 1, 10: 1, or 5: 1 for a target nucleic acid over a single nucleotide variant of the target nucleic acid. In some embodiments, an RNP may comprise a selectivity of at least 5: 1 for a target nucleic acid over a single nucleotide variant of the target nucleic acid.

[0355] By leveraging such effector protein selectivity, some methods described herein may detect a target nucleic acid present in the sample in various concentrations or amounts as a target nucleic acid population. In some embodiments, the method detects at least 2 target nucleic acid populations. In some embodiments, the method detects at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 target nucleic acid populations. In some embodiments, the method detects 3 to 50, 5 to 40, or 10 to 25 target nucleic acid populations. In some embodiments, the method detects at least 2 individual target nucleic acids. In some embodiments, the method detects at least 3, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 individual target nucleic acids. In some embodiments, the method detects 1 to 10,000, 100 to 8000, 400 to 6000, 500 to 5000, 1000 to 4000, or 2000 to 3000 individual target nucleic acids. In some embodiments, the method detects target nucleic acid present at least at one copy per 10 non-target nucleic acids, 10² non-target nucleic acids, 10³ non-target nucleic acids, 10⁴ non-target nucleic acids, 10⁵ non-target nucleic acids, 10⁶ non- target nucleic acids, 10⁷ non-target nucleic acids, 10⁸ non-target nucleic acids, 10⁹ non-target nucleic acids, or 10¹⁰ non-target nucleic acids.

[0356] In some embodiments, compositions described herein exhibit indiscriminate trans-cleavage of a nucleic acid (e.g., ssRNA or ssDNA), enabling their use for detection of a nucleic acid (e.g, RNA or DNA, respectively) in samples. In some embodiments, target nucleic acids are generated from many nucleic acid templates (e.g., RNA) in order to achieve cleavage of a reporter (e.g., a FQ reporter) in a device (e.g., a DETECTR platform). Certain effector proteins may be activated by a nucleic acid (e.g., ssDNA or ssRNA), upon which they may exhibit trans-cleavage of the nucleic acid (e.g., ssDNA or ssRNA) and may, thereby, be used to cleave reporter molecules (e.g., ssDNA or ssRNA FQ reporter molecules) in a device (e.g., a DETECTR). These effector proteins may target nucleic acids present in the sample or nucleic acids generated and/or amplified from any number of nucleic acid templates (e.g., RNA). Described herein are reagents comprising a single stranded reporter nucleic acid comprising a detection moiety, wherein the reporter nucleic acid (e.g., a ssDNA-FQ reporter described herein) is capable of being cleaved by the effector protein, upon generation (e.g., cDNA) and amplification of nucleic acids from a nucleic acid template (e.g., ssRNA) using the methods disclosed herein, thereby generating a first detectable signal. While DNA and RNA are used as an exemplary reporter in the foregoing, any suitable reporter may be used.

[0357] In some embodiments, a target nucleic acid is an amplified nucleic acid of interest. In some embodiments, the nucleic acid of interest is any nucleic acid disclosed herein or from any sample as disclosed herein. In some embodiments, the nucleic acid of interest is an RNA that is reverse transcribed before amplification. In some embodiments, the nucleic acid of interest is amplified then the amplicons is transcribed into RNA.

[0358] In some embodiments, target nucleic acids may activate an effector protein to initiate sequenceindependent cleavage of a nucleic acid-based reporter (e.g., a reporter comprising an RNA sequence, or a reporter comprising DNA and RNA). For example, an effector protein of the present disclosure is activated by a target nucleic acid to cleave reporters having an RNA (also referred to herein as an “RNA reporter”). Alternatively, an effector protein of the present disclosure is activated by a target nucleic acid to cleave reporters having an RNA. Alternatively, an effector protein of the present disclosure is activated by a target RNA to cleave reporters having an RNA (also referred to herein as a “RNA reporter”). The RNA reporter may comprise a single-stranded RNA labelled with a detection moiety or may be any RNA reporter as disclosed herein. [0359] Further description of editing or detecting a target nucleic acid in a gene of interest can be found in more detail in Kim et al., “Enhancement of target specificity of CRISPR-Casl2aby using a chimeric DNA-RNA guide”, Nucleic Acids Res. 2020 Sep 4;48(15):8601-8616; Wang et al., “Specificity profiling of CRISPR system reveals greatly enhanced off-target gene editing”, Scientific Reports volume 10, Article number: 2269 (2020); Tuladhar et al., “CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation”, Nature Communications volume 10, Article number: 4056 (2019); Dong et al., “Genome-Wide Off-Target Analysis in CRISPR-Cas9 Modified Mice and Their Offspring”, G3, Volume 9, Issue 11, 1 November 2019, Pages 3645-3651; Winter et al., “Genomewide CRISPR screen reveals novel host factors required for Staphylococcus aureus a-hemolysin- mediated toxicity”, Scientific Reports volume 6, Article number: 24242 (2016); and Ma et al., “A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death”, Cell Rep. 2015 Jul 28;12(4):673-83, which are hereby incorporated by reference in their entirety.

Certain Samples

[0360] Various sample types comprising a target nucleic acid of interest are consistent with the present disclosure. These samples may comprise a target nucleic acid for detection. In some embodiments, the detection of the target nucleic indicates an ailment, such as a disease, cancer, or genetic disorder, or genetic information, such as for phenotyping, genotyping, or determining ancestry and are compatible with the reagents and support mediums as described herein. Generally, a sample from an individual or an animal or an environmental sample may be obtained to test for presence of a disease, cancer, genetic disorder, or any mutation of interest.

[0361] In some embodiments, a sample comprises a target nucleic acid from 0.05% to 20% of total nucleic acids in the sample. In some embodiments, the target nucleic acid is 0. 1% to 10% of the total nucleic acids in the sample. In some embodiments, the target nucleic acid is 0.1% to 5% of the total nucleic acids in the sample. In some embodiments, the target nucleic acid is 0.1% to 1% of the total nucleic acids in the sample. In some embodiments, the target nucleic acid is in any amount less than

100% of the total nucleic acids in the sample. In some embodiments, the target nucleic acid is 100% of the total nucleic acids in the sample. In some embodiments, the sample comprises a portion of the target nucleic acid and at least one nucleic acid comprising less than 100% sequence identity to the portion of the target nucleic acid but no less than 50% sequence identity to the portion of the target nucleic acid. For example, the portion of the target nucleic acid comprises a mutation as compared to at least one nucleic acid comprising less than 100% sequence identity to the portion of the target nucleic acid but no less than 50% sequence identity to the portion of the target nucleic acid. In some embodiments, the portion of the target nucleic acid comprises a single nucleotide mutation as compared to at least one nucleic acid comprising less than 100% sequence identity to the portion of the target nucleic acid but no less than 50% sequence identity to the portion of the target nucleic acid. [0362] In some embodiments, a sample comprises target nucleic acid populations at different concentrations or amounts. In some embodiments, the sample has at least 2 target nucleic acid populations. In some embodiments, the sample has at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 target nucleic acid populations. In some embodiments, the sample has 3 to 50, 5 to 40, or 10 to 25 target nucleic acid populations.

[0363] In some embodiments, a sample has at least 2 individual target nucleic acids. In some embodiments, the sample has at least 3, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 individual target nucleic acids. In some embodiments, the sample comprises 1 to 10,000, 100 to 8000, 400 to 6000, 500 to 5000, 1000 to 4000, or 2000 to 3000 individual target nucleic acids.

[0364] In some embodiments, a sample comprises one copy of target nucleic acid per 10 non-target nucleic acids, IO² non-target nucleic acids, IO³ non-target nucleic acids, IO⁴ non-target nucleic acids, 10⁵ non-target nucleic acids, IO⁶ non-target nucleic acids, IO⁷ non-target nucleic acids, IO⁸ non-target nucleic acids, IO⁹ non-target nucleic acids, or 10¹⁰ non-target nucleic acids.

[0365] In some embodiments, samples comprise a target nucleic acid at a concentration of less than 1 nM, less than 2 nM, less than 3 nM, less than 4 nM, less than 5 nM, less than 6 nM, less than 7 nM, less than 8 nM, less than 9 nM, less than 10 nM, less than 20 nM, less than 30 nM, less than 40 nM, less than 50 nM, less than 60 nM, less than 70 nM, less than 80 nM, less than 90 nM, less than 100 nM, less than 200 nM, less than 300 nM, less than 400 nM, less than 500 nM, less than 600 nM, less than 700 nM, less than 800 nM, less than 900 nM, less than 1 pM, less than 2 pM, less than 3 pM, less than 4 pM, less than 5 pM, less than 6 pM, less than 7 pM, less than 8 pM, less than 9 pM, less than 10 pM, less than 100 pM, or less than 1 mM. In some embodiments, the sample comprises a target nucleic acid at a concentration of 1 nM to 2 nM, 2 nM to 3 nM, 3 nM to 4 nM, 4 nM to 5 nM, 5 nM to 6 nM, 6 nM to 7 nM, 7 nM to 8 nM, 8 nM to 9 nM, 9 nM to 10 nM, 10 nM to 20 nM, 20 nM to 30 nM, 30 nM to 40 nM, 40 nM to 50 nM, 50 nM to 60 nM, 60 nM to 70 nM, 70 nM to 80 nM, 80 nM to 90 nM, 90 nM to 100 nM, 100 nM to 200 nM, 200 nM to 300 nM, 300 nM to 400 nM, 400 nM to 500 nM, 500 nM to 600 nM, 600 nM to 700 nM, 700 nM to 800 nM, 800 nM to 900 nM, 900 nM to 1 pM, 1 pM to 2 pM, 2 pM to 3 pM, 3 pM to 4 pM, 4 pM to 5 pM, 5 pM to 6 pM, 6 pM to 7 pM, 7 pM to 8 pM, 8 pM to 9 pM, 9 pM to 10 pM, 10 pM to 100 pM, 100 pM to 1 mM, 1 nM to 10 nM, 1 nM to 100 nM, 1 nM to 1 pM, 1 nM to 10 pM, 1 nM to 100 pM, 1 nM to 1 mM, 10 nM to 100 nM, 10 nM to 1 pM, 10 nM to 10 pM, 10 nM to 100 pM, 10 nM to 1 mM, 100 nM to 1 pM, 100 nM to 10 pM, 100 nM to 100 pM, 100 nM to 1 mM, 1 pM to 10 pM, 1 pM to 100 pM, 1 pM to 1 mM, 10 pM to 100 pM, 10 pM to 1 mM, or 100 pM to 1 mM. In some embodiments, the sample comprises a target nucleic acid at a concentration of 20 nM to 200 pM, 50 nM to 100 pM, 200 nM to 50 pM, 500 nM to 20 pM, or 2 pM to 10 pM. In some embodiments, the target nucleic acid is not present in the sample.

[0366] In some embodiments, samples comprise fewer than 10 copies, fewer than 100 copies, fewer than 1000 copies, fewer than 10,000 copies, fewer than 100,000 copies, or fewer than 1,000,000 copies of a target nucleic acid. In some embodiments, the sample comprises 10 copies to 100 copies, 100 copies to 1000 copies, 1000 copies to 10,000 copies, 10,000 copies to 100,000 copies, 100,000 copies to 1,000,000 copies, 10 copies to 1000 copies, 10 copies to 10,000 copies, 10 copies to 100,000 copies, 10 copies to 1,000,000 copies, 100 copies to 10,000 copies, 100 copies to 100,000 copies, 100 copies to 1,000,000 copies, 1,000 copies to 100,000 copies, or 1,000 copies to 1,000,000 copies of a target nucleic acid. In some embodiments, the sample comprises 10 copies to 500,000 copies, 200 copies to 200,000 copies, 500 copies to 100,000 copies, 1000 copies to 50,000 copies, 2000 copies to 20,000 copies, 3000 copies to 10,000 copies, or 4000 copies to 8000 copies. In some embodiments, the target nucleic acid is not present in the sample.

[0367] In some embodiments, the sample is a biological sample, an environmental sample, or a combination thereof. Non-limiting examples of biological samples are blood, serum, plasma, saliva, urine, mucosal sample, peritoneal sample, cerebrospinal fluid, gastric secretions, nasal secretions, sputum, pharyngeal exudates, urethral or vaginal secretions, an exudate, an effusion, and a tissue sample (e.g., a biopsy sample). A tissue sample from a subject may be dissociated or liquified prior to application to detection system of the present disclosure. Non-limiting examples of environmental samples are soil, air, or water. In some embodiments, an environmental sample is taken as a swab from a surface of interest or taken directly from the surface of interest.

[0368] In some embodiments, the sample is a raw (unprocessed, unedited, unmodified) sample. Raw samples may be applied to a system for detecting or editing a target nucleic acid, such as those described herein. In some embodiments, the sample is diluted with a buffer or a fluid or concentrated prior to its application to the system or be applied neat to the detection system. Sometimes, the sample contains no more 20 pl of buffer or fluid. The sample, in some embodiments, is contained in no more than 1, 5, 10, 15, 20, 25, 30, 35 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 200, 300, 400, 500 pl, or any of value 1 pl to 500 pl, preferably 10 pLto 200 pL, ormore preferably 50 pLto 100 pL ofbuffer or fluid. Sometimes, the sample is contained in more than 500 pl. In some embodiments, the systems, devices, kits, and methods disclosed herein are compatible with the buffers or fluid disclosed herein.

[0369] In some embodiments, the sample is taken from a single-cell eukaryotic organism; a plant or a plant cell; an algal cell; a fungal cell; an animal cell, tissue, or organ; a cell, tissue, or organ from an invertebrate animal; a cell, tissue, fluid, or organ from a vertebrate animal such as fish, amphibian, reptile, bird, and mammal; a cell, tissue, fluid, or organ from a mammal such as a human, a non-human primate, an ungulate, a feline, a bovine, an ovine, and a caprine. In some embodiments, the sample is taken from nematodes, protozoans, helminths, or malarial parasites. In some embodiments, the sample comprises nucleic acids from a cell lysate from a eukaryotic cell, a mammalian cell, a human cell, a prokaryotic cell, or a plant cell. In some embodiments, the sample comprises nucleic acids expressed from a cell.

[0370] In some embodiments, samples are used for diagnosing a disease. The disease may comprise, at least in part, a cancer, an inherited disorder, an ophthalmological disorder, a neurological disorder, a neurodegenerative disease, a blood disorder, a metabolic disorder, a genetic disorder, an infection, or a combination thereof. In some embodiments the disease is cancer. The sample used for cancer testing may comprise at least one target nucleic acid that may hybridize to a guide nucleic acid of the reagents described herein. The target nucleic acid, in some cases, comprises a portion of a gene comprising a mutation associated with a disease, such as cancer, a gene whose overexpression is associated with cancer, a tumor suppressor gene, an oncogene, a checkpoint inhibitor gene, a gene associated with cellular growth, a gene associated with cellular metabolism, or a gene associated with cell cycle. Sometimes, the target nucleic acid encodes a cancer biomarker. In some embodiments, the assay may be used to detect “hotspots” in target nucleic acids that may be predictive of a cancer. In some embodiments, the target nucleic acid comprises a portion of a nucleic acid that is associated with a cancer. In some embodiments, the cancer is a solid cancer (i.e., a tumor). In some embodiments, the cancer is a blood cell cancer, including a leukemia or lymphoma. In some embodiments, the cancer is colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, bladder cancer, cancer of the kidney or ureter, lung cancer, cancer of the small intestine, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, brain cancer (e.g., glioblastoma), cancer of the head or neck, melanoma, uterine cancer, ovarian cancer, breast cancer, testicular cancer, cervical cancer, stomach cancer, Hodgkin's Disease, non-Hodgkin's lymphoma, thyroid cancer. The cancer may be a leukemia, such as, by way of non-limiting example, acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), and chronic lymphocytic leukemia (CLL).

[0371] The target nucleic acid, in some embodiments, comprises a portion of a gene comprising a mutation associated with cancer, a gene whose overexpression is associated with cancer, a tumor suppressor gene, an oncogene, a checkpoint inhibitor gene, a gene associated with cellular growth, a gene associated with cellular metabolism, or a gene associated with cell cycle. Sometimes, the target nucleic acid encodes a cancer biomarker, such as a prostate cancer biomarker or non-small cell lung cancer. In some cases, the assay may be used to detect “hotspots” in target nucleic acids that may be predictive of lung cancer. In some cases, the target nucleic acid comprises a portion of a nucleic acid that is associated with a hemorrhagic fever.

[0372] In some cases, the target nucleic acid is a portion of a nucleic acid from a genomic locus, any DNA amplicon of, a reverse transcribed mRNA, or a cDNA from a locus of at least one of: a gene set forth in TABLE 8. Any region of the aforementioned gene loci may be probed for a mutation or deletion using the compositions and methods disclosed herein. For example, in the EGFR gene locus, the compositions and methods for detection disclosed herein may be used to detect a single nucleotide polymorphism or a deletion.

[0373] In some embodiments, samples are used to diagnose a genetic disorder, also referred to as genetic disorder testing. The sample used for genetic disorder testing may comprise at least one target nucleic acid that may hybridize to a guide nucleic acid of the reagents described herein. The target nucleic acid, in some embodiments, is from a gene with a mutation associated with a genetic disorder, from a gene whose overexpression is associated with a genetic disorder, from a gene associated with abnormal cellular growth resulting in a genetic disorder, or from a gene associated with abnormal cellular metabolism resulting in a genetic disorder. In some embodiments, the target nucleic acid is a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed mRNA, a DNA amplicon of or a cDNA from a locus of at least one of a gene set forth in TABLE 8.

[0374] A sample used for phenotyping testing may comprise at least one target nucleic acid that may hybridize to a guide nucleic acid of the reagents described herein. The target nucleic acid, in some embodiments, is a nucleic acid encoding a sequence associated with a phenotypic trait. A sample used for genotyping testing may comprise at least one target nucleic acid that may hybridize to a guide nucleic acid of the reagents described herein. A target nucleic acid, in some embodiments, is a nucleic acid encoding a sequence associated with a genotype of interest. A sample used for ancestral testing may comprise at least one target nucleic acid that may hybridize to a guide nucleic acid of the reagents described herein. A target nucleic acid, in some embodiments, is a nucleic acid encoding a sequence associated with a geographic region of origin or ethnic group. A sample may be used for identifying a disease status. For example, a sample is any sample described herein, and is obtained from a subject for use in identifying a disease status of a subject. In some embodiments, the disease is cancer. In some embodiments, the disease is a genetic disorder. In some embodiments, a method comprises obtaining a serum sample from a subject; and identifying a disease status of the subject.

[0375]

VII. Compositions

[0376] Disclosed herein are compositions comprising one or more effector proteins described herein or nucleic acids encoding the one or more effector proteins, one or more guide nucleic acids described herein or nucleic acids encoding the one or more guide nucleic acids described herein, or combinations thereof. In some embodiments, one or more of a repeat sequence, a handle sequence, and intermediary sequence of the one or more guide nucleic acids are capable of interacting with the one or more of the effector proteins. In some embodiments, spacer sequences of the one or more guide nucleic acids hybridizes with a target sequence of a target nucleic acid. In some embodiments, the compositions comprise one or more donor nucleic acids described herein. In some embodiments, the compositions are capable of editing a target nucleic acid in a cell or a subject. In some embodiments, the compositions are capable of editing a target nucleic acid or the expression thereof in a cell, in a tissue, in an organ, in vitro, in vivo, or ex vivo. In some embodiments, the compositions are capable of editing a target nucleic acid in a sample comprising the target nucleic.

[0377] In some embodiments, compositions described herein comprise plasmids described herein, viral vectors described herein, non-viral vectors described herein, or combinations thereof. In some embodiments, compositions described herein comprise the viral vectors. In some embodiments, compositions described herein comprise an AAV. In some embodiments, compositions described herein comprise liposomes (e.g. , cationic lipids or neutral lipids), dendrimers, lipid nanoparticle (LNP), or cellpenetrating peptides. In some embodiments, compositions described herein comprise an LNP.

Certain Compositions

[0378] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some instances, the guide nucleic acid comprises any one of nucleotide sequences recited in TABLE 10. In some instances the guide nucleic acid comprises any two nucleotide sequences recited in TABLE 10, wherein the two nucleotide sequences are connected directly or via a linker. In some instances, the linker comprises a nucleotide sequence of 5’-GAAA-3’. In some instances, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of nucleotide sequences recited in TABLE 10. In some instances, the nucleotide sequence of the guide nucleic acid is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of nucleotide sequences recited in TABLE 10.

[0379] In some instances guide nucleic acids comprise at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of any one of nucleotide sequences recited in TABLE 10. In some instances, guide nucleic acids comprise at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, or at least 220 contiguous nucleotides of any one of nucleotide sequences recited in TABLE 10.

[0380] In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 1. In some embodiments, a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NOS: 19-22, 47-50, 75, 176-178, and 187-188. In some embodiments, a PAM is SEQ ID NO: 10 In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 1 that recognizes the PAM sequence of SEQ ID NO: 10, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 47-50, 75, 176-178, 187, and 188.

[0381] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 2. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NOS: 23-28, 51-56, 76-79, and 179-183. In some embodiments, a PAM is SEQ ID NO: 11 In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 2 that recognizes the PAM sequence of SEQ ID NO: 11, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 51-56, 76-79, and 179-183.

[0382] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 3. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NOS: 29-30, 57-58, and 80. In some embodiments, a PAM is SEQ ID NO: 12. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 3 that recognizes the PAM sequence of SEQ ID NO: 12, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 57-58, and 80.

[0383] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 4. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NOS: 31-32, 59-60, and 81. In some embodiments, a PAM is SEQ ID NO: 13. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 4 that recognizes the PAM sequence of SEQ ID NO: 13, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 59-60, and 81.

[0384] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 5. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NOS: 33-35, 61-63 and 82. In some embodiments, a PAM is SEQ ID NO: 14. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 5 that recognizes the PAM sequence of SEQ ID NO: 14, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 61-63, and 82.

[0385] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 6. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NOS: 36-40, 64-68, 83, 184-186, and 189-190. In some embodiments, a PAM is SEQ ID NO: 15. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 6 that recognizes the PAM sequence of SEQ ID NO: 15, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 64-68, 83, 184-186, and 189-190.

[0386] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 7. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 41-42, 69-70, and 84. In some embodiments, a PAM is SEQ ID NO: 16 In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 7 that recognizes the PAM sequence of SEQ ID NO: 16, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 69-70, and 84.

[0387] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 8. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 43-44, 71-72, and 85. In some embodiments, a PAM is SEQ ID NO: 17. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 8 that recognizes the PAM sequence of SEQ ID NO: 17, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 71-72, and 85.

[0388] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 9. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 45-46, 73-74, and 86. In some embodiments, a PAM is SEQ ID NO: 18 In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 9 that recognizes the PAM sequence of SEQ ID NO: 18, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 73-74, and 86.

[0389] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 87. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 109, 118 and 127. In some embodiments, a PAM is SEQ ID NO: 18 In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 87 that recognizes the PAM sequence of SEQ ID NO: 98, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 109, 118, and 127.

[0390] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 88. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 110, 119, and 128-129. In some embodiments, a PAM is SEQ ID NO: 98 or 99. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 88 that recognizes the PAM sequence of SEQ ID NO: 98 or 99, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 110, 119, and 128-129.

[0391] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 89. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 111, 120, and 130-131. In some embodiments, a PAM is SEQ ID NO: 98 or 99. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 89 that recognizes the PAM sequence of SEQ ID NO: 98 or 99, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 111, 120, and 130-131.

[0392] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 90. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 112, 121, and 132. In some embodiments, a PAM is SEQ ID NO: 98 or 100. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 90 that recognizes the PAM sequence of SEQ ID NO: 98 or 100, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 112, 121, and 132.

[0393] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 91. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 113, 122 and 133. In some embodiments, a PAM is SEQ ID NO: 98 or 101. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 91 that recognizes the PAM sequence of SEQ ID NO: 98 or 101, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 113, 122, and 133.

[0394] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 92. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 114, 123, and 134-135. In some embodiments, a PAM is of SEQ ID NO: 98 or 102. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 92 that recognizes the PAM sequence of SEQ ID NO: 98 or 102, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 114, 123, and 134-135.

[0395] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 93. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to an equal length portion of any one of SEQ ID NO: 115, 124, and 136-137. In some embodiments, a PAM is of SEQ ID NO: 98 or 102. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 93 that recognizes the PAM sequence of SEQ ID NO: 98 or 102, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to an equal length portion of any one of SEQ ID NO: 115, 124, and 136-137.

[0396] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 94. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to an equal length portion of any one of SEQ ID NO: 114, 123, and 138. In some embodiments, a PAM is of SEQ ID NO: 103 or 104 In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 94 that recognizes the PAM sequence of SEQ ID NO: 103 or 104, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to an equal length portion of any one of SEQ ID NO: 114, 123, and 138.

[0397] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 95. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to an equal length portion of any one of SEQ ID NO: 116, 125, and 139. In some embodiments, a PAM is of SEQ ID NO: 105 or 106. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 95 that recognizes the PAM sequence of SEQ ID NO: 105 or 106, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to an equal length portion of any one of SEQ ID NO: 116, 125, and 139.

[0398] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 96. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 114, 123, and 140. In some embodiments, a PAM is SEQ ID NO: 98. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 96 that recognizes the PAM sequence of SEQ ID NO: 98, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 114, 123, and 140.

[0399] In some embodiments, a composition comprises an effector protein and a guide nucleic acid. In some embodiments, the guide nucleic is not transactivating or transactivated. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 97. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 117, 126, and 141. In some embodiments, a PAM is SEQ ID NO: 107 or 108. In some embodiments, the effector protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 97 that recognizes the PAM sequence of SEQ ID NO: 107 or 108, and the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of SEQ ID NO: 117, 126, and 141.

Pharmaceutical Compositions and Modes of Administration

[0400] Disclosed herein, in some aspects, are pharmaceutical compositions for modifying a target nucleic acid in a cell or a subject, comprising any one of the effector proteins, engineered effector proteins, or fusion effector proteins described herein. Also disclosed herein, in some aspects, are pharmaceutical compositions comprising a nucleic acid encoding any one of the effector proteins, engineered effector proteins, or fusion effector proteins described herein. In some embodiments, pharmaceutical compositions comprise a guide nucleic acid. In some embodiments, pharmaceutical compositions comprise a plurality of guide nucleic acids. Pharmaceutical compositions may be used to modify a target nucleic acid or the expression thereof in a cell in vitro, in vivo or ex vivo.

[0401] In some embodiments, the pharmaceutically acceptable excipient, carrier or diluent allows the active ingredient to retain biological activity. In some embodiments, the pharmaceutically acceptable excipient, carrier or diluent is non-reactive with the subject's immune system. In some embodiments, the pharmaceutically acceptable excipient, carrier or diluent provides for long-term stabilization of the composition. In some embodiments, the pharmaceutically acceptable excipient, carrier or diluent is provided as a bulking agent in solid formulations that contain potent active ingredients in small amounts. In some embodiments, the pharmaceutically acceptable excipient, carrier or diluent confers a therapeutic enhancement on the active ingredient in the final dosage form. In some embodiments, the pharmaceutically acceptable excipient, carrier or diluent facilitates absorption, reduces viscosity, or enhances solubility. In some embodiments, the pharmaceutically acceptable excipient, carrier or diluent is selected based upon the route of administration, dosage form, active ingredient, other factors, or any combination thereof. In some embodiments, the pharmaceutically acceptable excipient, carrier or diluent can be formulated by well-known conventional methods (see, e.g, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005).

[0402] In some embodiments, pharmaceutical compositions comprise one or more nucleic acids encoding an effector protein, fusion effector protein, fusion partner, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent. The effector protein, fusion effector protein, fusion partner protein, or combination thereof may be any one of those described herein. The one or more nucleic acids may comprise a plasmid. The one or more nucleic acids may comprise a nucleic acid expression vector. The one or more nucleic acids may comprise a viral vector. A viral vector delivered by such viruses or viral particles may be referred to by the type of virus to deliver the viral vector (e.g., an AAV viral vector is a viral vector that is to be delivered by an adeno- associated virus). A viral vector referred to by the type of virus to be delivered by the viral vector can contain viral elements (e.g., nucleotide sequences) necessary for packaging of the viral vector into the virus or viral particle, replicating the virus, or other desired viral activities. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the vector is an adeno-associated viral (AAV) vector. In some instances, compositions, including pharmaceutical compositions, comprise a viral vector encoding a fusion effector protein and a guide nucleic acid, wherein at least a portion of the guide nucleic acid binds to the effector protein of the fusion effector protein.

[0403] In some embodiments, pharmaceutical compositions comprise a virus comprising a viral vector encoding a fusion effector protein, an effector protein, a fusion partner, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent. The virus may be a lentivirus. The virus may be an adenovirus. The virus may be a non-replicating virus. The virus may be an adeno- associated virus (AAV). The viral vector may be a retroviral vector. Retroviral vectors may include gamma-retroviral vectors such as vectors derived from the Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV) or the Murine Stem cell Virus (MSCV) genome. Retroviral vectors may include lentiviral vectors such as those derived from the human immunodeficiency virus (HIV) genome. In some embodiments, the viral vector is a chimeric viral vector, comprising viral portions from two or more viruses. In some embodiments, the viral vector is a recombinant viral vector. [0404] In some embodiments, the viral vector is an AAV. The AAV may be any AAV known in the art. In some embodiments, the viral vector corresponds to a virus of a specific serotype. In some examples, the serotype is selected from an AAV1 serotype, an AAV2 serotype, AAV3 serotype, an AAV4 serotype, AAV5 serotype, an AAV6 serotype, AAV7 serotype, an AAV8 serotype, an AAV9 serotype, an AAV 10 serotype, an AAV11 serotype, and an AAV 12 serotype. In some embodiments the AAV vector is a recombinant vector, a hybrid AAV vector, a chimeric AAV vector, a self- complementary AAV (scAAV) vector, a single-stranded AAV or any combination thereof. scAAV genomes are generally known in the art and contain both DNA strands which can anneal together to form double-stranded DNA.

[0405] In some embodiments, methods of producing delivery vectors herein comprise packaging a nucleic acid encoding an effector protein and a guide nucleic acid, or a combination thereof, into an AAV vector. In some instances, methods of producing the delivery vector comprises, (a) contacting a cell with at least one nucleic acid encoding: (i) a guide nucleic acid; (ii) a Replication (Rep) gene; and (iii) a Capsid (Cap) gene that encodes an AAV capsid protein; (b) expressing the AAV capsid protein in the cell; (c) assembling an AAV particle; and (d) packaging a Cas effector encoding nucleic acid into the AAV particle, thereby generating an AAV delivery vector. In some embodiments, promoters, staffer sequences, and any combination thereof may be packaged in the AAV vector. In some examples, the AAV vector can package 1, 2, 3, 4, or 5 guide nucleic acids or copies thereof. In some embodiments, the AAV vector comprises inverted terminal repeats, e.g. , a 5 ’ inverted terminal repeat and a 3 ’ inverted terminal repeat. In some instances, the AAV vector comprises a mutated inverted terminal repeat that lacks a terminal resolution site.

[0406] In some embodiments, a hybrid AAV vector is produced by transcapsidation, e.g., packaging an inverted terminal repeat (ITR) from a first serotype into a capsid of a second serotype, wherein the first and second serotypes may be not the same. In some examples, the Rep gene and ITR from a first AAV serotype (e.g., AAV2) may be used in a capsid from a second AAV serotype (e.g., AAV9), wherein the first and second AAV serotypes may be not the same. As a non-limiting example, a hybrid AAV serotype comprising the AAV2 ITRs and AAV9 capsid protein may be indicated AAV2/9. In some examples, the hybrid AAV delivery vector comprises an AAV2/1, AAV2/2, AAV 2/4, AAV2/5, AAV2/8, or AAV2/9 vector.

[0407] In some embodiments, the AAV vector may be a chimeric AAV vector. In some embodiments, the chimeric AAV vector comprises an exogenous amino acid or an amino acid substitution, or capsid proteins from two or more serotypes. In some examples, a chimeric AAV vector may be genetically engineered to increase transduction efficiency, selectivity, or a combination thereof.

[0408] In some examples, the delivery vector may be a eukaryotic vector, a prokaryotic vector (e.g. , a bacterial vector) a viral vector, or any combination thereof. In some embodiments, the delivery vehicle may be a non-viral vector. In some embodiments, the delivery vehicle may be a plasmid. In some embodiments, the plasmid comprises DNA. In some embodiments, the plasmid comprises RNA. In some examples, the plasmid comprises circular double-stranded DNA. In some examples, the plasmid may be linear. In some examples, the plasmid comprises one or more genes of interest and one or more regulatory elements. In some examples, the plasmid comprises a bacterial backbone containing an origin of replication and an antibiotic resistance gene or other selectable marker for plasmid amplification in bacteria. In some examples, the plasmid may be a minicircle plasmid. In some examples, the plasmid contains one or more genes that provide a selective marker to induce a target cell to retain the plasmid. In some examples, the plasmid may be formulated for delivery through injection by a needle carrying syringe. In some examples, the plasmid may be formulated for delivery via electroporation. In some examples, the plasmids may be engineered through synthetic or other suitable means known in the art. For example, in some embodiments, the genetic elements may be assembled by restriction digest of the desired genetic sequence from a donor plasmid or organism to produce ends of the DNA which may then be readily ligated to another genetic sequence.

[0409] In some embodiments, the vector is a non-viral vector, and a physical method or a chemical method is employed for delivery into the somatic cell. Exemplary physical methods include electroporation, gene gun, sonoporation, magnetofection, or hydrodynamic delivery. Exemplary chemical methods include delivery of the recombinant polynucleotide via liposomes such as, cationic lipids or neutral lipids; dendrimers; nanoparticles; or cell-penetrating peptides.

[0410] In some embodiments, a fusion effector protein as described herein is inserted into a vector. In some embodiments, the vector optionally comprises one or more promoters, enhancers, ribosome binding sites, RNA splice sites, polyadenylation sites, a replication origin, and/or transcriptional terminator sequences.

[0411] In general, plasmids and vectors described herein comprise at least one promoter. In some embodiments, the promoters are constitutive promoters. In other embodiments, the promoters are inducible promoters. In additional embodiments, the promoters are prokaryotic promoters (e.g., drive expression of a gene in a prokaryotic cell). In some embodiments, the promoters are eukaryotic promoters, (e.g., drive expression of a gene in a eukaryotic cell). Exemplary promoters include, but are not limited to, ApoE, TBG, CMV, 7SK, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GALI-10, TEF1, GDS, ADH1, CaMV35S, Ubi, Hl, U6, CaMV35S, SV40, and HSV TK promoter. In some embodiments, the promoter is CMV. In some embodiments, the promoter is EFla. In some embodiments, the promoter is ubiquitin. In some instances, vectors are bicistronic or polycistronic vector (e.g. , having or involving two or more loci responsible for generating a protein) having an internal ribosome entry site (IRES) is for translation initiation in a cap-independent manner.

[0412] In some instances, vectors comprise an enhancer. Enhancers are nucleotide sequences that have the effect of enhancing promoter activity. In some embodiments, enhancers augment transcription regardless of the orientation of their sequence. In some embodiments, enhancers activate transcription from a distance of several kilo basepairs. Furthermore, enhancers are located optionally upstream or downstream of a gene region to be transcribed, and/or located within the gene, to activate the transcription. Exemplary enhancers include, but are not limited to, WPRE; CMV enhancers; the R-U5' segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; the intron sequence between exons 2 and 3 of rabbit [3-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527- 31, 1981); and the genome region of human growth hormone (J Immunol., Vol. 155(3), p. 1286-95, 1995).

[0413] Pharmaceutical compositions described herein may comprise a salt. In some instances, the salt is a sodium salt. In some instances, the salt is a potassium salt. In some instances, the salt is a magnesium salt. In some embodiments, the salt is NaCl. In some embodiments, the salt is KNO3. In some embodiments, the salt is Mg²⁺ SO4² .

[0414] Non-limiting examples of pharmaceutically acceptable carriers and diluents suitable for the pharmaceutical compositions disclosed herein include buffers (e.g., neutral buffered saline, phosphate buffered saline); carbohydrates (e.g., glucose, mannose, sucrose, dextran, mannitol); polypeptides or amino acids (e.g, glycine); antioxidants; chelating agents (e.g., EDTA, glutathione); adjuvants (e.g, aluminum hydroxide); surfactants (Polysorbate 80, Polysorbate 20, or Pluronic F68); glycerol; sorbitol; mannitol; polyethyleneglycol; and preservatives.

[0415] In some embodiments, the vector is formulated for delivery through injection by a needle carrying syringe. In some embodiments, the composition is formulated for delivery by electroporation. In some embodiments, the composition is formulated for delivery by chemical method. In some embodiments, the pharmaceutical compositions comprise a virus vector or a non-viral vector.

[0416] In some instances, pharmaceutical compositions are in the form of a solution (e.g, a liquid). The solution may be formulated for injection, e.g, intravenous or subcutaneous injection. In some instances, the pH of the solution is about 7, about 7. 1, about 7.2, about 7.3, about 7.4, about 7.5, about

7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about

8.6, about 8.7, about 8.8, about 8.9, or about 9. In some embodiments, the pH is 7 to 7.5, 7.5 to 8, 8 to 8.5, 8.5 to 9, or 7 to 8.5. In some cases, the pH of the solution is less than 7. In some cases, the pH is greater than 7.

VIII. Systems

[0417] Disclosed herein, in some aspects, are systems for detecting and/or editing target nucleic acids, comprising any one of the effector proteins described herein or a nucleic acid encoding the same. In some instances, systems comprise a guide nucleic acid or a nucleic acid encoding the same (e.g. , a DNA molecule). In instances embodiments, systems comprise components comprising one or more of: compositions described herein; a solution or buffer; a reagent; a support medium; other components or appurtenances as described herein; or combinations thereof. In some embodiments, systems for detecting and/or modifying a target nucleic acid described herein comprise at least two components each individually comprising one of the following: effector proteins described herein or a nucleic acid encoding the same, and a guide nucleic acid or a nucleic acid encoding the same (e.g. , a DNA molecule). [0418] Systems may be used to detect a target nucleic acid. In some embodiments, systems comprise an effector protein described herein, a reagent, support medium, or a combination thereof. In some instances, systems comprise a fusion protein described herein. In some instances, effector proteins comprise an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the amino acid sequences recited in TABLE 1. In some instances, the amino acid sequence of the effector protein is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the amino acid sequences recited in TABLE 1.

[0419] Systems may be used for detecting the presence of a target nucleic acid associated with or causative of a disease, such as cancer, a genetic disorder, or an infection. In some instances, systems are useful for phenotyping, genotyping, or determining ancestry. Unless specified otherwise, systems include kits and may be referred to as kits. Unless specified otherwise, systems include devices and may also be referred to as devices. Systems described herein may be provided in the form of a companion diagnostic assay or device, a point-of-care assay or device, or an over-the-counter diagnostic assay/device.

[0420] In some embodiments, in vitro can be used to describe an event that takes places in a container for holding laboratory reagents such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed. In some cases, in vivo can be used to describe an event that takes place in a subject’s body. In some cases, ex vivo can be used to describe an event that takes place outside of a subject’s body. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample is an in vitro assay.

[0421] Reagents and effector proteins of various systems may be provided in a reagent chamber or on a support medium. Alternatively, the reagent and/or effector protein may be contacted with the reagent chamber or the support medium by the individual using the system. An exemplary reagent chamber is a test well or container. The opening of the reagent chamber may be large enough to accommodate the support medium. Optionally, the system comprises a buffer and a dropper. The buffer may be provided in a dropper bottle for ease of dispensing. The dropper may be disposable and transfer a fixed volume. The dropper may be used to place a sample into the reagent chamber or on the support medium.

System solutions

[0422] In general, systems comprise a solution in which the activity of an effector protein occurs. Often, the solution comprises or consists essentially of a buffer. The solution or buffer may comprise a buffering agent, a salt, a crowding agent, a detergent, a reducing agent, a competitor, or a combination thereof. Often the buffer is the primary component or the basis for the solution in which the activity occurs. Thus, concentrations for components of buffers described herein (e.g., buffering agents, salts, crowding agents, detergents, reducing agents, and competitors) are the same or essentially the same as the concentration of these components in the solution in which the activity occurs. In some instances, a buffer is required for cell lysis activity or viral lysis activity.

[0423] In some instances, systems comprise a buffer, wherein the buffer comprise at least one buffering agent. Exemplary buffering agents include HEPES, TRIS, MES, ADA, PIPES, ACES, MOPSO, BISTRIS propane, BES, MOPS, TES, DISO, Trizma, TRICINE, GLY-GLY, HEPPS, BICINE, TAPS, A MPD, A MPSO, CHES, CAPSO, AMP, CAPS, phosphate, citrate, acetate, imidazole, or any combination thereof.

[0424] In some instances, the concentration of the buffering agent in the buffer is 1 mM to 200 mM. A buffer compatible with an effector protein may comprise a buffering agent at a concentration of 10 mM to 30 mM. A buffer compatible with an effector protein may comprise a buffering agent at a concentration of about 20 mM. A buffering agent may provide a pH for the buffer or the solution in which the activity of the effector protein occurs. The pH may be 3 to 4, 3.5 to 4.5, 4 to 5, 4.5 to 5.5, 5 to 6, 5.5 to 6.5, 6 to 7, 6.5 to 7.5, 7 to 8, 7.5 to 8.5, 8 to 9, 8.5 to 9.5, 9 to 10, or 9.5 to 10.5.

[0425] In some instances, systems comprise a solution, wherein the solution comprises at least one salt. In some instances, the at least one salt is selected from potassium acetate, magnesium acetate, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, and any combination thereof. In some instances, the concentration of the at least one salt in the solution is 5 mM to 100 mM, 5 mM to 10 mM, 1 mM to 60 mM, or 1 mM to 10 mM. In some instances, the concentration of the at least one salt is about 105 mM. In some instances, the concentration of the at least one salt is about 55 mM. In some instances, the concentration of the at least one salt is about 7 mM. In some instances, the solution comprises potassium acetate and magnesium acetate. In some instances, the solution comprises sodium chloride and magnesium chloride. In some instances, the solution comprises potassium chloride and magnesium chloride. In some instances, the salt is a magnesium salt and the concentration of magnesium in the solution is at least 5 mM, 7 mM, at least 9 mM, at least 11 mM, at least 13 mM, or at least 15 mM. In some instances, the concentration of magnesium is less than 20mM, less than 18 mM, or less than 16 mM.

[0426] In some instances, systems comprise a solution, wherein the solution comprises at least one crowding agent. A crowding agent may reduce the volume of solvent available for other molecules in the solution, thereby increasing the effective concentrations of said molecules. Exemplary crowding agents include glycerol and bovine serum albumin. In some instances the crowding agent is glycerol. In some instances, the concentration of the crowding agent in the solution is 0.01% (v/v) to 10% (v/v). In some instances, the concentration of the crowding agent in the solution is 0.5% (v/v) to 10% (v/v). [0427] In some instances, systems comprise a solution, wherein the solution comprises at least one detergent. Exemplary detergents include Tween, Triton-X, and IGEPAL. A solution may comprise Tween, Triton-X, or any combination thereof. A solution may comprise Triton-X. A solution may comprise IGEPAL CA-630. In some instances, the concentration of the detergent in the solution is 2% (v/v) or less. In some instances, the concentration of the detergent in the solution is 1% (v/v) or less. In some instances, the concentration of the detergent in the solution is 0.00001% (v/v) to 0.01% (v/v). In some instances, the concentration of the detergent in the solution is about 0.01% (v/v).

[0428] In some instances, systems comprise a solution, wherein the solution comprises at least one reducing agent. Exemplary reducing agents comprise dithiothreitol (DTT), B-mercaptoethanol (BME), or tris(2 -carboxyethyl) phosphine (TCEP). In some instances, the reducing agent is DTT. In some instances, the concentration of the reducing agent in the solution is 0.01 mM to 100 mM. In some instances, the concentration of the reducing agent in the solution is 0.1 mM to 10 mM. In some instances, the concentration of the reducing agent in the solution is 0.5 mM to 2 mM. In some instances, the concentration of the reducing agent in the solution is 0.01 mM to 100 mM. In some instances, the concentration of the reducing agent in the solution is 0.1 mM to 10 mM. In some instances, the concentration of the reducing agent in the solution is about 1 mM.

[0429] In some instances, systems comprise a solution, wherein the solution comprises a competitor. In general, competitors compete with the target nucleic acid or the reporter nucleic acid for cleavage by the effector protein or a dimer thereof. Exemplary competitors include heparin, and imidazole, and salmon sperm DNA. In some instances, the concentration of the competitor in the solution is 1 pg/mL to 100 pg/mL. In some instances, the concentration of the competitor in the solution is 40 pg/mL to 60 pg/mL.

[0430] In some instances, systems comprise a solution, wherein the solution comprises a co-factor. In some instances, the co-factor allows an effector protein or a multimeric complex thereof to perform a function, including pre-crRNA processing and/or target nucleic acid cleavage. The suitability of a cofactor for an effector protein or a multimeric complex thereof may be assessed, such as by methods based on those described by Sundaresan et al. (Cell Rep. 2017 Dec 26; 21(13): 3728-3739). In some instances, an effector or a multimeric complex thereof forms a complex with a co-factor. In some instances, the co-factor is a divalent metal ion. In some instances, the divalent metal ion is selected from Mg2⁺, Mir , Zn2⁺, Ca2⁺, Cu2⁺. In some instances, the divalent metal ion is Mg2⁺. In some instances, the co-factor is Mg2⁺.

Reporters

[0431] In some instances, systems disclosed herein comprise a reporter. By way of non-limiting and illustrative example, a reporter may comprise a single stranded nucleic acid and a detection moiety (e.g. , a labeled single stranded RNA reporter), wherein the nucleic acid is capable of being cleaved by an effector protein (e.g., a CRISPR/Cas protein as disclosed herein) or a multimeric complex thereof, releasing the detection moiety, and generating a detectable signal or a detectable product. In some embodiments, cleavage of the reporter is effective to produce a detectable product comprising a detectable moiety or a detectable signal.

[0432] As used herein, “reporter” is used interchangeably with “reporter nucleic acid” or “reporter molecule”. In some embodiments, a detectable signal comprises a signal that can be detected using optical, fluorescent, chemiluminescent, electrochemical and other detection methods known in the art. The effector proteins disclosed herein, activated upon hybridization of a guide nucleic acid to a target nucleic acid, may cleave the reporter. Cleaving the “reporter” may be referred to herein as cleaving the “reporter nucleic acid,” the “reporter molecule,” or the “nucleic acid of the reporter.” Reporters may comprise RNA. Reporters may comprise DNA. Reporters may be double-stranded. Reporters may be single-stranded.

[0433] In some instances, reporters comprise a protein capable of generating a signal. In some embodiments, reporters are operably linked to the protein capable of generating a signal. A signal may be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezoelectric signal. In some instances, the reporter comprises a detection moiety. In some embodiments, the reporter is configured to release a detection moiety or generate a signal indicative of a presence or absence of the target nucleic acid. Suitable detectable labels and/or moieties that may provide a signal include, but are not limited to, an enzyme, a radioisotope, a member of a specific binding pair; a fluorophore; a fluorescent protein; a quantum dot; and the like.

[0434] In some instances, the reporter comprises a detection moiety and a quenching moiety. In some instances, the reporter comprises a cleavage site, wherein the detection moiety is located at a first site on the reporter and the quenching moiety is located at a second site on the reporter, wherein the first site and the second site are separated by the cleavage site. Sometimes the quenching moiety is a fluorescence quenching moiety. In some instances, the quenching moiety is 5 ’ to the cleavage site and the detection moiety is 3’ to the cleavage site. In some instances, the detection moiety is 5’ to the cleavage site and the quenching moiety is 3’ to the cleavage site. Sometimes the quenching moiety is at the 5 ’ terminus of the nucleic acid of a reporter. Sometimes the detection moiety is at the 3 ’ terminus of the nucleic acid of a reporter. In some instances, the detection moiety is at the 5 ’ terminus of the nucleic acid of a reporter. In some instances, the quenching moiety is at the 3 ’ terminus of the nucleic acid of a reporter.

[0435] Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, Phycobiliproteins and Phycobiliprotein conjugates including B -Phycoerythrin, R-Phycoerythrin and Allophycocyanin. Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N- acetylglucosaminidase, P-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase, and glucose oxidase (GO).

[0436] In some instances, the detection moiety comprises an invertase. The substrate of the invertase may be sucrose. A DNS reagent may be included in the system to produce a colorimetric change when the invertase converts sucrose to glucose. In some instances, the reporter nucleic acid and invertase are conjugated using a heterobifimctional linker via sulfo-SMCC chemistry.

[0437] Suitable fluorophores may provide a detectable fluorescence signal in the same range as 6- Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies). Non-limiting examples of fluorophores are fluorescein amidite, 6-Fluorescein, IRDye 700, TYE 665, Alex Fluor 594, or ATTO TM 633 (NHS Ester). The fluorophore may be an infrared fluorophore. The fluorophore may emit fluorescence in the range of 500 nm and 720 nm. In some instances, the fluorophore emits fluorescence at a wavelength of 700 nm or higher. In some instances, the fluorophore emits fluorescence at about 665 nm. In some instances, the fluorophore emits fluorescence in the range of 500 nm to 520 nm, 500 nm to 540 nm, 500 nm to 590 nm, 590 nm to 600 nm, 600 nm to 610 nm, 610 nm to 620 nm, 620 nm to 630 nm, 630 nm to 640 nm, 640 nm to 650 nm, 650 nm to 660 nm, 660 nm to 670 nm, 670 nm to 680 nm, 690 nm to 690 nm, 690 nm to 700 nm, 700 nm to 710 nm, 710 nm to 720 nm, or 720 nm to 730 nm. In some instances, the fluorophore emits fluorescence in the range 450 nm to 750 nm, 500 nm to 650 nm, or 550 to 650 nm. [0438] Systems may comprise a quenching moiety. A quenching moiety may be chosen based on its ability to quench the detection moiety. A quenching moiety may be a non-fluorescent fluorescence quencher. A quenching moiety may quench a detection moiety that emits fluorescence in the range of 500 nm and 720 nm. A quenching moiety may quench a detection moiety that emits fluorescence in the range of 500 nm and 720 nm. In some instances, the quenching moiety quenches a detection moiety that emits fluorescence at a wavelength of 700 nm or higher. In some instances the quenching moiety quenches a detection moiety that emits fluorescence at about 660 nm or about 670 nm. In some instances, the quenching moiety quenches a detection moiety that emits fluorescence in the range of 500 to 520, 500 to 540, 500 to 590, 590 to 600, 600 to 610, 610 to 620, 620 to 630, 630 to 640, 640 to 650, 650 to 660, 660 to 670, 670 to 680, 690 to 690, 690 to 700, 700 to 710, 710 to 720, or 720 to 730 nm. In some instances, the quenching moiety quenches a detection moiety that emits fluorescence in the range 450 nm to 750 nm, 500 nm to 650 nm, or 550 to 650 nm. A quenching moiety may quench fluorescein amidite, 6-Fluorescein, IRDye 700, TYE 665, Alex Fluor 594, or ATTO TM 633 (NHS Ester). A quenching moiety may be Iowa Black RQ, Iowa Black FQ or IRDye QC-1 Quencher. A quenching moiety may quench fluorescein amidite, 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies). A quenching moiety may be Iowa Black RQ (Integrated DNA Technologies), Iowa Black FQ (Integrated DNA Technologies) or IRDye QC-1 Quencher (Li Cor). Any of the quenching moieties described herein may be from any commercially available source, may be an alternative with a similar function, a generic, or a non-tradename of the quenching moieties listed.

[0439] The generation of the detectable signal or detectable product from the release of the detection moiety may indicate that cleavage by the effector protein has occurred and that the sample contains the target nucleic acid. In some instances, the detection moiety comprises a fluorescent dye. Sometimes the detection moiety comprises a fluorescence resonance energy transfer (FRET) pair. In some instances, the detection moiety comprises an infrared (IR) dye. In some instances, the detection moiety comprises an ultraviolet (UV) dye. Alternatively, or in combination, the detection moiety comprises a protein. Sometimes the detection moiety comprises an antigen. Sometimes the detection moiety comprises a biotin. Sometimes the detection moiety comprises at least one of avidin or streptavidin. In some instances, the detection moiety comprises a polysaccharide, a polymer, or a nanoparticle. In some instances, the detection moiety comprises a gold nanoparticle or a latex nanoparticle.

[0440] A detection moiety may be any moiety capable of generating a detectable product or detectable signal upon cleavage of the reporter by the effector protein. The detectable product may be a detectable unit generated from the detectable moiety and capable of emitting a detectable signal as described herein. In some embodiments, the detectable product further comprises a detectable label, a fluorophore, a reporter, or a combination thereof. In some embodiments, the detectable product comprises RNA, DNA, or both. In some embodiments, the detectable product is configured to generate a signal indicative of the presence or absence of the target nucleic acid in, for instance, a cell or a sample.

[0441] A detection moiety may be any moiety capable of generating a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. A nucleic acid of a reporter, sometimes, is protein-nucleic acid that is capable of generating a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal upon cleavage of the nucleic acid. Often a calorimetric signal is heat produced after cleavage of the nucleic acids of a reporter. Sometimes, a calorimetric signal is heat absorbed after cleavage of the nucleic acids of a reporter. A potentiometric signal, for example, is electrical potential produced after cleavage of the nucleic acids of a reporter. An amperometric signal may be movement of electrons produced after the cleavage of nucleic acid of a reporter. Often, the signal is an optical signal, such as a colorimetric signal or a fluorescence signal. An optical signal is, for example, a light output produced after the cleavage of the nucleic acids of a reporter. Sometimes, an optical signal is a change in light absorbance between before and after the cleavage of nucleic acids of a reporter. Often, a piezo-electric signal is a change in mass between before and after the cleavage of the nucleic acid of a reporter.

[0442] The detectable signal may be a colorimetric signal or a signal visible by eye. In some instances, the detectable signal may be fluorescent, electrical, chemical, electrochemical, or magnetic. In some instances, the first detection signal may be generated by interaction (e.g., binding) of the detection moiety to the capture molecule in the detection region, where the first detection signal indicates that the sample contained the target nucleic acid. Sometimes systems are capable of detecting more than one type of target nucleic acid, wherein the system comprises more than one type of guide nucleic acid and more than one type of reporter nucleic acid. In some instances, the detectable signal may be generated directly by the cleavage event. Alternatively, or in combination, the detectable signal may be generated indirectly by the signal event. Sometimes the detectable signal is not a fluorescent signal. In some instances, the detectable signal may be a colorimetric or color-based signal. In some instances, the detected target nucleic acid may be identified based on its spatial location on the detection region of the support medium. In some instances, the second detectable signal may be generated in a spatially distinct location than the first generated signal.

[0443] In some instances, the reporter nucleic acid is a single-stranded nucleic acid sequence comprising ribonucleotides. The nucleic acid of a reporter may be a single-stranded nucleic acid sequence comprising at least one ribonucleotide. In some instances, the nucleic acid of a reporter is a single-stranded nucleic acid comprising at least one ribonucleotide residue at an internal position that functions as a cleavage site. In some instances, the nucleic acid of a reporter comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 ribonucleotide residues at an internal position. In some instances, the nucleic acid of a reporter comprises from 2 to 10, from 3 to 9, from 4 to 8, or from 5 to 7 ribonucleotide residues at an internal position. Sometimes the ribonucleotide residues are continuous. Alternatively, the ribonucleotide residues are interspersed in between non-ribonucleotide residues. In some instances, the nucleic acid of a reporter has only ribonucleotide residues. In some instances, the nucleic acid of a reporter has only deoxyribonucleotide residues. In some instances, the nucleic acid comprises nucleotides resistant to cleavage by the effector protein described herein. In some instances, the nucleic acid of a reporter comprises synthetic nucleotides. In some instances, the nucleic acid of a reporter comprises at least one ribonucleotide residue and at least one non-ribonucleotide residue.

[0444] In some instances, the nucleic acid of a reporter comprises at least one uracil ribonucleotide. In some instances, the nucleic acid of a reporter comprises at least two uracil ribonucleotides. Sometimes the nucleic acid of a reporter has only uracil ribonucleotides. In some instances, the nucleic acid of a reporter comprises at least one adenine ribonucleotide. In some instances, the nucleic acid of a reporter comprises at least two adenine ribonucleotides. In some instances, the nucleic acid of a reporter has only adenine ribonucleotides. In some instances, the nucleic acid of a reporter comprises at least one cytosine ribonucleotide. In some instances, the nucleic acid of a reporter comprises at least two cytosine ribonucleotides. In some instances, the nucleic acid of a reporter comprises at least one guanine ribonucleotide. In some instances, the nucleic acid of a reporter comprises at least two guanine ribonucleotides. In some instances, a nucleic acid of a reporter comprises a single unmodified ribonucleotide. In some instances, a nucleic acid of a reporter comprises only unmodified deoxyribonucleotides .

[0445] In some instances, the nucleic acid of a reporter is 5 to 20, 5 to 15, 5 to 10, 7 to 20, 7 to 15, or 7 to 10 nucleotides in length. In some instances, the nucleic acid of a reporter is 3 to 20, 4 to 10, 5 to 10, or 5 to 8 nucleotides in length. In some instances, the nucleic acid of a reporter is 5 to 12 nucleotides in length. In some instances, the reporter nucleic acid is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 nucleotides in length. In some instances, the reporter nucleic acid is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0446] In some instances, systems comprise a plurality of reporters. The plurality of reporters may comprise a plurality of signals. In some instances, systems comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 30, at least 40, or at least 50 reporters. In some instances, there are 2 to 50, 3 to 40, 4 to 30, 5 to 20, or 6 to 10 different reporters.

[0447] In some instances, systems comprise an effector protein and a reporter nucleic acid configured to undergo trans cleavage by the effector protein. Trans cleavage of the reporter may generate a signal from the reporter or alter a signal from the reporter. In some instances, the signal is an optical signal, such as a fluorescence signal or absorbance band. Trans cleavage of the reporter may alter the wavelength, intensity, or polarization of the optical signal. For example, the reporter may comprise a fluorophore and a quencher, such that trans cleavage of the reporter separates the fluorophore and the quencher thereby increasing a fluorescence signal from the fluorophore. Herein, detection of reporter cleavage to determine the presence of a target nucleic acid sequence may be referred to as ‘DETECTR’. In some instances, described herein is a method of assaying for a target nucleic acid in a sample comprising contacting the target nucleic acid with an effector protein, a non-naturally occurring guide nucleic acid that hybridizes to a segment of the target nucleic acid, and a reporter nucleic acid, and assaying for a change in a signal, wherein the change in the signal is produced by cleavage of the reporter nucleic acid.

[0448] In the presence of a large amount of non-target nucleic acids, an activity of an effector protein (e.g., an effector protein as disclosed herein) may be inhibited. This is because the activated effector proteins collaterally cleave any nucleic acids. If total nucleic acids are present in large amounts, they may outcompete reporters for the effector proteins. In some instances, systems comprise an excess of reporter(s), such that when the system is operated and a solution of the system comprising the reporter is combined with a sample comprising a target nucleic acid, the concentration of the reporter in the combined solution-sample is greater than the concentration of the target nucleic acid. In some instances, the sample comprises amplified target nucleic acid. In some instances, the sample comprises an unamplified target nucleic acid. In some instances, the concentration of the reporter is greater than the concentration of target nucleic acids and non-target nucleic acids. The non-target nucleic acids may be from the original sample, either lysed or unlysed. The non-target nucleic acids may comprise byproducts of amplification. In some instances, systems comprise a reporter wherein the concentration of the reporter in a solution 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold excess of total nucleic acids. In some embodiments, systems comprise a reporter wherein the concentration of the reporter in a solution is 1.5 fold to 100 fold, 2 fold to 10 fold, 10 fold to 20 fold, 20 fold to 30 fold, 30 fold to 40 fold, 40 fold to 50 fold, 50 fold to 60 fold, 60 fold to 70 fold, 70 fold to 80 fold, 80 fold to 90 fold, 90 fold to 100 fold, 1.5 fold to 10 fold, 1.5 fold to 20 fold, 10 fold to 40 fold, 20 fold to 60 fold, or 10 fold to 80 fold excess of total nucleic acids.

Detection Reagents/Components

[0449] In some embodiments, systems described herein comprise a reagent or component for detecting a nucleic acid. Non-limiting examples of reagents for detecting a nucleic acid include a reporter nucleic acid, a detection moiety, and additional polypeptides. In some embodiments, systems comprise reagents for nucleic acid detection of a target nucleic acid in a sample. Nucleic acid amplification of the target nucleic acid may improve at least one of sensitivity, specificity, or accuracy of the assay in detecting the target nucleic acid. Accordingly, in some embodiments, nucleic acid detection involves PCR or isothermal nucleic acid amplification, providing improved sensitive, specific, or rapid detection.

[0450] The reagents or components for nucleic acid detection may comprise recombinases, primers, polypeptides, buffers, and signal reagents suitable for a detection reaction. In some embodiments, a reagent for detecting is operably linked to the effector protein capable of being activated.

[0451] In some embodiments, systems described herein comprise a PCR tube, a PCR well or a PCR plate. In some embodiments, the wells of the PCR plate may be pre-aliquoted with the reagent for detecting a nucleic acid, as well as a guide nucleic acid, an effector protein, a multimeric complex, an amplification reagent, or any combination thereof. In some embodiments, the pre-aliquoted guide nucleic acid targeting a target sequence, and an effector protein capable of being activated when complexed with the guide nucleic acid and the target sequence. A user may thus add a sample of interest to a well of the pre-aliquoted PCR plate.

[0452] In some embodiments, nucleic acid detection is performed in a nucleic acid detection region on a support medium, or sample interface. Alternatively, or in combination, the nucleic acid detection is performed in a reagent chamber, and the resulting sample is applied to the support medium, sample interface, or surface within a reagent chamber. [0453] In some embodiments, the reporter nucleic acid is capable of being cleaved by the activated nuclease, thereby generating a detectable signal. A user may thus add a sample of interest to a well of the pre-aliquoted PCR plate and measure for the detectable signal with a fluorescent light reader or a visible light reader.

[0454] In some embodiments, detection reaction of nucleic acid as described herein is performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes, or any value 1 to 60 minutes. In some embodiments, the detection reaction is performed for 1 to 60, 5 to 55, 10 to 50, 15 to 45, 20 to 40, or 25 to 35 minutes. In some embodiments, the detection reaction is performed at a temperature of around 20-45°C. In some embodiments, the detection reaction is performed at a temperature no greater than 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, or any value 20 °C to 45 °C. In some embodiments, the detection reaction is performed at a temperature of at least 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, or 45°C, or any value 20 °C to 45 °C. In some embodiments, the detection reaction is performed at a temperature of 20°C to 45°C, 25°C to 40°C, 30°C to 40°C, or 35°C to 40°C.

[0455] In some embodiments, the reagents or components for detecting a nucleic acid are, for example, consistent for use within various fluidic devices disclosed herein for detection of a target nucleic acid within the sample, wherein the fluidic device may comprise multiple pumps, valves, reservoirs, and chambers for sample preparation, amplification of a target nucleic acid within the sample, mixing with an effector protein, and detection of a detectable signal arising from cleavage of detector nucleic acids by the effector protein within the fluidic system itself. These reagents are compatible with the samples, devices, fluidic devices, methods of detection, and support mediums as described herein for detection of an ailment, such as a disease, cancer, or genetic disorder, or genetic information, such as for phenotyping, genotyping, or determining ancestry. The reagents described herein for detecting a disease, cancer, or genetic disorder comprise a guide nucleic acid targeting the target nucleic acid segment indicative of a disease, cancer, or genetic disorder.

Amplification Reagents/Components

[0456] In some instances, systems described herein comprise a reagent or component for amplifying a nucleic acid. Non-limiting examples of reagents for amplifying a nucleic acid include polymerases, primers, and nucleotides. In some instances, systems comprise reagents for nucleic acid amplification of a target nucleic acid in a sample. Nucleic acid amplification of the target nucleic acid may improve at least one of sensitivity, specificity, or accuracy of the assay in detecting the target nucleic acid. In some instances, nucleic acid amplification is isothermal nucleic acid amplification, providing for the use of the system or system in remote regions or low resource settings without specialized equipment for amplification. In some instances, amplification of the target nucleic acid increases the concentration of the target nucleic acid in the sample relative to the concentration of nucleic acids that do not correspond to the target nucleic acid. [0457] The reagents for nucleic acid amplification may comprise a recombinase, an oligonucleotide primer, a single-stranded DNA binding (SSB) protein, a polymerase, or a combination thereof that is suitable for an amplification reaction. In some embodiments, amplification reagents comprise a primer, an activator, a dNTP, an rNTP, or combinations thereof. Non-limiting examples of amplification reactions are transcription mediated amplification (TMA), helicase dependent amplification (HD A), or circular helicase dependent amplification (cHDA), strand displacement amplification (SDA), recombinase polymerase amplification (RPA), loop mediated amplification (LAMP), exponential amplification reaction (EXPAR), rolling circle amplification (RCA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence based amplification (NASBA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), and improved multiple displacement amplification (IMDA).

[0458] In some instances, systems comprise a PCR tube, a PCR well or a PCR plate. The wells of the PCR plate may be pre-aliquoted with the reagent for amplifying a nucleic acid, as well as a guide nucleic acid, an effector protein, a multimeric complex, or any combination thereof. The wells of the PCR plate may be pre-aliquoted with a guide nucleic acid targeting a target sequence, an effector protein capable of being activated when complexed with the guide nucleic acid and the target sequence. The wells of the PCR plate may be pre-aliquoted with a guide nucleic acid targeting a target sequence, an effector protein capable of being activated when complexed with the guide nucleic acid and the target sequence, and at least one population of a single stranded reporter nucleic acid comprising a detection moiety. A user may thus add the biological sample of interest to a well of the pre-aliquoted PCR plate and measure for the detectable signal with a fluorescent light reader or a visible light reader.

[0459] In some instances, systems comprise a PCR plate; a guide nucleic acid targeting a target sequence; an effector protein capable of being activated when complexed with the guide nucleic acid and the target sequence; and a single stranded reporter nucleic acid comprising a detection moiety, wherein the reporter nucleic acid is capable of being cleaved by the activated nuclease, thereby generating a detectable signal.

[0460] In some instances, systems comprise a support medium; a guide nucleic acid targeting a target sequence; and an effector protein capable of being activated when complexed with the guide nucleic acid and the target sequence. In some instances, nucleic acid amplification is performed in a nucleic acid amplification region on the support medium. Alternatively, or in combination, the nucleic acid amplification is performed in a reagent chamber, and the resulting sample is applied to the support medium.

[0461] In some instances, a system for modifying a target nucleic acid comprises a PCR plate; a guide nucleic acid targeting a target sequence; and an effector protein capable of being activated when complexed with the guide nucleic acid and the target sequence. The wells of the PCR plate may be pre- aliquoted with the guide nucleic acid targeting a target sequence, and an effector protein capable of being activated when complexed with the guide nucleic acid and the target sequence. A user may thus add the biological sample of interest to a well of the pre-aliquoted PCR plate.

[0462] Often, the nucleic acid amplification is performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes, or any value 1 to 60 minutes. Sometimes, the nucleic acid amplification is performed for 1 to 60, 5 to 55, 10 to 50, 15 to 45, 20 to 40, or 25 to 35 minutes. Sometimes, the nucleic acid amplification reaction is performed at a temperature of around 20-45°C. In some instances, the nucleic acid amplification reaction is performed at a temperature no greater than 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, or any value 20 °C to 45 °C. In some instances, the nucleic acid amplification reaction is performed at a temperature of at least 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, or 45°C, or any value 20 °C to 45 °C. In some instances, the nucleic acid amplification reaction is performed at a temperature of 20°C to 45°C, 25°C to 40°C, 30°C to 40°C, or 35°C to 40°C.

[0463] Often, systems comprise primers for amplifying a target nucleic acid to produce an amplification product comprising the target nucleic acid and a PAM. For instance, at least one of the primers may comprise the PAM that is incorporated into the amplification product during amplification. The compositions for amplification of target nucleic acids and methods of use thereof, as described herein, are compatible with any of the methods disclosed herein including methods of assaying for at least one base difference (e.g. , assaying for a SNP or a base mutation) in a target nucleic acid sequence, methods of assaying for a target nucleic acid that lacks a PAM by amplifying the target nucleic acid sequence to introduce a PAM, and compositions used in introducing a PAM via amplification into the target nucleic acid sequence.

Additional System Components

[0464] In some instances, systems include a package, carrier, or container that is compartmentalized to receive one or more containers such as vials, syringes, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, test wells, bottles, vials, syringes and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass, plastic, or polymers. The system or systems described herein contain packaging materials. Examples of packaging materials include, but are not limited to, pouches, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for intended mode of use.

[0465] A system may include labels listing contents and/or instructions for use, or package inserts with instructions for use. A set of instructions will also typically be included. In one embodiment, a label is on or associated with the container. In some instances, a label is on a container when letters, numbers or other characters forming the label are attached, molded, or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein. After packaging the formed product and wrapping or boxing to maintain a sterile barrier, the product may be terminally sterilized by heat sterilization, gas sterilization, gamma irradiation, or by electron beam sterilization. Alternatively, the product may be prepared and packaged by aseptic processing.

[0466] In some instances, systems comprise a solid support. An RNP or effector protein may be attached to a solid support. The solid support may be an electrode or a bead. The bead may be a magnetic bead. Upon cleavage, the RNP is liberated from the solid support and interacts with other mixtures. For example, upon cleavage of the nucleic acid of the RNP, the effector protein of the RNP flows through a chamber into a mixture comprising a substrate. When the effector protein meets the substrate, a reaction occurs, such as a colorimetric reaction, which is then detected. As another example, the protein is an enzyme substrate, and upon cleavage of the nucleic acid of the enzyme substrate-nucleic acid, the enzyme flows through a chamber into a mixture comprising the enzyme. When the enzyme substrate meets the enzyme, a reaction occurs, such as a calorimetric reaction, which is then detected.

Certain System Conditions

[0467] In some instances, systems and methods are employed under certain conditions that enhance an activity of the effector protein relative to alternative conditions, as measured by a detectable signal released from cleavage of a reporter in the presence of the target nucleic acid. The detectable signal may be generated at about the rate of trans cleavage of a reporter nucleic acid. In some instances, the reporter nucleic acid is a homopolymeric reporter nucleic acid comprising 5 to 20 consecutive adenines, 5 to 20 consecutive thymines, 5 to 20 consecutive cytosines, or 5 to 20 consecutive guanines. In some instances, the reporter is an RNA-FQ reporter.

[0468] In some instances, effector proteins disclosed herein recognize, bind, or are activated by, different target nucleic acids having different sequences, but are active toward the same reporter nucleic acid, allowing for facile multiplexing in a single assay having a single ssRNA-FQ reporter.

[0469] In some instances, systems are employed under certain conditions that enhance trans cleavage activity of an effector protein. In some instances, under certain conditions, trans cleavage occurs at a rate of at least 0.005 mmol/min, at least 0.01 mmol/min, at least 0.05 mmol/min, at least 0. 1 mmol/min, at least 0.2 mmol/min, at least 0.5 mmol/min, or at least 1 mmol/min. In some instances, systems and methods are employed under certain conditions that enhance cis-cleavage activity of the effector protein.

[0470] Certain conditions that may enhance the activity of an effector protein include a certain salt presence or salt concentration of the solution in which the activity occurs. For example, cis-cleavage activity of an effector protein may be inhibited or halted by a high salt concentration. The salt may be a sodium salt, a potassium salt, or a magnesium salt. In some instances, the salt is NaCl. In some instances, the salt is KNO3. In some instances, the salt concentration is less than 150 mM, less than 125 mM, less than 100 mM, less than 75 mM, less than 50 mM, or less than 25 mM.

[0471] Certain conditions that may enhance the activity of an effector protein include the pH of a solution in which the activity. For example, increasing pH may enhance trans cleavage activity. For example, the rate of trans cleavage activity may increase with increase in pH up to pH 9. In some instances, the pH is about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9. In some instances, the pH is 7 to 7.5, 7.5 to 8, 8 to 8.5, 8.5 to 9, or 7 to 8.5. In some instances, the pH is less than 7. In some instances, the pH is greater than 7.

[0472] Certain conditions that may enhance the activity of an effector protein includes the temperature at which the activity is performed. In some instances, the temperature is about 25°C to about 50°C. In some instances, the temperature is about 20°C to about 40°C, about 30°C to about 50°C, or about 40°C to about 60°C. In some instances, the temperature is about 25 °C, about 30°C, about 35 °C, about 40°C, about 45°C, or about 50°C.

IX. Methods and Formulations for Introducing System Components and Compositions into a Target Cell

[0473] A guide nucleic acid (or a nucleic acid comprising a nucleotide sequence encoding same) and/or an effector protein described herein may be introduced into a host cell by any of a variety of well-known methods. As a non-limiting example, a guide nucleic acid and/or effector protein may be combined with a lipid. As another non-limiting example, a guide nucleic acid and/or effector protein may be combined with a particle or formulated into a particle.

Methods of Introduction of System Components and Compositions to a Host

[0474] Described herein are methods of introducing various components described herein to a host. A host can be any suitable host, such as a host cell. When described herein, a host cell can be an in vivo or in vitro eukaryotic cell, a prokaryotic cell (e.g., bacterial or archaeal cell), or a cell from a multicellular organism (e.g. , a cell line) cultured as a unicellular entity, which eukaryotic or prokaryotic cells can be, or have been, used as recipients for methods of introduction described herein, and include the progeny of the original cell which has been transformed by the methods of introduction described herein. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. A host cell can be a recombinant host cell or a genetically modified host cell, if a heterologous nucleic acid, e.g., an expression vector, has been introduced into the cell.

[0475] Methods of introducing a nucleic acid and/or protein into a host cell are known in the art, and any convenient method may be used to introduce a subject nucleic acid (e.g., an expression construct/vector) into a target cell (e.g., a human cell, and the like). Suitable methods include, e.g., viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al. Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9. doi: 10.1016/j.addr.2012.09.023), and the like. In some embodiments, the nucleic acid and/or protein are introduced into a disease cell comprised in a pharmaceutical composition comprising the guide nucleic acid and/or effector protein and a pharmaceutically acceptable excipient.

[0476] In certain embodiments, molecules of interest, such as nucleic acids of interest, are introduced to a host. In certain embodiments, polypeptides, such as an effector protein, are introduced to a host. In certain embodiments, vectors, such as lipid particles and/or viral vectors can be introduced to a host. Introduction can be for contact with a host or for assimilation into the host, for example, introduction into a host cell.

[0477] In some embodiments, described herein are methods of introducing one or more nucleic acids, such as a nucleic acid encoding an effector protein, a nucleic acid encoding an engineered guide nucleic acid, and/or a donor nucleic acid, or combinations thereof, into a host cell. Any suitable method can be used to introduce a nucleic acid into a cell. Suitable methods include, for example, viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)- mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like. Further methods are described throughout.

[0478] Introducing one or more nucleic acids into a host cell can occur in any culture media and under any culture conditions that promote the survival of the cells. Introducing one or more nucleic acids into a host cell can be carried out in vivo or ex vivo. Introducing one or more nucleic acids into a host cell can be carried out in vitro.

[0479] In some embodiments, an effector protein can be provided as RNA. The RNA can be provided by direct chemical synthesis or may be transcribed in vitro from a DNA (e.g., encoding the effector protein). Once synthesized, the RNA may be introduced into a cell by way of any suitable technique for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, etc.). In some embodiments, introduction of one or more nucleic acid can be through the use of a vector and/or a vector system, accordingly, in some embodiments, compositions and system described herein comprise a vector and/or a vector system.

[0480] Vectors may be introduced directly to a host. In certain embodiments, host cells can be contacted with one or more vectors as described herein, and in certain embodiments, said vectors are taken up by the cells. Methods for contacting cells with vectors include, but are not limited to, electroporation, calcium chloride transfection, microinjection, lipofection, micro-injection, contact with the cell or particle that comprises a molecule of interest, or a package of cells or particles that comprise molecules of interest.

[0481] Components described herein can also be introduced directly to a host. For example, an engineered guide nucleic acid can be introduced to a host, specifically introduced into a host cell. Methods of introducing nucleic acids, such as RNA into cells include, but are not limited to direct injection, transfection, or any other method used for the introduction of nucleic acids.

[0482] Polypeptides (e.g. , effector proteins) described herein can also be introduced directly to a host. In some embodiments, polypeptides described herein can be modified to promote introduction to a host. For example, polypeptides described herein can be modified to increase the solubility of the polypeptide. Such a polypeptide may optionally be fused to a polypeptide domain that increases solubility. The domain may be linked to the polypeptide through a defined protease cleavage site, such as TEV sequence which is cleaved by TEV protease. The linker may also include one or more flexible sequences, e.g. from 1 to 10 glycine residues. In some embodiments, the cleavage of the polypeptide is performed in a buffer that maintains solubility of the product, e.g. in the presence of from 0.5 to 2 M urea, in the presence of polypeptides and/or polynucleotides that increase solubility, and the like. Domains of interest include endosomolytic domains, e.g. influenza HA domain; and other polypeptides that aid in production, e.g. IF2 domain, GST domain, GRPE domain, and the like. In another example, the polypeptide can be modified to improve stability. For example, the polypeptides may be PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream. Polypeptides can also be modified to promote uptake by a host, such as a host cell. For example, a polypeptide described herein can be fused to a polypeptide permeant domain to promote uptake by a host cell. Any suitable permeant domains can be used in the non-integrating polypeptides of the present disclosure, including peptides, peptidomimetics, and non-peptide carriers. Examples include penetratin, a permeant peptide may be derived from the third alpha helix of Drosophila melanogaster transcription factor Antennapaedia; the HIV-1 tat basic region amino acid sequence, e.g. , amino acids 49-57 of a naturally- occurring tat protein; and poly-arginine motifs, for example, the region of amino acids 34-56 of HIV-1 rev protein, nonaarginine, octa-arginine, and the like. The site at which the fusion is made may be selected in order to optimize the biological activity, secretion or binding characteristics of the polypeptide. The optimal site can be determined by suitable methods.

Formulations for Introducing System Components and Compositions to a Host

[0483] Described herein are formulations of introducing compositions or components of a system described herein to a host. In some embodiments, such formulations, systems and compositions described herein comprise an effector protein and a carrier (e.g., excipient, diluent, vehicle, or filling agent). In some aspects of the present disclosure, the effector protein is provided in a pharmaceutical composition comprising the effector protein and any pharmaceutically acceptable excipient, carrier, or diluent. X. Modification of Target Nucleic Acids

Methods of Modifying Nucleic Acids

[0484] Provided herein are methods, systems and compositions of modifying target nucleic acids or the expression thereof. In some instances, methods comprise editing a target nucleic acid. In general, editing refers to modifying the nucleotide sequence of a target nucleic acid. In some embodiments, modifying refers to changing the physical composition of a target nucleic acid. However, in some embodiments, compositions, methods, and systems disclosed herein may also be capable of modifying target nucleic acids, such as making epigenetic modifications of target nucleic acids, which does not change the nucleotide sequence of the target nucleic acids per se.

[0485] Also provided herein are methods of modulating the expression of a target nucleic acid. Fusion effector proteins and systems described herein may be used for such methods. Methods of editing a target nucleic acid may comprise one or more of cleaving the target nucleic acid, deleting one or more nucleotides of the target nucleic acid, inserting one or more nucleotides into the target nucleic acid, modifying, mutating, or otherwise changing, one or more nucleotides of the target nucleic acid. Modifying a target nucleic acid may comprise one or more of: methylating, demethylating, deaminating, or oxidizing one or more nucleotides of the target nucleic acid. Methods of modulating expression of target nucleic acids may comprise modifying the target nucleic acid or a protein associated with the target nucleic acid, e.g., a histone.

[0486] In some instances, methods comprise contacting a target nucleic acid with a composition described herein. In some instances, methods comprise contacting a target nucleic acid with an effector protein described herein. In some instances, methods comprise contacting a target nucleic acid with a fusion effector protein described herein. The effector protein may be any one of effector proteins provided in TABLE 1 or a catalytically inactive variant thereof. The effector protein may comprise an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of sequences described in TABLE 1. In some instances, the amino acid sequence of the effector protein is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of sequences described in TABLE 1.

[0487] In some instances, methods comprise contacting a target nucleic acid with a donor nucleic acid. In some instances, compositions and systems described herein comprise a donor nucleic acid. Methods may comprise contacting a target nucleic acid, including but not limited to a cell comprising the target nucleic acid, with such compositions. In some instances, the donor nucleic acid is inserted at a site that has been cleaved by a composition disclosed herein, for example, an effector protein, resulting in a nick or double strand break. In some instances, the donor nucleic acid comprises a sequence that serves as a template in the process of homologous recombination. The sequence may carry one or more nucleobase modifications that are to be introduced into the target nucleic acid. By using this donor nucleic acid as a template, the genetic information, including the modification(s), is copied into the target nucleic acid by way of homologous recombination. In reference to a viral vector, the term donor nucleic acid refers to a sequence of nucleotides that will be or has been introduced into a cell following transfection of the viral vector. The donor nucleic acid may be introduced into the cell by any mechanism of the transfecting viral vector, including, but not limited to, integration into the genome of the cell or introduction of an episomal plasmid or viral genome.

[0488] In some instances, methods, systems, and compositions described herein comprise base editing. In some instances, base editing comprises contacting a target nucleic acid with a fusion effector protein comprising an effector protein fused to a base editing enzyme, such as a deaminase, thereby changing a nucleobase of the target nucleic acid to an alternative nucleobase. In some instances, the nucleobase of the target nucleic acid is adenine (A) and the method comprises changing A to guanine (G). In some instances, the nucleobase of the target nucleic acid is cytosine (C) and the method comprises changing C to thymine (T). In some instances, the nucleobase of the target nucleic acid is C and the method comprises changing C to G. In some instances, the nucleobase of the target nucleic acid is A and the method comprises changing A to G.

[0489] In some instances, methods, systems, and compositions described herein introduce a nucleobase change in a target nucleic acid relative to a corresponding wildtype or mutant nucleobase sequence. In some instances, methods, systems, and compositions described herein remove or correct a diseasecausing mutation in a nucleic acid sequence, e.g., to produce a corresponding wildtype nucleobase sequence. In some instances, methods, systems, and compositions described herein remove/correct point mutations, deletions, null mutations, or tissue-specific mutations in a target nucleic acid. In some instances, methods, systems, and compositions described herein generate gene knock-out, gene knock- in, gene editing, gene tagging, or a combination thereof. Methods, systems, and compositions described herein of the disclosure may be targeted to a locus in a genome of a cell.

[0490] Compositions, methods, and systems described herein may modify a coding portion of a gene, a non-coding portion of a gene, or a combination thereof. Modifying at least one gene using the compositions and methods described herein can, in some embodiments, induce a reduction or increase in expression of the one or more genes. In some embodiments, the at least one modified gene results in a reduction in expression, also referred to as gene silencing. In some embodiments, the gene silencing reduces expression of one or more genes by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, gene silencing is accomplished by transcriptional silencing, post-transcriptional silencing, or meiotic silencing. In some embodiments, transcriptional silencing is by genomic imprinting, paramutation, transposon silencing, position effect, or RNA-directed DNA methylation. In some embodiments, post- transcriptional silencing is by RNA interference, RNA silencing, or nonsense mediated decay. In some embodiments, meiotic silencing is by transfection or meiotic silencing of unpaired DNA. In some embodiments, the at least one modified gene results in removing all expression, also referred to as the gene being knocked out (KO). In some embodiments, the compositions, methods or systems increase expression of one or more genes by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%.

[0491] In some embodiments, the compositions, methods or systems comprise a nucleic acid expression vector, or use thereof, to introduce an effector protein, guide nucleic acid, donor template or any combination thereof to a cell. In some embodiments, the nucleic acid expression vector is a viral vector. Viral vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses. In some embodiments, the viral vector is a replication-defective viral vector, comprising an insertion of a therapeutic gene inserted in genes essential to the lytic cycle, preventing the virus from replicating and exerting cytotoxic effects. In some embodiments, the viral vector is an adeno associated viral (AAV) vector. In some embodiments, the nucleic acid expression vector is a non-viral vector. In some embodiments, compositions and methods comprise a lipid, polymer, nanoparticle, or a combination thereof, or use thereof, to introduce a Cas protein, guide nucleic acid, donor template or any combination thereof to a cell. Non-limiting examples of lipids and polymers are cationic polymers, cationic lipids, or bio-responsive polymers. In some embodiments, the bio- responsive polymer exploits chemical -physical properties of the endosomal environment (e.g., pH) to preferentially release the genetic material in the intracellular space.

[0492] Methods of modifying may comprise contacting a target nucleic acid with one or more components, compositions or systems described herein. In some embodiments, a method of modifying comprises contacting a target nucleic acid with at least one of: a) one or more effector proteins, or one or more nucleic acids encoding one or more effector proteins; or b) one or more guide nucleic acids, or one or more nucleic acids encoding one or more guide nucleic acids. In some embodiments, a method of modifying comprises contacting a target nucleic acid with a system described herein wherein the system comprises components comprising at least one of: a) one or more effector proteins, or one or more nucleic acids encoding one or more effector proteins; or b) one or more guide nucleic acids, or one or more nucleic acids encoding one or more guide nucleic acids. In some embodiments, a method of modifying comprises contacting a target nucleic acid with a composition described herein comprising at least one of: a) one or more effector proteins, or one or more nucleic acids encoding one or more effector proteins; or b) one or more guide nucleic acids, or one or more nucleic acids encoding one or more guide nucleic acids; in a composition. In some embodiments, a method of modifying as described herein produces a modified target nucleic acid.

[0493] Editing a target nucleic acid sequence may introduce a mutation (e.g, point mutations, deletions) in a target nucleic acid relative to a corresponding wildtype nucleotide sequence. Editing may remove or correct a disease-causing mutation in a nucleic acid sequence to produce a corresponding wildtype nucleotide sequence. Editing a target nucleic acid sequence may remove/correct point mutations, deletions, null mutations, or tissue-specific mutations in a target nucleic acid. Editing a target nucleic acid sequence may be used to generate gene knock-out, gene knock-in, gene editing, gene tagging, or a combination thereof. Methods of the disclosure may be targeted to any locus in a genome of a cell.

[0494] Modifying may comprise single stranded cleavage, double stranded cleavage, donor nucleic acid insertion, epigenetic modification (e.g., methylation, demethylation, acetylation, or deacetylation), or a combination thereof. In some embodiments, cleavage (single-stranded or double-stranded) is sitespecific, meaning cleavage occurs at a specific site in the target nucleic acid, often within the region of the target nucleic acid that hybridizes with the guide nucleic acid spacer sequence. In some embodiments, the effector proteins introduce a single-stranded break in a target nucleic acid to produce a cleaved nucleic acid. In some embodiments, the effector protein is capable of introducing a break in a single stranded RNA (ssRNA). The effector protein may be coupled to a guide nucleic acid that targets a particular region of interest in the ssRNA. In some embodiments, the target nucleic acid, and the resulting cleaved nucleic acid is contacted with a nucleic acid for homologous recombination (e.g, homology directed repair (HDR)) or non-homologous end joining (NHEJ). In some embodiments, a double-stranded break in the target nucleic acid may be repaired (e.g., by NHEJ or HDR) without insertion of a donor template, such that the repair results in an indel in the target nucleic acid at or near the site of the double-stranded break. In some embodiments, an indel, sometimes referred to as an insertion-deletion or indel mutation, is a type of genetic mutation that results from the insertion and/or deletion of one or more nucleotide in a target nucleic acid. An indel may vary in length (e.g., 1 to 1,000 nucleotides in length) and be detected using methods well known in the art, including sequencing. If the number of nucleotides in the insertion/deletion is not divisible by three, and it occurs in a protein coding region, it is also a frameshift mutation. Indel percentage is the percentage of sequencing reads that show at least one nucleotide has been mutation that results from the insertion and/or deletion of nucleotides regardless of the size of insertion or deletion, or number of nucleotides mutated. For example, if there is at least one nucleotide deletion detected in a given target nucleic acid, it counts towards the percent indel value. As another example, if one copy of the target nucleic acid has one nucleotide deleted, and another copy of the target nucleic acid has 10 nucleotides deleted, they are counted the same. This number reflects the percentage of target nucleic acids that are edited by a given effector protein.

[0495] In some embodiments, methods of modifying described herein cleave a target nucleic acid at one or more locations to generate a cleaved target nucleic acid. In some embodiments, the cleaved target nucleic acid undergoes recombination (e.g., NHEJ or HDR). In some embodiments, cleavage in the target nucleic acid may be repaired (e.g., by NHEJ or HDR) without insertion of a donor nucleic acid, such that the repair results in an indel in the target nucleic acid at or near the site of the cleavage site. In some embodiments, cleavage in the target nucleic acid may be repaired (e.g. , by NHEJ or HDR) with insertion of a donor nucleic acid, such that the repair results in an indel in the target nucleic acid at or near the site of the cleavage site. [0496] In some embodiments, wherein the compositions, systems, and methods of the present disclosure comprise an additional guide nucleic acid or a use thereof, and such dual-guided compositions, systems, and methods described herein may modify the target nucleic acid in two locations. In some embodiments, dual -guided modifying may comprise cleavage of the target nucleic acid in the two locations targeted by the guide nucleic acids. In some embodiments, upon removal of the sequence between the guide nucleic acids, the wild-type reading frame is restored. A wild-type reading frame may be a reading frame that produces at least a partially, or fully, functional protein. A non-wild-type reading frame may be a reading frame that produces a non-functional or partially nonfunctional protein.

[0497] Accordingly, in some embodiments, compositions, systems, and methods described herein may edit 1 to 1,000 nucleotides or any integer in between, in a target nucleic acid. In some embodiments, 1 to 1,000, 2 to 900, 3 to 800, 4 to 700, 5 to 600, 6 to 500, 7 to 400, 8 to 300, 9 to 200, or 10 to 100 nucleotides, or any integer in between, may be edited by the compositions, systems, and methods described herein. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides may be edited by the compositions, systems, and methods described herein. In some embodiments, 10, 20, 30, 40, 50, 60, 70, 80 90, 100 or more nucleotides, or any integer in between, may be edited by the compositions, systems, and methods described herein. In some embodiments, 100, 200, 300, 400, 500, 600, 700, 800, 900 or more nucleotides, or any integer in between, may be edited by the compositions, systems, and methods described herein.

[0498] Methods may comprise use of two or more effector proteins. An illustrative method for introducing a break in a target nucleic acid comprises contacting the target nucleic acid with: (a) a first engineered guide nucleic acid comprising a region that binds to a first effector protein described herein; and (b) a second engineered guide nucleic acid comprising a region that binds to a second effector protein described herein, wherein the first engineered guide nucleic acid comprises an additional region that hybridizes to the target nucleic acid and wherein the second engineered guide nucleic acid comprises an additional region that hybridizes to the target nucleic acid. In some embodiments, the first and second effector protein are identical. In some embodiments, the first and second effector protein are not identical.

[0499] In some instances methods of editing a target nucleic acid or modulating the expression of a target nucleic acid are performed in vivo. In some instances, methods of editing a target nucleic acid or modulating the expression of a target nucleic acid are performed in vitro. For example, a plasmid may be modified in vitro using a composition described herein and introduced into a cell or organism. In some instances, methods of editing a target nucleic acid or modulating the expression of a target nucleic acid are performed ex vivo. For example, methods may comprise obtaining a cell from a subject, modifying a target nucleic acid in the cell with methods and compositions described herein, and returning the cell to the subject. Methods of editing performed ex vivo may be particularly advantageous to produce CAR T-cells. [0500] In some instances, methods comprise editing a target nucleic acid or modulating the expression of the target nucleic acid in a cell or a subject. The cell may be a dividing cell. The cell may be a terminally differentiated cell. In some instances, the target nucleic acid is a gene.

[0501] In some embodiments, editing a target nucleic acid comprises genome editing. Genome editing may comprise editing a genome, chromosome, plasmid, or other genetic material of a cell or organism. In some embodiments, the genome, chromosome, plasmid, or other genetic material of the cell or organism is modified in vivo. In some embodiments, the genome, chromosome, plasmid, or other genetic material of the cell or organism is modified in a cell. In some embodiments, the genome, chromosome, plasmid, or other genetic material of the cell or organism is modified in vitro. For example, a plasmid may be edited in vitro using a composition described herein and introduced into a cell or organism.

[0502] In some embodiments, editing a target nucleic acid may comprise deleting a sequence from a target nucleic acid. For example, a mutated sequence or a sequence associated with a disease may be removed from a target nucleic acid. In some embodiments, editing a target nucleic acid may comprise replacing a sequence in a target nucleic acid with a second sequence. For example, a mutated sequence or a sequence associated with a disease may be replaced with a second sequence lacking the mutation or that is not associated with the disease. In some embodiments, editing a target nucleic acid may comprise deleting or replacing a sequence comprising markers associated with a disease or disorder. In some embodiments, editing a target nucleic acid may comprise introducing a sequence into a target nucleic acid. For example, a beneficial sequence or a sequence that may reduce or eliminate a disease may be inserted into the target nucleic acid.

[0503] In some embodiments, methods comprise inserting a donor nucleic acid into a cleaved target nucleic acid. The donor nucleic acid may be inserted at a specified (e.g. , effector protein targeted) point within the target nucleic acid. In some embodiments, the cleaved target nucleic acid is cleaved at a single location. In such embodiments, the methods comprise contacting a target nucleic acid with an effector protein described herein, thereby introducing a single-stranded break in the target nucleic acid; and contacting the target nucleic acid with a donor nucleic acid for homologous recombination, optionally by HDR or NHEJ, thereby introducing a new sequence into the target nucleic acid (e.g., at a cleavage site). In some embodiments, the cleaved target nucleic acid is cleaved at two locations. In such embodiments, the methods comprise contacting a target nucleic acid with an effector protein described herein, thereby introducing a single-stranded break in the target nucleic acid; contacting the target nucleic acid with a second effector protein described herein, to generate a second cleavage site in the target nucleic acid, ligating the regions flanking the first and second cleavage site, optionally through NHEJ or single-strand annealing, thereby resulting in the excision of a portion of the target nucleic acid between the first and second cleavage sites from the target nucleic acid; and contacting the target nucleic acid with a donor nucleic acid for homologous recombination, optionally by HDR or NHEJ, thereby introducing a new sequence into the target nucleic acid (e.g., in between two cleavage sites). [0504] In some embodiments, methods comprise editing a target nucleic acid with two or more effector proteins. Editing a target nucleic acid may comprise introducing a two or more single-stranded breaks in a target nucleic acid. In some embodiments, a break may be introduced by contacting a target nucleic acid with an effector protein and a guide nucleic acid. The guide nucleic acid may bind to the effector protein and hybridize to a region of the target nucleic acid, thereby recruiting the effector protein to the region of the target nucleic acid. Binding of the effector protein to the guide nucleic acid and the region of the target nucleic acid may activate the effector protein, and the effector protein may introduce a break (e.g., a single stranded break) in the region of the target nucleic acid. In some embodiments, editing a target nucleic acid may comprise introducing a first break in a first region of the target nucleic acid and a second break in a second region of the target nucleic acid. For example, editing a target nucleic acid may comprise contacting a target nucleic acid with a first guide nucleic acid that binds to a first effector protein and hybridizes to a first region of the target nucleic acid and a second guide nucleic acid that binds to a second programmable nickase and hybridizes to a second region of the target nucleic acid. The first effector protein may introduce a first break in a first strand at the first region of the target nucleic acid, and the second effector protein may introduce a second break in a second strand at the second region of the target nucleic acid. In some embodiments, a segment of the target nucleic acid between the first break and the second break may be removed, thereby editing the target nucleic acid. In some embodiments, a segment of the target nucleic acid between the first break and the second break may be replaced (e.g., with donor nucleic acid), thereby editing the target nucleic acid.

[0505] Methods, systems and compositions described herein may edit a target nucleic acid wherein such editing may effect one or more indels. In some embodiments, where compositions, systems, and/or methods described herein effect one or more indels, the impact on the transcription and/or translation of the target nucleic acid may be predicted depending on: 1) the amount of indels generated; and 2) the location of the indel on the target nucleic acid. For example, as described herein, in some embodiments, if the amount of indels is not divisible by three, and the indels occur within or along a protein coding region, then the edit or mutation may be a frameshift mutation. In some embodiments, if the amount of indels is divisible by three, then a frameshift mutation may not be effected, but a splicing disruption mutation and/or sequence skip mutation may be effected, such as an exon skip mutation. In some embodiments, if the amount of indels is not evenly divisible by three, then a frameshift mutation may be effected.

[0506] Methods, systems and compositions described herein may edit a target nucleic acid wherein such editing may be measured by indel activity. Indel activity measures the amount of change in a target nucleic acid (e.g., nucleotide deletion(s) and/or insertion(s)) compared to a target nucleic acid that has not been contacted by a polypeptide described in compositions, systems, and methods described herein. For example, indel activity may be detected by next generation sequencing of one or more target loci of a target nucleic acid where indel percentage is calculated as the fraction of sequencing reads containing insertions or deletions relative to an unedited reference sequence. In some embodiments, methods, systems, and compositions comprising an effector protein and guide nucleic acid described herein may exhibit about 0.0001% to about 65% or more indel activity upon contact to a target nucleic acid compared to a target nucleic acid non-contacted with compositions, systems, or by methods described herein. For example, methods, systems, and compositions comprising an effector protein and guide nucleic acid described herein may exhibit about 0.0001%, about 0.001%, about 0.01%, about 0.1%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% or more indel activity.

[0507] In some embodiments, editing of a target nucleic acid as described herein effects one or more mutations comprising splicing disruption mutations, frameshift mutations (e.g., 1+ or 2+ frameshift mutation), sequence deletion, sequence skipping, sequence reframing, sequence knock-in, or any combination thereof. In some embodiments, the splicing disruption can be an editing that disrupts a splicing of a target nucleic acid or a splicing of a sequence that is transcribed from a target nucleic acid relative to a target nucleic acid without the splicing disruption. In some embodiments, the frameshift mutation can be an editing that alters the reading frame of a target nucleic acid relative to a target nucleic acid without the frameshift mutation. In some embodiments, the frameshift mutation can be a +2 frameshift mutation, wherein a reading frame is edited by 2 bases. In some embodiments, the frameshift mutation can be a +1 frameshift mutation, wherein a reading frame is edited by 1 base. In some embodiments, the frameshift mutation is an editing that alters the number of bases in a target nucleic acid so that it is not divisible by three. In some embodiments, the frameshift mutation can be an editing that is not a splicing disruption. In some embodiments a sequence as described in reference to the sequence deletion, sequence skipping, sequence reframing, and sequence knock-in can be a DNA sequence, a RNA sequence, an edited DNA or RNA sequence, a mutated sequence, a wild-type sequence, a coding sequence, a non-coding sequence, an exonic sequence (exon), an intronic sequence (intron), or any combination thereof. In some embodiments, the sequence deletion is an editing where one or more sequences in a target nucleic acid are deleted relative to a target nucleic acid without the sequence deletion. In some embodiments, the sequence deletion can result in or effect a splicing disruption or a frameshift mutation. In some embodiments, the sequence deletion result in or effect a splicing disruption. In some embodiments, the sequence skipping is an editing where one or more sequences in a target nucleic acid are skipped upon transcription or translation of the target nucleic acid relative to a target nucleic acid without the sequence skipping. In some embodiments, the sequence skipping can result in or effect a splicing disruption or a frameshift mutation. In some embodiments, the sequence skipping can result in or effect a splicing disruption. In some embodiments, the sequence reframing is an editing where one or more bases in a target are edited so that the reading frame of the sequence is reframed relative to a target nucleic acid without the sequence reframing. In some embodiments, the sequence reframing can result in or effect a splicing disruption or a frameshift mutation. In some embodiments, the sequence reframing can result in or effect a frameshift mutation. In some embodiments, the sequence knock-in is an editing where one or more sequences is inserted into a target nucleic acid relative to a target nucleic acid without the sequence knock-in. In some embodiments, the sequence knock-in can result in or effect a splicing disruption or a frameshift mutation. In some embodiments, the sequence knock-in can result in or effect a splicing disruption.

[0508] In some embodiments, editing of a target nucleic acid can be locus specific, wherein compositions, systems, and methods described herein can edit a target nucleic acid at one or more specific loci to effect one or more specific mutations comprising splicing disruption mutations, frameshift mutations, sequence deletion, sequence skipping, sequence reframing, sequence knock-in, or any combination thereof. For example, editing of a specific locus can affect any one of a splicing disruption, frameshift (e.g., 1+ or 2+ frameshift), sequence deletion, sequence skipping, sequence reframing, sequence knock-in, or any combination thereof. In some embodiments, editing of a target nucleic acid can be locus specific, modification specific, or both. In some embodiments, editing of a target nucleic acid can be locus specific, modification specific, or both, wherein compositions, systems, and methods described herein comprise an effector protein described herein and a guide nucleic acid described herein.

[0509] Methods of editing a target nucleic acid or modulating the expression of a target nucleic acid may be performed in vivo. Methods of editing a target nucleic acid or modulating the expression of a target nucleic acid may be performed in vitro. For example, a plasmid may be edited in vitro using a composition described herein and introduced into a cell or organism. Methods of editing a target nucleic acid or modulating the expression of a target nucleic acid may be performed ex vivo. For example, methods may comprise obtaining a cell from a subject, editing a target nucleic acid in the cell with methods described herein, and returning the cell to the subject.

[0510] In some embodiments, methods of modifying described herein comprise contacting a target nucleic acid with one or more components, compositions or systems described herein. In some embodiments, the one or more components, compositions or systems described herein comprise at least one of: a) one or more effector proteins, or one or more nucleic acids encoding one or more effector proteins; and b) one or more guide nucleic acids, or one or more nucleic acids encoding one or more guide nucleic acids. In some embodiments, the one or more effector proteins introduce a single-stranded break or a double-stranded break in the target nucleic acid.

[0511] In some embodiments, methods of modifying described herein comprise using one or more guide nucleic acids or uses thereof, wherein the methods modify a target nucleic acid at a single location. In some embodiments, the methods comprise contacting an RNP comprising an effector protein and a guide nucleic acid to the target nucleic acid. In some embodiments, the methods introduce a mutation (e.g., point mutations, deletions) in the target nucleic acid relative to a corresponding wildtype nucleotide sequence. In some embodiments, the methods remove or correct a disease-causing mutation in a nucleic acid sequence to produce a corresponding wildtype nucleotide sequence. In some embodiments, the methods remove/correct point mutations, deletions, null mutations, or tissue-specific mutations in a target nucleic acid. In some embodiments, the methods introduce a single stranded cleavage, a nick, a deletion of one or two nucleotides, an insertion of one or two nucleotides, a substitution of one or two nucleotides, an epigenetic modification (e.g., methylation, demethylation, acetylation, or deacetylation), or a combination thereof to the target nucleic acid. In some embodiments, the methods comprise using an effector protein and two guide nucleic acids, wherein two RNPs cleave the target nucleic acid at the same location, wherein a first RNP comprises the effector protein and a first guide nucleic acid, and wherein a second RNP comprises the effector protein and a second guide nucleic acid. In some embodiments, methods comprising using two effector protein and two guide nucleic acids, wherein both RNPs cleave the target nucleic acid at the same location, wherein a first RNP comprises a first effector protein and a first target nucleic acid, and wherein a second RNP comprises a second effector protein and a second target nucleic acid.

[0512] In some embodiments, methods of modifying described herein comprise using one or more guide nucleic acids or uses thereof, wherein the methods modify a target nucleic acid at two different locations. In some embodiments, the methods introduce two cleavage sites in the target nucleic acid, wherein a first cleavage site and a second cleavage site comprise one or more nucleotides therebetween. In some embodiments, the methods cause deletion of the one or more nucleotides. In some embodiments, the deletion restores a wild-type reading frame. In some embodiments, the wild-type reading frame produces at least a partially functional protein. In some embodiments, the deletion causes a non-wild-type reading frame. In some embodiments, a non-wild-type reading frame produces a partially functional protein or non-fimctional protein. In some embodiments, the at least partially functional protein has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 180%, at least 200%, at least 300%, at least 400% activity compared to a corresponding wildtype protein. In some embodiments, the methods comprise using an effector protein and two guide nucleic acids, wherein two RNPs cleave the target nucleic acid at different locations, wherein a first RNP comprises the effector protein and a first guide nucleic acid, and wherein a second RNP comprises the effector protein and a second guide nucleic acid. In some embodiments, methods comprising using two effector protein and two guide nucleic acids, wherein both RNPs cleave the target nucleic acid at the same location, wherein a first RNP comprises a first effector protein and a first target nucleic acid, and wherein a second RNP comprises a second effector protein and a second target nucleic acid.

[0513] In some embodiments, methods of editing described herein comprise inserting a donor nucleic acid into a cleaved target nucleic acid. In some embodiments, the cleaved target nucleic acid formed by introducing a single-stranded break into a target nucleic acid. The donor nucleic acid may be inserted at a specified (e.g. , effector protein targeted) point within the target nucleic acid. In some embodiments, the cleaved target nucleic acid is cleaved at a single location. In such embodiments, the methods comprise contacting a target nucleic acid with an effector protein described herein, thereby introducing a single-stranded break in the target nucleic acid; and contacting the target nucleic acid with a donor nucleic acid for homologous recombination, optionally by HDR or NHEJ, thereby introducing a new sequence into the target nucleic acid (e.g., at a cleavage site). In some embodiments, the cleaved target nucleic acid is cleaved at two locations. In such embodiments, the methods comprise contacting a target nucleic acid with an effector protein described herein, thereby introducing a single-stranded break in the target nucleic acid; contacting the target nucleic acid with a second effector protein described herein, to generate a second cleavage site in the target nucleic acid, ligating the regions flanking the first and second cleavage site, optionally through NHEJ or single-strand annealing, thereby resulting in the excision of a portion of the target nucleic acid between the first and second cleavage sites from the target nucleic acid; and contacting the target nucleic acid with a donor nucleic acid for homologous recombination, optionally by HDR or NHEJ, thereby introducing a new sequence into the target nucleic acid (e.g., in between two cleavage sites).

Donor Nucleic Acids

[0514] In some embodiments, a donor nucleic acid comprises a nucleic acid that is incorporated into a target nucleic acid or genome. In some embodiments, a donor nucleic acid comprises a sequence that is derived from a plant, bacteria, fungi, virus, or an animal. In some embodiments, the animal is a nonhuman animal, such as, by way of non-limiting example, a mouse, rat, hamster, rabbit, pig, bovine, deer, sheep, goat, chicken, cat, dog, ferret, a bird, non-human primate (e.g., marmoset, rhesus monkey). In some embodiments, the non-human animal is a domesticated mammal or an agricultural mammal. In some embodiments, the animal is a human. In some embodiments, the sequence comprises a human wild-type (WT) gene or a portion thereof. In some embodiments, the human WT gene or the portion thereof comprises a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% identical to an equal length portion of the WT sequence of any one of the sequences recited in TABLE 9. In some embodiments, the donor nucleic acid is incorporated into an insertion site of a target nucleic acid.

[0515] In some embodiments, the donor nucleic acid comprises single-stranded DNA or linear doublestranded DNA. In some embodiments, the donor nucleic acid comprises a nucleotide sequence encoding a functional polypeptide and/or wherein the donor nucleic acid comprises a wildtype sequence. In some embodiments, the donor nucleic acid comprises a protein coding sequence, a gene, a gene fragment, an exon, an intron, an exon fragment, an intron fragment, a gene regulatory fragment, a gene regulatory region fragment, coding sequences thereof, or combinations thereof. In some embodiments, the donor nucleic acid comprises a naturally occurring sequence. In some embodiments, the naturally occurring sequence does not contain a mutation.

[0516] In some embodiments, the donor nucleic acid comprises a gene fragment, an exon fragment, an intron fragment, a gene regulatory region fragment, or combinations thereof. In some embodiments, the fragment is at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, or at least 80 contiguous nucleotides. [0517] In some embodiments, a donor nucleic acid of any suitable size is integrated into a target nucleic acid or a genome. In some embodiments, the donor nucleic acid integrated into the target nucleic acid or the genome is less than 3, about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 kilobases in length. In some embodiments, the donor nucleic acid is more than 500 kilobases (kb) in length.

[0518] In some embodiments, a viral vector comprising a donor nucleic acid introduces the donor nucleic acid into a cell following transfection. In some embodiments, the donor nucleic acid is introduced into the cell by any mechanism of the transfecting viral vector, including, but not limited to, integration into the genome of the cell or introduction of an episomal plasmid or viral genome.

[0519] In some embodiments, an effector protein as described herein facilitates insertion of a donor nucleic acid at a site of cleavage or between two cleavage sites by cleaving (hydrolysis of a phosphodiester bond) of a nucleic acid resulting in a nick or double strand break - nuclease activity.

[0520] In some embodiments, a donor nucleic acid serves as a template in the process of homologous recombination, which may carry an alteration that is to be or has been introduced into a target nucleic acid. By using the donor nucleic acid as a template, the genetic information, including the alteration, is copied into the target nucleic acid by way of homologous recombination.

Genetically modified cells and organisms

[0521] Methods of editing (e.g., editing a target nucleic acid or modulating the expression of a target nucleic acid) described herein may be employed to generate a genetically modified cell. The cell may be a eukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g., an archaeal cell). The cell may be a human cell. The cell may be a T cell. The cell may be a hematopoietic stem cell. The cell may be a bone marrow derived cell, a white blood cell, a blood cell progenitor, or a combination thereof. The cell may be derived from a multicellular organism and cultured as a unicellular entity. The cell may comprise a heritable genetic modification, such that progeny cells derived therefrom comprise the heritable genetic mutation. The cell may be progeny of a genetically modified cell comprising a genetic modification of the genetically modified parent cell. A genetically modified cell may comprise a deletion, insertion, mutation, or non-native sequence relative to a wild-type version of the cell or the organism from which the cell was derived.

[0522] In some aspects, disclosed herein are modified cells or populations of modified cells, wherein the modified cell comprises an effector protein described herein, a nucleic acid encoding an effector protein described herein, or a combination thereof. In some instances, the modified cell comprises a fusion effector protein described herein, a nucleic acid encoding an effector protein described herein, or a combination thereof. In some instances, the modified cell is a modified prokaryotic cell. In some instances, the modified cell is a modified eukaryotic cell. A modified cell may be a modified fungal cell. In some instances, the modified cell is a modified vertebrate cell. In some instances, the modified cell is a modified invertebrate cell. In some instances, the modified cell is a modified mammalian cell. In some instances the modified cell is a modified human cell. In some instances, the modified cell is in a subject. A modified cell may be in vitro. A modified cell may be in vivo. A modified cell may be ex vivo. A modified cell may be a cell in a cell culture. A modified cell may be a cell obtained from a biological fluid, organ, or tissue of a subject and modified with a composition and/or method described herein. Non-limiting examples of biological fluids are blood, plasma, serum, and cerebrospinal fluid. Non-limiting examples of tissues and organs are bone marrow, adipose tissue, skeletal muscle, smooth muscle, spleen, thymus, brain, lymph node, adrenal gland, prostate gland, intestine, colon, liver, kidney, pancreas, heart, lung, bladder, ovary, uterus, breast, and testes. Non-limiting examples of cells that may be obtained from a subject are hepatocytes, epithelial cells, endothelial cells, neurons, cardiomyocytes, muscle cells and adipocytes. Non-limiting examples of cells that may be modified with compositions and methods described herein include immune cells, such as CAR T-cells, T-cells, B-cells, NK cells, granulocytes, basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages, dendritic cells, microglia, Kupffer cells, antigen-presenting cells (APC), or adaptive cells.

[0523] Methods may comprise contacting a cell with a nucleic acid (e.g., a plasmid or mRNA) comprising a nucleotide sequence encoding an effector protein, wherein the effector protein comprise comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to any one of sequences recited in TABLE 1. Methods may comprise contacting cells with a nucleic acid (e.g, a plasmid or mRNA) comprising a nucleotide sequence encoding a guide nucleic acid, an intermediary RNA, a tracrRNA, a crRNA, or any combination thereof. Contacting may comprise electroporation, acoustic poration, optoporation, viral vector-based delivery, iTOP, nanoparticle delivery (e.g., lipid or gold nanoparticle delivery), cell-penetrating peptide (CPP) delivery, DNA nanostructure delivery, or any combination thereof. In some cases, the nanoparticle delivery comprises lipid nanoparticle delivery or gold nanoparticle delivery. In some cases, the nanoparticle delivery comprises lipid nanoparticle delivery. In some cases, the nanoparticle delivery comprises gold nanoparticle delivery.

[0524] Methods may comprise contacting a cell with an effector protein or a multimeric complex thereof, wherein the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to any one of sequences recited in TABLE 1. Methods may comprise contacting a cell with an effector protein, wherein the amino acid sequence of the effector protein is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to any one of sequences recited in TABLE 1.

[0525] Methods may comprise cell line engineering (e.g., engineering a cell from a cell line for bioproduction). Generally, cell line engineering comprises modifying a pre-existing cell (e.g., naturally- occurring or engineered) or pre-existing cell line to produce a novel cell line or modified cell line. In some instances, modifying the pre-existing cell or cell line comprises contacting the pre-existing cell or cell line with an effector protein or fusion effector protein described herein and a guide nucleic acid. The resulting modified cell line may be useful for production of a protein of interest.

[0526] Cell lines may be used to produce a desired protein. In some embodiments, target nucleic acids comprise a genomic sequence. In some embodiments, the cell line is a Chinese hamster ovary cell line (CHO), human embryonic kidney cell line (HEK), cell lines derived from cancer cells, cell lines derived from lymphocytes, and the like. Non-limiting examples of cell lines includes: C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL-2, CIR, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.0I, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP- 1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfir -/-, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML Tl, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KY01, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI- H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, and YAR.

[0527] Non-limiting examples of cells that may be engineered or modified with compositions, systems and methods described herein include immune cells, such as CART, T-cells, B-cells, NK cells, granulocytes, basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages, dendritic cells, antigen-presenting cells (APC), or adaptive cells. Non-limiting examples of cells that may be engineered or modified with compositions and methods described herein include include plant cells, such as parenchyma, sclerenchyma, collenchyma, xylem, phloem, germline (e.g., pollen). Cells from lycophytes, fems, gymnosperms, angiosperms, bryophytes, charophytes, chloropytes, rhodophytes, or glaucophytes. Non-limiting examples of cells that may be engineered or modified with compositions and methods described herein include stem cells, such as human stem cells, animal stem cells, stem cells that are not derived from human embryonic stem cells, embryonic stem cells, mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells (iPS), somatic stem cells, adult stem cells, hematopoietic stem cells, tissue-specific stem cells. A cell may be a pluripotent cell.

[0528] Methods of the disclosure may be performed in a subject. Compositions of the disclosure may be administered to a subject. A subject may be a human. A subject may be a mammal (e.g., rat, mouse, cow, dog, pig, sheep, horse). A subject may be a vertebrate or an invertebrate. A subject may be a laboratory animal. A subject may be a patient. A subject may be suffering from a disease. A subject may display symptoms of a disease. A subject may not display symptoms of a disease, but still have a disease. A subject may be under medical care of a caregiver (e.g., the subject is hospitalized and is treated by a physician). Methods of the disclosure may be performed in a plant, bacteria, or a fungus.

[0529] Methods of the disclosure may be performed in a cell. A cell may be in vitro. A cell may be in vivo. A cell may be ex vivo. A cell may be an isolated cell. A cell may be a cell inside of an organism. A cell may be an organism. A cell may be a cell in a cell culture. A cell may be one of a collection of cells. A cell may be a mammalian cell or derived from a mammalian cell. A cell may be a rodent cell or derived from a rodent cell. A cell may be a human cell or derived from a human cell. A cell may be a prokaryotic cell or derived from a prokaryotic cell. A cell may be a bacterial cell or may be derived from a bacterial cell. A cell may be an archaeal cell or derived from an archaeal cell. A cell may be a eukaryotic cell or derived from a eukaryotic cell. A cell may be a pluripotent stem cell. A cell may be a plant cell or derived from a plant cell. A cell may be an animal cell or derived from an animal cell. A cell may be an invertebrate cell or derived from an invertebrate cell. A cell may be a vertebrate cell or derived from a vertebrate cell. A cell may be a microbe cell or derived from a microbe cell. A cell may be a fungi cell or derived from a fungi cell. A cell may be from a specific organ or tissue.

[0530] In some embodiments, the cell is a hepatocyte. In some embodiments, the tissue is a subject’s blood, bone marrow, or cord blood. In some embodiments, the tissue is a heterologous donor blood, cord blood, or bone marrow. In some embodiments, the tissue is an allogenic blood, cord blood, or bone marrow. In some embodiments, the tissue may be muscle. In some embodiments, the muscle may be a skeletal muscle. In some embodiments, skeletal muscles include the following: abductor digiti minimi (foot), abductor digiti minimi (hand), abductor hallucis, abductor pollicis brevis, abductor pollicis longus, adductor brevis, adductor hallucis, adductor longus, adductor magnus, adductor pollicis, anconeus, articularis cubiti, articularis genu, aryepiglotticus, auricularis, biceps brachii, biceps femoris, brachialis, brachioradialis, buccinator, bulbospongiosus, constrictor of pharynx -inferior, constrictor of pharynx -middle, constrictor of pharynx -superior, coracobrachialis, corrugator supercilii, cremaster, cricothyroid, dartos, deep transverse perinei, deltoid, depressor anguli oris, depressor labii inferioris, diaphragm, digastric, digastric (anterior view), erector spinae - spinalis, erector spinae - iliocostalis, erector spinae - longissimus, extensor carpi radialis brevis, extensor carpi radialis longus, extensor carpi ulnaris, extensor digiti minimi (hand), extensor digitorum (hand), extensor digitorum brevis (foot), extensor digitorum longus (foot), extensor hallucis brevis, extensor hallucis longus, extensor indicis, extensor pollicis brevis, extensor pollicis longus, external oblique abdominis, flexor carpi radialis, flexor carpi ulnaris, flexor digiti minimi brevis (foot), flexor digiti minimi brevis (hand), flexor digitorum brevis, flexor digitorum longus (foot), flexor digitorum profundus, flexor digitorum superficialis, flexor hallucis brevis, flexor hallucis longus, flexor pollicis brevis, flexor pollicis longus, frontalis, gastrocnemius, gemellus inferior, gemellus superior, genioglossus, geniohyoid, gluteus maximus, gluteus medius, gluteus minimus, gracilis, hyoglossus, iliacus, inferior oblique, inferior rectus, infraspinatus, intercostals external, intercostals innermost, intercostals internal, internal oblique abdominis, interossei - dorsal of hand, interossei -dorsal of foot, interossei- palmar of hand, interossei - plantar of foot, interspinales, intertransversarii, intrinsic muscles of tongue, ishiocavemosus, lateral cricoarytenoid, lateral pterygoid, lateral rectus, latissimus dorsi, levator anguli oris, levator ani- coccygeus, levator ani - iliococcygeus, levator ani-pubococcygeus, levator ani-puborectalis, levator ani- pubovaginalis, levator labii superioris, levator labii superioris, alaeque nasi, levator palpebrae superioris, levator scapulae, levator veli palatini, levatores costarum, longus capitis, longus colli, lumbricals of foot, lumbricals of hand, masseter, medial pterygoid, medial rectus, mentalis, m. uvulae, mylohyoid, nasalis, oblique arytenoid, obliquus capitis inferior, obliquus capitis superior, obturator extemus, obturator intemus (A), obturator intemus (B), omohyoid, opponens digiti minimi (hand), opponens pollicis, orbicularis oculi, orbicularis oris, palatoglossus, palatopharyngeus, palmaris brevis, palmaris longus, pectineus, pectoralis major, pectoralis minor, peroneus brevis, peroneus longus, peroneus tertius, piriformis (A), piriformis (B), plantaris, platysma, popliteus, posterior cricoarytenoid, procerus, pronator quadratus, pronator teres, psoas major, psoas minor, pyramidalis, quadratus femoris, quadratus lumborum, quadratus plantae, rectus abdominis, rectus capitus anterior, rectus capitus lateralis, rectus capitus posterior major, rectus capitus posterior minor, rectus femoris, rhomboid major, rhomboid minor, risorius, salpingopharyngeus, sartorius, scalenus anterior, scalenus medius, scalenus minimus, scalenus posterior, semimembranosus, semitendinosus, serratus anterior, serratus posterior inferior, serratus posterior superior, soleus, sphincter ani, sphincter urethrae, splenius capitis, splenius cervicis, stapedius, sternocleidomastoid, sternohyoid, sternothyroid, styloglossus, stylohyoid, stylohyoid (anterior view), stylopharyngeus, subclavius, subcostalis, subscapularis, superficial transverse perinei, superior oblique, superior rectus, supinator, supraspinatus, temporalis, temporoparietalis, tensor fasciae lata, tensor tympani, tensor veli palatini, teres major, teres minor, thyro-arytenoid & vocalis, thyro-epiglotticus, thyrohyoid, tibialis anterior, tibialis posterior, transverse arytenoid, transversospinalis -multifidus, transversospinalis -rotatores, transversospinalis -semispinalis, transversus abdominis, transversus thoracis, trapezius, triceps, vastus intermedins, vastus lateralis, vastus medialis, zygomaticus major, or zygomaticus minor. In some embodiments, the cell is a myocyte. In some embodiments, the cell is a muscle cell. In some embodiments, the muscle cell is a skeletal muscle cell. In some embodiments, the skeletal muscle cell is a red (slow) skeletal muscle cell, a white (fast) skeletal muscle cell or an intermediate skeletal muscle cell.

[0531] Methods of the disclosure may be performed in a eukaryotic cell or cell line. In some embodiments, the eukaryotic cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the eukaryotic cell is a Human embryonic kidney 293 cells (also referred to as HEK or HEK 293) cell. Nonlimiting examples of cell lines that may be used with compositions, systems and methods of the present disclosure include C8I6I, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL-2, CIR, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calul, SW480, SW620, SK0V3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK- 52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B 16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H- 10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr -/-, COR- L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML Tl, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, and YAR. Non-limiting examples of other cells that may be used with the disclosure include immune cells, such as CART, T-cells, B-cells, NK cells, granulocytes, basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages, dendritic cells, antigen-presenting cells (APC), or adaptive cells. Non-limiting examples of cells that may be used with this disclosure also include plant cells, such as Parenchyma, sclerenchyma, collenchyma, xylem, phloem, germline (e.g., pollen). Cells from lycophytes, fems, gymnosperms, angiosperms, bryophytes, charophytes, chloropytes, rhodophytes, or glaucophytes. Non-limiting examples of cells that may be used with this disclosure also include stem cells, such as human stem cells, animal stem cells, stem cells that are not derived from human embryonic stem cells, embryonic stem cells, mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells (iPS), somatic stem cells, adult stem cells, hematopoietic stem cells, tissue-specific stem cells.

Agricultural Engineering

[0532] Compositions and methods of the disclosure may be used for agricultural engineering. For example, compositions and methods of the disclosure may be used to confer desired traits on a plant. A plant may be engineered for the desired physiological and agronomic characteristic using the present disclosure. In some embodiments, the target nucleic acid sequence comprises a nucleic acid sequence of a plant. In some embodiments, the target nucleic acid sequence comprises a genomic nucleic acid sequence of a plant cell. In some embodiments, the target nucleic acid sequence comprises a nucleic acid sequence of an organelle of a plant cell. In some embodiments, the target nucleic acid sequence comprises a nucleic acid sequence of a chloroplast of a plant cell. [0533] The plant may be a dicotyledonous plant. Non-limiting examples of orders of dicotyledonous plants include Magniolales, Illiciales, Laurales, Piperales, Aristochiales, Nymphaeales, Ranunculales, Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales, Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales, Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, Urticales, Lecythidales, Violates, Salicales, Capparales, Ericales, Diapensales, Ebenales, Primulales, Rosales, Fabales, Podostemales, Haloragales, Myrtales, Comates, Proteales, San tales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales, Sapindales, Juglandales, Geraniales, Polygalales, Umbellales, Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales, Campanulales, Rubiales, Dipsacales, and Asterales.

[0534] The plant may be a monocotyledonous plant. Non-limiting examples of orders of monocotyledonous plants include Alismatales, Hydrocharitales, Najadales, Triuridales, Commelinales, Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales, Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales, Lilliales, and Orchid ales. A plant may belong to the order, for example, Gymnospermae, Pinales, Ginkgoales, Cycadales, Araucariales, Cupressales and Gnetales.

[0535] Non-limiting examples of plants include plant crops, fruits, vegetables, grains, soy bean, com, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, fems, clubmosses, homworts, liverworts, mosses, wheat, maize, rice, millet, barley, tomato, apple, pear, strawberry, orange, acacia, carrot, potato, sugar beets, yam, lettuce, spinach, sunflower, rape seed, Arabidopsis, alfalfa, amaranth, apple, apricot, artichoke, ash tree, asparagus, avocado, banana, barley, beans, beet, birch, beech, blackberry, blueberry, broccoli, Bmssel's sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, cedar, a cereal, celery, chestnut, cherry, Chinese cabbage, citms, clementine, clover, coffee, com, cotton, cowpea, cucumber, cypress, eggplant, elm, endive, eucalyptus, fennel, figs, fir, geranium, grape, grapefruit, groundnuts, ground cherry, gum hemlock, hickory, kale, kiwifmit, kohlrabi, larch, lettuce, leek, lemon, lime, locust, pine, maidenhair, maize, mango, maple, melon, millet, mushroom, mustard, nuts, oak, oats, oil palm, okra, onion, orange, an ornamental plant or flower or tree, papaya, palm, parsley, parsnip, pea, peach, peanut, pear, peat, pepper, persimmon, pigeon pea, pine, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, safflower, sallow, soybean, spinach, spmce, squash, strawberry, sugar beet, sugarcane, sunflower, sweet potato, sweet com, tangerine, tea, tobacco, tomato, trees, triticale, turf grasses, turnips, vine, walnut, watercress, watermelon, wheat, yams, yew, and zucchini. A plant may include algae.

XL Detection of Target Nucleic Acids

Methods of Detecting a Target Nucleic Acid

[0536] Provided herein are methods of detecting target nucleic acids. Methods may comprise detecting target nucleic acids with compositions or systems described herein. Methods may comprise detecting a target nucleic acid in a sample, e.g, a cell lysate, a biological fluid, or environmental sample. Methods may comprise detecting a target nucleic acid in a cell. In some instances, methods of detecting a target nucleic acid in a sample or cell comprises contacting the sample or cell with an effector protein or a multimeric complex thereof, a guide nucleic acid, wherein at least a portion of the guide nucleic acid is complementary to at least a portion of the target nucleic acid, and a reporter nucleic acid that is cleaved in the presence of the effector protein, the guide nucleic acid, and the target nucleic acid, and detecting a signal produced by cleavage of the reporter nucleic acid, thereby detecting the target nucleic acid in the sample. In some instances, methods result in trans cleavage of the reporter nucleic acid. In some instances, methods result in cis cleavage of the reporter nucleic acid.

[0537] In some instances, methods of detecting comprise contacting a target nucleic acid, a cell comprising the target nucleic acid, or a sample comprising a target nucleic acid with an effector protein that comprises an amino acid sequence that is at least is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of sequences recited in TABLE 1. In some instances, the amino acid sequence of the effector protein is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of sequences recited in TABLE 1. In some embodiments, a reporter and/or a reporter nucleic acid comprise a non-target nucleic acid molecule that can provide a detectable signal upon cleavage by an effector protein. Examples of detectable signals and detectable moieties that generate detectable signals are provided herein.

[0538] In some embodiments, target nucleic acid comprises a nucleic acid that is selected as the nucleic acid for modification, binding, hybridization or any other activity of or interaction with a nucleic acid, protein, polypeptide, or peptide described herein. A target nucleic acid may comprise RNA, DNA, or a combination thereof. A target nucleic acid may be single-stranded (e.g. , single-stranded RNA or singlestranded DNA) or double-stranded (e.g., double-stranded DNA). The target nucleic acid may be from any organism, including, but not limited to, a bacterium, a virus, a parasite, a protozoon, a fungus, a mammal, a plant, and an insect. As another non-limiting example, the target nucleic acid may be responsible for a disease, contain a mutation (e.g., single strand polymorphism, point mutation, insertion, or deletion), be contained in an amplicon, or be uniquely identifiable from the surrounding nucleic acids (e.g., contain a unique sequence of nucleotides).

[0539] Methods may comprise contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is a reverse complementary sequence to a segment of the target nucleic acid and an effector protein that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; and assaying for a signal indicating cleavage of at least some protein-nucleic acids of a population of protein-nucleic acids, wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample. [0540] Methods may comprise contacting the sample comprising the target nucleic acid with a guide nucleic acid targeting a target nucleic acid segment, an effector protein capable of being activated when complexed with the guide nucleic acid and the target nucleic acid segment, a single stranded nucleic acid of a reporter comprising a detection moiety, wherein the nucleic acid of a reporter is capable of being cleaved by the activated effector protein, thereby generating a first detectable signal, cleaving the single stranded nucleic acid of a reporter using the effector protein that cleaves as measured by a change in color, and measuring the first detectable signal on the support medium.

[0541] Methods may comprise contacting the sample or cell with an effector protein or a multimeric complex thereof and a guide nucleic acid at a temperature of at least about 25 °C, at least about 30°C, at least about 35°C, at least about 40°C, at least about 50°C, or at least about 65°C. In some instances, the temperature is not greater than 80°C. In some instances, the temperature is about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, or about 70°C. In some instances, the temperature is about 25 °C to about 45 °C, about 35 °C to about 55 °C, or about 55 °C to about 65 °C.

[0542] In some cases, there is a threshold of detection for methods of detecting target nucleic acids. In some instances, methods are not capable of detecting target nucleic acids that are present in a sample or solution at a concentration less than or equal to 10 nM. The term "threshold of detection" is used herein to describe the minimal amount of target nucleic acid that must be present in a sample in order for detection to occur. For example, when a threshold of detection is 10 nM, then a signal can be detected when a target nucleic acid is present in the sample at a concentration of 10 nM or more.

[0543] In some cases, the threshold of detection is less than or equal to 5 nM, 1 nM, 0.5 nM, 0. 1 nM, 0.05 nM, 0.01 nM, 0.005 nM, 0.001 nM, 0.0005 nM, 0.0001 nM, 0.00005 nM, 0.00001 nM, 10 pM, 1 pM, 500 fM, 250 fM, 100 fM, 50 fM, 10 fM, 5 fM, 1 fM, 500 attomole (aM), 100 aM, 50 aM, 10 aM, or 1 aM. In some cases, the threshold of detection is in a range of from 1 aM to 1 nM, 1 aM to 500 pM, 1 aM to 200 pM, 1 aM to 100 pM, 1 aM to 10 pM, 1 aM to 1 pM, 1 aM to 500 fM, 1 aM to 100 fM, 1 aM to 1 fM, 1 aM to 500 aM, 1 aM to 100 aM, 1 aM to 50 aM, 1 aM to 10 aM, 10 aM to 1 nM, 10 aM to 500 pM, 10 aM to 200 pM, 10 aM to 100 pM, 10 aM to 10 pM, 10 aM to 1 pM, 10 aM to 500 fM, 10 aM to 100 fM, 10 aM to 1 fM, 10 aM to 500 aM, 10 aM to 100 aM, 10 aM to 50 aM, 100 aM to 1 nM, 100 aM to 500 pM, 100 pM to 200 pM, 100 aM to 100 pM, 100 aM to 10 pM, 100 aM to 1 pM, 100 aM to 500 fM, 100 aM to 100 fM, 100 aM to 1 fM, 100 aM to 500 aM, 500 aM to 1 nM, 500 aM to 500 pM, 500 aM to 200 pM, 500 aM to 100 pM, 500 aM to 10 pM, 500 aM to 1 pM, 500 aM to 500 fM, 500 aM to 100 fM, 500 aM to 1 fM, 1 fM to 1 nM, 1 fM to 500 pM, 1 fM to 200 pM, 1 fM to 100 pM, 1 fM to 10 pM, 1 fM to 1 pM, 10 fM to 1 nM, 10 fM to 500 pM, 10 fM to 200 pM, 10 fM to 100 pM, 10 fM to 10 pM, 10 fM to 1 pM, 500 fM to 1 nM, 500 fM to 500 pM, 500 fM to 200 pM, 500 fM to 100 pM, 500 fM to 10 pM, 500 fM to 1 pM, 800 fM to 1 nM, 800 fM to 500 pM, 800 fM to 200 pM, 800 fM to 100 pM, 800 fM to 10 pM, 800 fM to 1 pM, 1 pM to 1 nM, 1 pM to 500 pM, 1 pM to 200 pM, 1 pM to 100 pM, or 1 pM to 10 pM. In some cases, the threshold of detection in a range of from 800 fM to 100 pM, 1 pM to 10 pM, 10 fM to 500 fM, 10 fM to 50 fM, 50 fM to 100 fM, 100 fM to 250 fM, or 250 fM to 500 fM. In some cases, the threshold of detection is in a range of from 2 aM to 100 pM, from 20 aM to 50 pM, from 50 aM to 20 pM, from 200 aM to 5 pM, or from 500 aM to 2 pM. [0544] In some instances, the target nucleic acid is present in a cleavage reaction at a concentration of about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 pM, about 10 pM, or about 100 pM. In some instances, the target nucleic acid is present in the cleavage reaction at a concentration of from 10 nM to 20 nM, from 20 nM to 30 nM, from 30 nM to 40 nM, from 40 nM to 50 nM, from 50 nM to 60 nM, from 60 nM to 70 nM, from 70 nM to 80 nM, from 80 nM to 90 nM, from 90 nM to 100 nM, from 100 nM to 200 nM, from 200 nM to 300 nM, from 300 nM to 400 nM, from 400 nM to 500 nM, from 500 nM to 600 nM, from 600 nM to 700 nM, from 700 nM to 800 nM, from 800 nM to 900 nM, from 900 nM to 1 pM, from 1 pM to 10 pM, from 10 pM to 100 pM, from 10 nM to 100 nM, from 10 nM to 1 pM, from 10 nM to 10 pM, from 10 nM to 100 pM, from 100 nM to 1 pM, from 100 nM to 10 pM, from 100 nM to 100 pM, or from 1 pM to 100 pM. In some instances, the target nucleic acid is present in the cleavage reaction at a concentration of from 20 nM to 50 pM, from 50 nM to 20 pM, or from 200 nM to 5 pM.

[0545] In some cases, methods detect a target nucleic acid in less than 60 minutes. In some cases, methods detect a target nucleic acid in less than about 120 minutes, less than about 110 minutes, less than about 100 minutes, less than about 90 minutes, less than about 80 minutes, less than about 70 minutes, less than about 60 minutes, less than about 55 minutes, less than about 50 minutes, less than about 45 minutes, less than about 40 minutes, less than about 35 minutes, less than about 30 minutes, less than about 25 minutes, less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes, or less than about 1 minute.

[0546] Methods may comprise detecting a detectable signal within 5 minutes of contacting the sample and/or the target nucleic acid with the guide nucleic acid and/or the effector protein. In some cases, detecting occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, or 120 minutes of contacting the target nucleic acid. In some instances, detecting occurs within 1 to 120, 5 to 100, 10 to 90, 15 to 80, 20 to 60, or 30 to 45 minutes of contacting the target nucleic acid.

Amplification of a Target Nucleic Acid

[0547] Methods may comprise amplifying a target nucleic acid for detection using any of the compositions or systems described herein. Amplifying may comprise changing the temperature of the amplification reaction, also known as thermal amplification (e.g., PCR). Amplifying may be performed at essentially one temperature, also known as isothermal amplification. Amplifying may improve at least one of sensitivity, specificity, or accuracy of the detection of the target nucleic acid.

[0548] Amplifying may comprise subjecting a target nucleic acid to an amplification reaction selected from transcription mediated amplification (TMA), helicase dependent amplification (HD A), or circular helicase dependent amplification (cHDA), strand displacement amplification (SDA), recombinase polymerase amplification (RPA), loop mediated amplification (LAMP), exponential amplification reaction (EXPAR), rolling circle amplification (RCA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence based amplification (NASBA), hinge- initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), and improved multiple displacement amplification (IMDA).

[0549] In some instances, amplification of the target nucleic acid comprises modifying the sequence of the target nucleic acid. For example, amplification may be used to insert a PAM sequence into a target nucleic acid that lacks a PAM sequence. In some cases, amplification may be used to increase the homogeneity of a target nucleic acid in a sample. For example, amplification may be used to remove a nucleic acid variation that is not of interest in the target nucleic acid sequence.

[0550] Amplifying may take 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes. Amplifying may be performed at a temperature of around 20-45°C. Amplifying may be performed at a temperature of less than about 20°C, less than about 25°C, less than about 30°C, 35°C, less than about 37°C, less than about 40°C, or less than about 45°C. The nucleic acid amplification reaction may be performed at a temperature of at least about 20°C, at least about 25°C, at least about 30°C, at least about 35°C, at least about 37°C, at least about 40°C, or at least about 45°C.

XII. Methods of Treatment

[0551] Described herein are methods for treating a disease in a subject by editing a target nucleic acid associated with a gene or expression of a gene related to the disease. In some embodiments, the methods comprise methods of editing nucleic acid described herein.

[0552] In some embodiments, methods for treating a disease in a subject comprises administration of a composition(s) or component(s) of a system described herein. In some embodiments, the composition(s) or component(s) of the system comprises use of a recombinant nucleic acid (DNA or RNA), administered for the purpose to edit a nucleic acid. In some embodiments, the composition or component of the system comprises use of a vector to introduce a functional gene or transgene. In some embodiments, when describing a transgene, as used herein, refers to a nucleotide sequence that is inserted into a cell for expression of said nucleotide sequence in the cell. In some embodiments, a transgene includes (1) a nucleotide sequence that is not naturally found in the cell (e.g. , a heterologous nucleotide sequence); (2) a nucleotide sequence that is a mutant form of a nucleotide sequence naturally found in the cell into which it has been introduced; (3) a nucleotide sequence that serves to add additional copies of the same (e.g., exogenous or homologous) or a similar nucleotide sequence naturally occurring in the cell into which it has been introduced; or (4) a silent naturally occurring or homologous nucleotide sequence whose expression is induced in the cell into which it has been introduced. In some embodiments, a donor nucleic acid can comprise a transgene. In some embodiments, the cell in which transgene expression occurs can be a target cell, such as a host cell.

[0553] In some embodiments, vectors comprise nonviral vectors, including cationic polymers, cationic lipids, or bio-responsive polymers. In some embodiments, the bio-responsive polymer exploits chemical -physical properties of the endosomal environment (e.g., pH) to preferentially release the genetic material in the intracellular space. In some embodiments, vectors comprise viral vectors, including retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses. In some embodiments, the vector comprises a replication-defective viral vector, comprising an insertion of a therapeutic gene inserted in genes essential to the lytic cycle, preventing the virus from replicating and exerting cytotoxic effects. Methods of gene therapy that are applicable to the compositions and systems described herein are described in more detail in Ingusci et al., “Gene Therapy Tools for Brain Diseases”, Front. Pharmacol. 10:724 (2019), which is hereby incorporated by reference in its entirety.

[0554] In some embodiments, treating, preventing, or inhibiting disease or disorder in a subject may comprise contacting a target nucleic acid associated with a particular ailment with a composition described herein. In some aspects, the methods of treating, preventing, or inhibiting a disease or disorder may involve removing, editing, modifying, replacing, transposing, or affecting the regulation of a genomic sequence of a patient in need thereof. In some embodiments, the methods of treating, preventing, or inhibiting a disease or disorder may involve modulating gene expression.

[0555] Described herein are compositions and methods for treating a disease in a subject by editing a target nucleic acid associated with a gene or expression of a gene related to the disease. In some embodiments, methods comprise administering a composition or cell described herein to a subject. By way of non-limiting example, the disease may be a cancer, an ophthalmological disorder, a neurological disorder, a neurodegenerative disease, a blood disorder, or a metabolic disorder, or a combination thereof. The disease may be an inherited disorder, also referred to as a genetic disorder. The disease may be the result of an infection or associated with an infection. Also, by way of non-limiting example, the compositions are pharmaceutical compositions described herein.

[0556] The compositions and methods described herein may be used to treat, prevent, or inhibit a disease or syndrome in a subject. In some embodiments, the disease is a liver disease, a lung disease, an eye disease, or a muscle disease. Exemplary diseases and syndromes include but are not limited to the diseases and syndromes listed in TABLE 9.

[0557] In some embodiments, the method for treating a disease comprises modifying at least one gene associated with the disease or modifying expression of the at least one gene such that the disease is treated. [0558] In some embodiments, the disease is Alzheimer’s disease and the gene is selected from APP, BACE-1, PSD95, MAPT, PSEN1, PSEN2, and APOEa4. In some embodiments, the disease is Parkinson’s disease and the gene is selected from SNCA, GDNF, and LRRK2. In some embodiments, the disease comprises Centronuclear myopathy and the gene is DNM2. In some embodiments, the disease is Huntington's disease and the gene is HTT. In some embodiments, the disease is Alpha- 1 antitrypsin deficiency (AATD) and the gene is SERPINA1. In some embodiments, the disease is amyotrophic lateral sclerosis (ALS) and the gene is selected from SOD1, FUS, C9ORF72, ATXN2, TARDBP, and CHCHD10. In some embodiments, the disease comprises Alexander Disease and the gene is GFAP. In some embodiments, the disease comprises anaplastic large cell lymphoma and the gene is CD30. In some embodiments, the disease comprises Angelman Syndrome and the gene is UBE3A. In some embodiments, the disease comprises Calcific Aortic Stenosis and the gene is Apo(a). In some embodiments, the disease comprises CD3Z-associated primary T-cell immunodeficiency and the gene is CD3Z or CD247. In some embodiments, the disease comprises CD 18 deficiency and the gene is ITGB2. In some embodiments, the disease comprises CD40L deficiency and the gene is CD40L. In some embodiments, the disease is congenital adrenal hyperplasia and the gene is CAH1. In some embodiments, the disease comprises CNS trauma and the gene is VEGF. In some embodiments, the disease comprises coronary heart disease and the gene is selected from FGA, FGB, and FGG. In some embodiments, the disease comprises MECP2 Duplication syndrome and Rett syndrome and the gene is MECP2. In some embodiments, the disease comprises a bleeding disorder (coagulation) and the gene is FXI. In some embodiments, the disease comprises fragile X syndrome and the gene is FMRI. In some embodiments, the disease comprises Fuchs Comeal Dystrophy and the gene is selected from ZEB1, SLC4A11, and LOXHD1. In some embodiments, the disease comprises GM2 -Gangliosidoses (e.g., Tay Sachs Disease, Sandhoff disease) and the gene is selected from HEXA and HEXB. In some embodiments, the disease comprises Hearing loss disorders and the gene is DFNA36. In some embodiments, the disease is Pompe disease, including infantile onset Pompe Disease (IOPD) and late onset Pompe Disease (LOPD) and the gene is GAA. In some embodiments, the disease is Retinitis pigmentosa and the gene is selected from PDE6B, RHO, RP1, RP2, RPGR, PRPH2, IMPDH1, PRPF31, CRB1, PRPF8, TULP1, CA4, HPRPF3, ABCA4, EYS, CERKL, FSCN2, TOPORS, SNRNP200, PRCD, NR2E3, MERTK, USH2A, PROMI, KLHL7, CNGB1, TTC8, ARL6, DHDDS, BEST1, LRAT, SPARA7, CRX, CLRN1, RPE65, and WDR19. In some embodiments, the disease comprises Leber Congenital Amaurosis Type 10 and the gene is CEP290.

[0559] In some embodiments, the disease is cardiovascular disease and/or lipodystrophies and the gene is selected from ABCG5, ABCG8, AGT, ANGPTL3, APOCIII, APOA1, APOL1, ARH, CDKN2B, CFB, CXCL12, FXI, FXII, GATA-4, MIA3, MKL2, MTHFD1L, MYH7, NKX2-5, NOTCH1, PKK, PCSK9, PSRC1, SMAD3, and TTR. In some embodiments, the disease is cardiovascular disease and/or lipodystrophies and the gene is ANGPTL3. In some embodiments, the disease is cardiovascular disease and/or lipodystrophies and the gene is PCSK9. In some embodiments, the disease is cardiovascular disease and/or lipodystrophies and the gene is TTR. In some embodiments, the disease is severe hypertriglyceridemia (SHTG) and the gene is APOCIII or ANGPTL4.

[0560] In some embodiments, the disease comprises acromegaly and the gene is GHR. In some embodiments, the disease comprises acute myeloid leukemia and the gene is CD22. In some embodiments, the disease is diabetes and the gene is GCGR. In some embodiments, the disease is NAFLD/NASH and the gene is selected from HSD17B13, PSD3, GPAM, CIDEB, DGAT2 and PNPLA3. In some embodiments, the disease is NASH/cirrhosis and the gene is MARC 1.

[0561] In some embodiments, the disease is cancer and the gene is selected from STAT3, YAP1, FOXP3, AR (Prostate cancer), and IRF4 (multiple myeloma). In some embodiments, the disease is cystic fibrosis and the gene is CFTR. In some embodiments, the disease is Duchenne Muscular Dystrophy and the gene is DMD. In some embodiments, the disease is ornithine transcarbamylase deficiency and the gene is OTC.

[0562] In some embodiments, the disease is congenital adrenal hyperplasia (CAH) and the gene is CYP21A2. In some embodiments, the disease is atherosclerotic cardiovascular disease (ASCVD) and the gene is LPA. In some embodiments, the disease is hepatitis B virus infection (CHB) and the gene is HBV covalently closed circular DNA (cccDNA). In some embodiments, the disease is citrullinemia type I and the gene is ASS1. In some embodiments, the disease is citrullinemia type I and the gene is SLC25A13. In some embodiments, the disease is citrullinemia type I and the gene is ASS1. In some embodiments, the disease is arginase-1 deficiency and the gene is ARG1. In some embodiments, the disease is carbamoyl phosphate synthetase I deficiency and the gene is CPS1. In some embodiments, the disease is argininosuccinic aciduria and the gene is ASL.

[0563] In some embodiments, the disease comprises angioedema and the gene is PKK. In some embodiments, the disease comprises thalassemia and the gene is TMPRSS6. In some embodiments, the disease comprises achondroplasia and the gene is FGFR3. In some embodiments, the disease comprises Cri du chat syndrome and the gene is selected from CTNND2. In some embodiments, the disease comprises sickle cell anemia and the gene is Beta globin gene. In some embodiments, the disease comprises Alagille Syndrome and the gene is selected from JAG1 and NOTCH2. In some embodiments, the disease comprises Charcot Marie Tooth Disease and the gene is selected from PMP22 and MFN2. In some embodiments, the disease comprises Crouzon syndrome and the gene is selected from FGFR2, FGFR3, and FGFR3. In some embodiments, the disease comprises Dravet Syndrome and the gene is selected from SCN1A and SCN2A. In some embodiments, the disease comprises Emery-Dreifuss syndrome and the gene is selected from EMD, LMNA, SYNE1, SYNE2, FHL1, and TMEM43. In some embodiments, the disease comprises Factor V Leiden Thrombophilia and the gene is F5. In some embodiments, the disease is fabry disease and the gene is GLA. In some embodiments, the disease is facioscapulohumeral muscular dystrophy and the gene is FSHD 1. In some embodiments, the disease comprises Fanconi anemia and the gene is selected from FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN, FANCP, FANCS, RAD51C, and XPF. In some embodiments, the disease comprises Familial Creutzfeld-Jakob Disease and the gene is PRNP. In some embodiments, the disease comprises Familial Mediterranean Fever and the gene is MEFV. In some embodiments, the disease comprises Friedreich's ataxia and the gene is FXN. In some embodiments, the disease comprises Gaucher disease and the gene is GBA. In some embodiments, the disease comprises human papilloma virus (HPV) infection and the gene is HPV E7. In some embodiments, the disease comprises Hemochromatosis and the gene is HFE, optionally comprising a C282Y mutation.

[0564] In some embodiments, the disease comprises Hemophilia A and the gene is FVIII. In some embodiments, the disease is hereditary angioedema and the gene is SERPING1 or KLKB1. In some embodiments, the disease comprises histiocytosis and the gene is CD1. In some embodiments, the disease comprises immunodeficiency 17 and the gene is CD3D. In some embodiments, the disease comprises immunodeficiency 13 and the gene is CD4. In some embodiments, the disease comprises Common Variable Immunodeficiency and the gene is selected from CD 19 and CD81. In some embodiments, the disease comprises Joubert syndrome and the gene is selected from INPP5E, TMEM216, AHI1, NPHP1, CEP290, TMEM67, RPGRIP1L, ARL13B, CC2D2A, OFD1, TMEM138, TCTN3, ZNF423, and AMRC9. In some embodiments, the disease comprises leukocyte adhesion deficiency and the gene is CD 18. In some embodiments, the disease comprises Li-Fraumeni syndrome and the gene is TP53. In some embodiments, the disease comprises lymphoproliferative syndrome and the gene is CD27. In some embodiments, the disease comprises Lynch syndrome and the gene is selected from MSH2, MLH1, MSH6, PMS2, PMS1, TGFBR2, and MLH3. In some embodiments, the disease comprises mantle cell lymphoma and the gene is CD5. In some embodiments, the disease comprises Marfan syndrome and the gene is FBN1. In some embodiments, the disease comprises mastocytosis and the gene is CD2. In some embodiments, the disease comprises methylmalonic acidemia and the gene is selected from MMAA, MMAB, and MUT. In some embodiments, the disease is mycosis fimgoides and the gene is CD7. In some embodiments, the disease is myotonic dystrophy and the gene is selected from CNBP and DMPK. In some embodiments, the disease comprises neurofibromatosis and the gene is selected from NF1, and NF2. In some embodiments, the disease comprises osteogenesis imperfecta and the gene is selected from COL1A1, COL1A2, and IFITM5. In some embodiments, the disease is non-small cell lung cancer and the gene is selected from KRAS, EGFR, ALK, METexl4, BRAF V600E, ROS1, RET, and NTRK. In some embodiments, the disease comprises Peutz-Jeghers syndrome and the gene is STK11. In some embodiments, the disease comprises polycystic kidney disease and the gene is selected from PKD1 and PKD2. In some embodiments, the disease comprises Severe Combined Immune Deficiency and the gene is selected from IL7R, RAG1, and JAK3. In some embodiments, the disease comprises PRKAG2 cardiac syndrome and the gene is PRKAG2. In some embodiments, the disease comprises Spinocerebellar ataxia and the gene is selected from ATXN1, ATXN2, ATXN3, PLEKHG4, SPTBN2, CACNA1A, ATXN7, ATXN8OS, ATXN10, TTBK2, PPP2R2B, KCNC3, PRKCG, ITPR1, TBP, KCND3, and FGF14. In some embodiments, the disease is thrombophilia due to antithrombin III deficiency and the gene is SERPINC1. In some embodiments the disease is spinal muscular atrophy and the gene is SMN1. In some embodiments, the disease comprises Usher Syndrome and the gene is selected from MY07A, USH1C, CDH23, PCDH15, USH1G, USH2A, GPR98, DFNB31, and CLRN1. In some embodiments, the disease comprises von Willebrand disease and the gene is VWF. In some embodiments, the disease comprises Waardenburg syndrome and the gene is selected from PAX3, MITF, WS2B, WS2C, SNAI2, EDNRB, EDN3, and SOXIO. In some embodiments, the disease comprises Wiskott-Aldrich Syndrome and the gene is WAS. In some embodiments, the disease comprises von Hippel-Lindau disease and the gene is VHL. In some embodiments, the disease comprises Wilson disease and the gene is ATP7B. In some embodiments, the disease comprises Zellweger syndrome and the gene is selected from PEX1, PEX2, PEX3, PEX5, PEX6, PEX10, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26. In some embodiments, the disease comprises infantile myofibromatosis and the gene is CD34. In some embodiments, the disease comprises platelet glycoprotein IV deficiency and the gene is CD36. In some embodiments, the disease comprises immunodeficiency with hyper-IgM type 3 and the gene is CD40. In some embodiments, the disease comprises hemolytic uremic syndrome and the gene is CD46. In some embodiments, the disease comprises complement hyperactivation, angiopathic thrombosis, or protein-losing enteropathy and the gene is CD55. In some embodiments, the disease comprises hemolytic anemia and the gene is CD59. In some embodiments, the disease comprises calcification of joints and arteries and the gene is CD73. In some embodiments, the disease comprises immunoglobulin alpha deficiency and the gene is CD79A. In some embodiments, the disease comprises C syndrome and the gene is CD96. In some embodiments, the disease comprises hairy cell leukemia and the gene is CD 123. In some embodiments, the disease comprises histiocytic sarcoma and the gene is CD 163. In some embodiments, the disease comprises autosomal dominant deafness and the gene is CD 164. In some embodiments, the disease comprises immunodeficiency 25 and the gene is CD247. In some embodiments, the disease comprises methymalonic acidemia due to transcobalamin receptor defect and the gene is CD320.

[0565] In some embodiments, treatment of a disease comprises administration of a gene therapy. “Gene therapy”, as used herein, comprises use of a recombinant nucleic acid (DNA or RNA), administered for the purpose to adjust, repair, replace, add, or remove a gene sequence. In some embodiments, a gene therapy comprises use of a vector to introduce a functional gene or transgene. In some embodiments, vectors comprise nonviral vectors, including cationic polymers, cationic lipids, or bio-responsive polymers. In some embodiments, the bio-responsive polymer exploits chemical-physical properties of the endosomal environment (e.g., pH) to preferentially release the genetic material in the intracellular space. In some embodiments, vectors comprise viral vectors, including retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses. In some embodiments, the vector comprises a replication-defective viral vector, comprising an insertion of a therapeutic gene inserted in genes essential to the lytic cycle, preventing the virus from replicating and exerting cytotoxic effects. Methods of gene therapy are described in more detail in Ingusci et al., “Gene Therapy Tools for Brain Diseases”, Front. Pharmacol. 10:724 (2019) which is hereby incorporated by reference in its entirety. In some embodiments, the disease is a genetic disease. The “genetic disease”, as used herein, refers to a disease, disorder, condition, or syndrome caused by one or more mutations in the DNA of an organism. Mutations can be due to several different cellular mechanisms, including, but not limited to, an error in DNA replication, recombination, or repair, or due to environmental factors. A genetic disease comprises, in some embodiments, a single gene disorder, a chromosome disorder, or a multifactorial disorder.

Cancer

[0566] In some embodiments, compositions, systems or methods described herein edit at least one gene associated with a cancer or the expression thereof. Non-limiting examples of cancers include: acute lymphoblastic leukemia; acute lymphoblastic lymphoma; acute lymphocytic leukemia; acute myelogenous leukemia; acute myeloid leukemia (adult / childhood); adrenocortical carcinoma; anal cancer; appendix cancer; astrocytoma; atypical teratoid/rhabdoid tumor; basal-cell carcinoma; bile duct cancer; bladder cancer; bone osteosarcoma; brain cancer; brain tumor,; brainstem glioma; breast cancer; bronchial adenoma, carcinoid, or tumor; Burkitt lymphoma; carcinomacervical cancer; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloid leukemia; colon cancer; colorectal cancer; emphysema; endometrial cancer; esophageal cancer; Ewing sarcoma; gallbladder cancer; gastric (stomach) cancer; gastrointestinal tumor; gliomahairy cell leukemia; head and neck cancer; liver cancer; Hodgkin’s lymphoma; hypopharyngeal cancer; Kaposi Sarcoma; kidney cancer lip and oral cavity cancer; liposarcoma; lung cancer, non-small cell lung cancer; Waldenstrom; melanoma; mesotheliomamyelogenous leukemia; myeloid leukemia; myeloma; nasopharyngeal carcinoma; neuroblastoma; non-Hodgkin’s lymphoma; ovarian cancer; pancreatic cancer; pineal cancer; pituitary tumor; prostate cancer; rectal cancer; renal cell carcinomaretinoblastoma; spinal cord tumor; squamous cell carcinoma; squamous neck cancer; T-cell lymphoma, cutaneous (Mycosis Fungoides and Sezary syndrome); testicular cancer; throat cancer; thyroid cancer; urethral cancer; uterine cancervaginal cancer; and Wilms Tumor. In some embodiments, the cancer is a solid cancer (i.e., a tumor). In some embodiments, the cancer is selected from a blood cell cancer, a leukemia, and a lymphoma. The cancer can be a leukemia, such as, by way of non-limiting example, acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), and chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is any one of colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, bladder cancer, cancer of the kidney or ureter, lung cancer, non-small cell lung cancer, cancer of the small intestine, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, brain cancer (e.g., glioblastoma), cancer of the head or neck, melanoma, uterine cancer, ovarian cancer, breast cancer, testicular cancer, cervical cancer, stomach cancer, Hodgkin's Disease, non-Hodgkin's lymphoma, and thyroid cancer. [0567] In some embodiments, compositions, systems or methods described herein edit at least one mutation in a target nucleic acid, wherein the at least one mutation is associated with cancer or causative of cancer. In some embodiments, the target nucleic acid comprises a gene associated with cancer, a gene whose overexpression is associated with cancer, a tumor suppressor gene, an oncogene, a checkpoint inhibitor gene, a gene associated with cellular growth, a gene associated with cellular metabolism, a gene associated with cell cycle, combinations thereof, or portions thereof. Non-limiting examples of genes comprising a mutation associated with cancer are ABL, ACE, AF4/HRX, AKT-2, ALK, ALK/NPM, AML1, AML1/MTG8, APC, ATM, AXIN2, AXL, BAP1, BARD1, BCL-2, BCL-3, BCL- 6, BCR/ABL, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, c-MYC, CASR, CCR5, CDC73, CDH1, CDK4, CDKN1B, CDKN1C, CDKN2A, CEBPA, CHEK2, CREBBP, CTNNA1, DBL, DEK/CAN, DICER1, DIS3L2, E2A/PBX1, EGFR, ENL/HRX, EPCAM, ERG/TLS, ERBB, ERBB-2, ETS-1, EWS/FLI-1, FH, FKRP, FLCN, FMS, FOS, FPS, GATA2, GCG, GLI, GPC3, GPGSP, GREM1, HER2/neu, H0X11, HOXB13, HRAS, HST, IL-3, INT-2, JAK1, JUN, KIT, KS3, K-SAM, LBC, LCK, LM01, LM02, L-MYC, LYL-1, LYT-10, LYT-10/Cal, MAS, MAX, MDM-2, MEN1, MET, MITF, MLH1, MLL, MOS, MSH1, MSH2, MSH3, MSH6, MTG8/AML1, MUTYH, MYB, MYH1 1/CBFB, NBN, NEU, NF1, NF2, N-MYC, NTHL1, OST, PALB2, PAX-5, PBX1/E2A, PCDC1, PDGFRA, PHOX2B, PIM-1, PMS2, POLDI, POLE, POTI, PPARG, PRAD-1, PRKAR1A, PTCHI, PTEN, RAD50, RAD51C, RAD51D, RAF, RAR/PML, RAS-H, RAS-K, RAS-N, RBI, RECQL4, REL/NRG, RET, RH0M1, RH0M2, ROS, RUNX1, SDHA, SDHAF, SDHAF2, SDHB, SDHC, SDHD, SET/CAN, SIS, SKI, SMAD4, SMARCA4, SMARCB1, SMARCE1, SRC, STK11, SUFU, TALI, TAL2, TAN-1, TIAM1, TERC, TERT, TIMP3, TMEM127, TNF, TP53, TRAC, TSC1, TSC2, TRK, VHL, WRN, and WT1. Non-limiting examples of oncogenes are KRAS, NRAS, BRAF, MYC, CTNNB1, and EGFR. In some embodiments, the oncogene is a gene that encodes a cyclin dependent kinase (CDK). Non-limiting examples of CDKs are Cdkl, Cdk4, Cdk5, Cdk7, Cdk8, Cdk9, Cdkl 1 and CDK20. Non-limiting examples of tumor suppressor genes are TP53, RBI, and PTEN.

Infections

[0568] In some embodiments, compositions, systems or methods described herein treats an infection in a subject. In some embodiments, the infections are caused by a pathogen (e.g., bacteria, viruses, fungi, and parasites). In some embodiments, compositions, systems or methods described herein modifies a target nucleic acid associated with the pathogen or parasite causing the infection. In some embodiments, the target nucleic acid may be in the pathogen or parasite itself or in a cell, tissue or organ of the subject that the pathogen or parasite infects. In some embodiments, the methods described herein include treating an infection caused by one or more bacterial pathogens. Non-limiting examples of bacterial pathogens include Acholeplasma laidlawii, Brucella abortus, Chlamydia psittaci, Chlamydia trachomatis, Cryptococcus neoformans, Escherichia coli, Legionella pneumophila, Lyme disease spirochetes, methicillin-resistant Staphylococcus aureus, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma arginini, Mycoplasma arthritidis, Mycoplasma genitalium, Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma pneumoniae, Mycoplasma salivarium, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Pseudomonas aeruginosa, sexually transmitted infection, Streptococcus agalactiae, Streptococcus pyogenes, and Treponema pallidum.

[0569] In some embodiments, compositions, systems or methods described herein treats an infection caused by one or more viral pathogens. Non-limiting examples of viral pathogens include adenovirus, blue tongue virus, chikungunya, coronavirus (e.g, SARS-CoV-2), cytomegalovirus, Dengue virus, Ebola, Epstein-Barr virus, feline leukemia virus, Hemophilus influenzae B, Hepatitis virus A, Hepatitis virus B, Hepatitis virus C, herpes simplex virus I, herpes simplex virus II, human papillomavirus (HPV) including HPV 16 and HPV 18, human serum parvo-like virus, human T-cell leukemia viruses, immunodeficiency virus (e.g, HIV), influenza virus, lymphocytic choriomeningitis virus, measles virus, mouse mammary tumor virus, mumps virus, murine leukemia virus, polio virus, rabies virus, Reovirus, respiratory syncytial virus (RSV), rubella vims, Sendai vims, simian vims 40, Sindbis vims, varicella-zoster vims, vesicular stomatitis vims, wart vims, West Nile vims, yellow fever vims, or any combination thereof.

[0570] In some embodiments, compositions, systems or methods described herein treats an infection caused by one or more parasites. Non-limiting examples of parasites include helminths, annelids, platyhelminthes, nematodes, and thorny-headed worms. In some embodiments, parasitic pathogens comprise, without limitation, Babesia bovis, Echinococcus granulosus, Eimeria tenella, Leishmania tropica, Mesocestoides corti, Onchocerca volvulus, Plasmodium falcipamm, Plasmodium vivax, Schistosoma japonicum, Schistosoma mansoni, Schistosoma spp., Taenia hydatigena, Taenia ovis, Taenia saginata, Theileria parva, Toxoplasma gondii, Toxoplasma spp., Trichinella spiralis, Trichomonas vaginalis, Trypanosoma brucei, Trypanosoma cruzi, Trypanosoma rangeli, Trypanosoma rhodesiense, Balantidium coli, Entamoeba histolytica, Giardia spp., Isospora spp., Trichomonas spp., or any combination thereof.

[0571] The target nucleic acid may be from any organism, including, but not limited to, a bacterium, a virus, a parasite, a protozoon, a fungus, a mammal, a plant, and an insect. As another non-limiting example, the target nucleic acid may be responsible for a disease, contain a mutation (e.g. , single strand polymorphism, point mutation, insertion, or deletion), be contained in an amplicon, or be uniquely identifiable from the surrounding nucleic acids (e.g. , contain a unique sequence of nucleotides). In some embodiments, the target nucleic acid is from a bacteria. In some embodiments, the bacteria is Acholeplasma laidlawii, Brucella abortus, Chlamydia psittaci, Chlamydia trachomatis, Cryptococcus neoformans, Escherichia coli, Legionella pneumophila, Lyme disease spirochetes, methicillin-resistant Staphylococcus aureus, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma arginini, Mycoplasma arthritidis, Mycoplasma genitalium, Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma pneumoniae, Mycoplasma salivarium, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Pseudomonas aeruginosa, Streptococcus agalactiae, Streptococcus pyogenes, Treponema pallidum, or any combination thereof.

[0572] In some embodiments, the target nucleic acid is from a virus. In some embodiments, the virus is adenovirus, blue tongue virus, chikungunya, coronavirus (e.g. SARS-CoV-2), cytomegalovirus, Dengue virus, Ebola, Epstein-Barr virus, feline leukemia virus, Hemophilus influenzae B, Hepatitis Virus A, Hepatitis Virus B, Hepatitis Virus C, herpes simplex virus I, herpes simplex virus II, human papillomavirus (HPV) including HPV16 and HPV18, human serum parvo-like virus, human T-cell leukemia viruses, immunodeficiency virus (e.g. HIV), influenza virus, lymphocytic choriomeningitis virus, measles virus, mouse mammary tumor virus, mumps virus, murine leukemia virus, polio virus, rabies virus, Reovirus, respiratory syncytial virus (RSV), rubella virus, Sendai virus, simian virus 40, Sindbis virus, varicella-zoster virus, vesicular stomatitis virus, wart virus, West Nile virus, yellow fever virus, or any combination thereof. In some embodiments, the target nucleic acid comprises a portion of a nucleic acid that is associated with a hemorrhagic fever.

[0573] In some embodiments, the target nucleic acid is from a parasite. In some embodiments, the parasite is a helminth, an annelid, a platyhelminth, a nematode, or a thorny-headed worms. In some embodiments, the parasite is Babesia bovis, Echinococcus granulosus, Eimeria tenella, Leishmania tropica, Mesocestoides corti, Onchocerca volvulus, Plasmodium falciparum, Plasmodium vivax, Schistosoma japonicum, Schistosoma mansoni, Schistosoma spp., Taenia hydatigena, Taenia ovis, Taenia saginata, Theileria parva, Toxoplasma gondii, Toxoplasma spp., Trichinella spiralis, Trichomonas vaginalis, Trypanosoma brucei, Trypanosoma cruzi, Trypanosoma rangeli, Trypanosoma rhodesiense, Balantidium coli, Entamoeba histolytica, Giardia spp., Isospora spp., Trichomonas spp., or any combination thereof.

SEQUENCES AND TABLES

[0574] TABLE 1 provides illustrative amino acid sequences of effector proteins that are useful in the compositions, systems and methods described herein.

TABLE 1. EXEMPLARY AMINO ACID SEQUENCE(S) OF EFFECTOR PROTEIN(S)

[0575] TABLE 2 provides illustrative sequences of exemplary heterologous polypeptide modifications of effector protein(s) that are useful in the compositions, systems and methods described herein.

TABLE 2. SEQUENCES OF EXEMPLARY HETEROLOGOUS POLYPEPTIDE MODIFICATIONS OF EFFECTOR PROTEIN(S)

[0576] TABLE 3 provides illustrative PAM sequences that are useful in the compositions, systems and methods described herein.

TABLE 2. EXEMPLARY PAM SEQUENCES

[0577] TABLE 4 provides illustrative repeat sequences for use in guide nucleic acids that are useful in the compositions, systems and methods described herein.

TABLE 4. EXEMPLARY REPEAT SEQUENCES FOR USE IN GUIDE NUCLEIC ACIDS

[0578] TABLE 5 provides illustrative handle sequences for use in guide nucleic acids that are useful in the compositions, systems and methods described herein.

TABLE 5. EXEMPLARY HANDLE SEQUENCES FOR USE IN GUIDE NUCLEIC ACIDS

[0579] TABLE 6 provides illustrative crRNA sequences that are useful in the compositions, systems and methods described herein.

TABLE 6. EXEMPLARY crRNA SEQUENCES FOR USE IN SINGLE GUIDE SYSTEMS

[0580] TABLE 7 provides illustrative sgRNA sequences that are useful in the compositions, systems and methods described herein.

TABLE 7. EXEMPLARY SGRNA SEQUENCES FOR USE IN SINGLE GUIDE SYSTEMS

[0581] TABLE 8 provides illustrative target nucleic acids that are useful in the compositions, systems and methods described herein.

TABLE 8. EXEMPLARY TARGET NUCLEIC ACIDS

[0582] TABLE 9 provides illustrative diseases and syndromes for compositions, systems and methods described herein.

TABLE 9. DISEASES AND SYNDROMES

[0583] TABLE 10 provides exemplary compositions comprising effector proteins.

TABLE 10. EXEMPLARY GUIDE NUCLEIC ACIDS & GUIDE NUCLEIC ACID COMPONENTS

EXAMPLES

[0584] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1. PAM Screening for Effector Proteins

[0585] Effector proteins and guide RNA combinations represented in TABLE 11 were screened by an in vitro enrichment (IVE) assay to determine PAM recognition by each effector protein-guide RNA complex. TABLE 11 shows the components of each effector protein-guide RNA complex assayed for PAM recognition. The amino acid sequences of the effector protein names in the second column of the table are shown in TABLE 1 herein. The nucleotide sequences of the guide RNAs (crRNA or sgRNA) or guide RNA components are shown in TABLE 10 herein. Where crRNA and tracrRNA are indicated, these separate components were used in combination with the respective effector protein. Where sgRNA is indicated, this was the sole polynucleotide used with the effector protein. Briefly, effector proteins were complexed with corresponding guide RNAs for 15 minutes at 37°C. The complexes were added to an IVE reaction mix. IVE reactions were carried out in lx Cutsmart buffer (New England Biolabs), using 10 pl of RNP in 100 pl reactions with 1,000 ng of a plasmid library containing a 7N PAM sequence 5’ of the target (protospacer) sequence. The reactions were carried out for 15 minutes at 25°C, followed by 45 minutes at 37°C and then 15 minutes at 45°C. Reactions were terminated with 1 pl of proteinase K and 5 pl of 500 mM EDTA for 30 minutes at 37°C. Next generation sequencing was performed on cut sequences to identify enriched PAMs. Cis cleavage was observed with all complexes in TABLE 11.

[0586] FIG. 1 shows the composition of the sequences derived from libraries digested with complexes comprising the denoted effector proteins. As shown in FIG. 1, examination of the position frequency matrix (PFM) derived WebLogos revealed the presence of enriched 5’ PAM consensus sequences for the various effector proteins. These WebLogos are representative of the WebLogos obtained with all guide RNAs shown in TABLE 11 and their respective effector protein.

TABLE 11. Effector Protein-Guide RNA complexes and PAMs Providing Cleavage of Nucleic Acids

*SEQ ID NO: 51 is functional with SEQ ID NO: 52 or 53

Example 2. Mammalian IVE Screening

[0587] Effector proteins, 288574, 294080, 21442, and 293378 (sequences provided in TABLE 1 above) were produced by transfecting seeded HEK293T cells (150,000 cells/ml density and seeded in 96 well plate at 30,000 cells per well and grown overnight prior to transfection) with complexed 300 ng of plasmid DNA and 0.6 pl Trans-iT reagent, both diluted with Opti-MEM media. Transfected cells were incubated at 37°C, 20% O2, 5% CO2 for three days. After three days of growth, the transfected cells were harvested and lysed with lysis buffer (1% Triton-100, 150 mM KC1, 20 mM HEPES pH 7.5, 5 mM MgCb. ImM DTT, 5% Glycerol). Cell lysates were complexed with 50 nM of indicated RNAs. The complexes were added to an IVE reaction mix. IVE reactions were carried out in lx Cutsmart buffer (New England Biolabs), using 10 pl of RNP in 100 pl reactions with 1,000 ng of a plasmid library containing a 7N PAM sequence 5’ of the target (protospacer) sequence. The reactions were carried out for 15 minutes at 25 °C, followed by 45 minutes at 37°C and then 15 minutes at 45 °C. Reactions were terminated with 1 pl of proteinase K and 5 pl of 500 mM EDTA for 30 minutes at 37°C. Next generation sequencing was performed on cut sequences to identify enriched PAMs. Cis cleavage was observed with all complexes in TABLE 12.

TABLE 12. Effector Protein-Guide RNA complexes and PAMs Providing Cleavage of Nucleic Acids

[0588] FIG. 2 shows the composition of the sequences derived from libraries digested with complexes comprising the denoted effector proteins. As shown in FIG. 2, examination of the position frequency matrix (PFM) derived WebLogos revealed the presence of enriched 5 ’ PAM consensus sequences for the various effector proteins. These WebLogos are representative of the WebLogos obtained with all guide RNAs shown in TABLE 12 and their respective effector protein.

Example 3. Detecting Target Nucleic Acids

[0589] Effector proteins are tested for trans cleavage. Briefly, partially purified (e.g., nickel-NTA purified) effector proteins are incubated with crRNA and tracrRNA, wherein the crRNA and the tracrRNA function as unlinked, independent molecule, or crRNA linked to a tracrRNA, wherein the wherein the crRNA and the tracrRNA function as a single, linked molecule (or an sgRNA) in a trans cleavage buffer (e.g., 20 mM Tricine, 15 mM MgC12, 0.2 mg/ml BSA, ImM TCEP (pH 9 at 37°C)) at room temperature for about 10 to about 30 minutes, followed by addition of a target nucleic acid to produce effector-protein guide complexes. Trans cleavage activity is detected by fluorescence signal upon cleavage of a fluorophore -quencher reporter. Dilutions of the effector-protein guide complexes are performed, and the assay repeated at various concentrations of the effector-protein guide complexes. Example 4. Effector proteins edit genomic DNA in mammalian cells

[0590] Effector proteins are tested for their ability to produce indels in a mammalian cell line (e.g., HEK293T cells). Briefly, a plasmid encoding the effector proteins and a guide RNA are delivered by lipofection to the mammalian cells. This is performed with a variety of guide RNAs targeting several loci adjacent to biochemically determined PAM sequences. Indels in the loci are detected by next generation sequencing of PCR amplicons at the targeted loci and indel percentage is calculated as the fraction of sequencing reads containing insertions or deletions relative to an unedited reference sequence. “No plasmid” and Cas9 are included as negative and positive controls, respectively.

Example 5. Base Editing

[0591] A nucleic acid vector encoding a fusion protein is constructed for base editing. The fusion protein comprises a catalytically inactive variant of an effector protein fused to a deaminase. The fusion protein and at least one guide nucleic acid is tested for its ability to edit a target sequence in eukaryotic cells. Cells are transfected with the nucleic acid vector and guide nucleic acid. After sufficient incubation, DNA is extracted from the transfected cells. Target sequences are PCR amplified and sequenced by NGS and MiSeq. The presence of base modifications are analyzed from sequencing data. Results are recorded as a change in % base call relative to the negative control.

Example 6. Activation of Gene Expression with Cas Effector Fusion Polypeptide

[0592] A single stranded reporter nucleic acid encoding a fluorescent protein (e.g., enhanced green fluorescent protein (EGFP)) and a eukaryotic promoter is generated with a target sequence that is known to be recognized by complexes of effector proteins disclosed herein and corresponding guide nucleic acids. A nucleic acid vector encoding the Cas effector fused to a transcriptional activator; a guide nucleic acid; and the single stranded reporter nucleic acid encoding EGFP are introduced to eukaryotic cells via lipofection and EGFP expression is quantified by flow cytometry. Relative amounts of RNA, indicative of relative gene expression, are quantified with RT-qPCR.

Example 7. Reduction of Gene Expression with Cas Effector Fusion Polypeptide

[0593] A single stranded reporter nucleic acid encoding a fluorescent protein (e.g., enhanced green fluorescent protein (EGFP)) and a pSV40 promoter that drives constitutive expression of EGFP is generated with a target sequence that is known to be recognized by complexes of effector proteins disclosed herein and corresponding guide nucleic acids. A nucleic acid vector encoding the Cas effector fused to a transcriptional repressor; a guide nucleic acid; and the single stranded reporter nucleic acid encoding EGFP are introduced to eukaryotic cells via lipofection and EGFP expression is quantified by flow cytometry. Relative amounts of RNA, indicative of relative gene expression, are quantified with RT-qPCR. Example 8. Generating a Catalytically Inactive Variant of a CRISPR Cas Effector Protein

[0594] Extensive work has been done to evaluate the overall domain structure of the CRISPR Cas enzymes in the last decade. These data can be an effective reference when trying to identify a catalytic residue of a Cas nuclease. By selecting the residue of a Cas nuclease of interest that aligns at the same relative location as the catalytic residue of a known nuclease when the Cas nuclease and known nuclease are aligned for maximal sequence identity, one can identify the catalytic residue of the Cas nuclease.

[0595] Sequence or structural analogs of a Cas nuclease provide an additional or supplemental way to predict the catalytic residues of the novel Cas nuclease relative to the previous description in this Example. Catalytic residues are usually highly conserved and can be identified in this manner.

[0596] Alternatively, or additionally to the description already provided in this Example, computational software may be used to predict the structure of a Cas nuclease.

Example 9. PAM Screening for Effector Proteins

[0597] Effector proteins as set forth by SEQ ID NOs: 87-97 in TABLE 1 were produced by transfecting seeded HEK293T cells (150,000 cells/ml density and seeded in 96 well plate at 30,000 cells per well and grown overnight prior to transfection) with complexed 300 ng of plasmid DNA and 0.6 pl Trans-iT reagent, both diluted with Opti-MEM media.

[0598] Transfected cells were incubated at 37°C, 20% O2, 5% CO2 for three days. After three days of growth, the transfected cells were harvested and lysed with lysis buffer (1% Triton- 100, 150 mM KC1, 20 mM HEPES pH 7.5, 5 mM MgCT. 1 mM DTT, 5% Glycerol). Cell lysates were complexed with 50 nM each of a crRNA and atracrRNA. The complexes were added to an IVE reaction mix. IVE reactions were carried out in lx Cutsmart buffer (New England Biolabs), using 10 pl of RNP in 100 pl reactions with 1,000 ng of a plasmid library containing a 7N PAM sequence 5’ of the target (protospacer) sequence. The reactions were carried out for 15 minutes at 25 °C, followed by 45 minutes at 37°C and then 15 minutes at 45 °C. Reactions were terminated with 1 pl of proteinase K and 5 pl of 500 mM EDTA for 30 minutes at 37°C.

[0599] Next generation sequencing was performed on cut sequences to identify enriched PAMs. Frequency of nucleotides at each PAM position was independently calculated using a position frequency matrix (PFM) and plotted as a WebLogo (see FIG. 3). Additional analysis resulted in a description of PAMs that are likely recognized by corresponding effector proteins. The Ipc PAM is evaluated with greater stringency than the 5pc PAM. In general, the Ipc PAM may represent the preferred PAM of the effector protein and the 5pc PAM may represent additional PAMs that are recognized by the effector protein. Cis cleavage was observed with all complexes in TABLE 13. TABLE 13. Effector Protein-Guide RNA complexes and PAMs Providing Cleavage of Nucleic Acids

Claims

CLAIMS What is claimed is:

1. A composition comprising: i) an effector protein, or a nucleic acid encoding an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of sequences recited in TABLE 1; and ii) a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid.

2. A composition comprising: i) an effector protein, or a nucleic acid encoding an effector protein, wherein the amino acid sequence of the effector protein is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of sequences recited in TABLE 1; and ii) a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid.

3. A composition comprising: i) an effector protein, or a nucleic acid encoding an effector protein, wherein the effector protein comprises about 100, about 120, about 140, about 160, about 180, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 340, about 360, about 380, about 400, about 420, about 440, about 460, about 480, about 500, about 520, about 540, about 560, about 580, about 600, or about 620 contiguous amino acids of any one of sequences recited in TABLE 1; and ii) a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid.

4. A composition comprising: i) an effector protein, or a nucleic acid encoding an effector protein, wherein the effector protein comprises the amino acid sequence located at positions 1-100, 150-250, 101-200, 250-350, 201-300, 350-450, 301-400, 350-450, 401-500, 450-550, 501-600, or 550-615 of any one of sequences recited in TABLE 1; and ii) a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid.

5. A composition comprising: i) an effector protein, or a nucleic acid encoding an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to an equal length portion of any one of sequences recited in TABLE 1, wherein the portion of the sequence is about 30%, about 40% about 50%, about 60%, about 70%, about 80%, or about 90% of any one of sequences recited in TABLE 1; and ii) a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid.

6. The composition of any one of claims 1-5, wherein at least a portion of the guide nucleic acid binds the effector protein. The composition of claim 6, wherein the portion of the guide nucleic acid that is bound by the effector protein comprises at least 10, at least 15, at least 20, at least 25, or at least 30 contiguous nucleotides of any one of sequences recited in TABLE 4. The composition of claim 6, wherein the portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to an equal length portion of any one of sequences recited in TABLE 4. The composition of any one of claims 1-8, wherein the guide nucleic acid comprises a sequence that hybridizes to a target sequence of a target nucleic acid, and wherein the target nucleic acid comprises a protospacer adjacent motif (PAM) selected from any one of sequences recited in TABLE 3 The composition of claim 9, wherein the PAM is located within 20, 40, 60, 80, or 100 nucleotides of the 5’ end of the target sequence. The composition of any one of claims 1-10, wherein the guide nucleic acid comprises a first sequence and a second sequence, wherein the first sequence is heterologous with the second sequence. The composition of claim 11, wherein the first sequence comprises at least five nucleotides and the second sequence comprises at least five nucleotides. The composition of any one of claims 1-12, wherein at least one of the effector protein, the guide nucleic acid, and the combination thereof, are not naturally occurring. The composition of any one of claims 1-13, wherein at least one of the effector protein and the guide nucleic acid is recombinant or engineered. The composition of any one of claims 1-14, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to any one of sequences recited in TABLE 4, TABLE 5, TABLE 6, or TABLE 7 The composition of any one of claims 1-14, wherein the guide nucleic acid comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 contiguous nucleotides of any one of sequences recited in TABLE 4, TABLE 5, TABLE 6, or TABLE 7. The composition of any one of claims 1-14, wherein the guide nucleic acid comprises at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, or at least 220 contiguous nucleotides of any one of sequences recited in TABLE 4, TABLE 5, TABLE 6, or TABLE 7 The composition of any one of claims 1-17, wherein the guide nucleic acid comprises a crRNA. The composition of claim 18, wherein the guide nucleic acid comprises an intermediary RNA. The composition of any one of claims 1-19, wherein the crRNA is covalently linked to the intermediary RNA. The composition of any one of claims 1-17, wherein the guide nucleic acid comprises a sgRNA. The composition of claim 21, wherein the sgRNA comprises a handle sequence. The composition of claim 22, wherein the handle sequence comprises the intermediary RNA. The composition of any one of claims 1-18, wherein the guide nucleic acid comprises a tracrRNA. The composition of any one of claims 1-18 and 21-23, wherein the guide nucleic acid does not comprise a tracrRNA. The composition of any one of claims 1-18, 21-22 and 24, wherein the guide nucleic acid comprises a crRNA covalently linked to a tracrRNA. The composition of any one of claims 1-26, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 1, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 75. The composition of claim 27, wherein the PAM is of SEQ ID NO: 10. The composition of claim 26 or 27, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 47. The composition of claim 29, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 48. The composition of claim 26 or 27, wherein the guide nucleic acid is sgRNA, and wherein the sgRNA is at least 90% identical to SEQ ID NO: 49 or 50. The composition of claim 28, wherein the guide nucleic acid comprises at least one sequence that is at least 90% identical to a sequence selected from SEQ ID NOS: 19-22. The composition of claim 32, comprising a crRNA and a tracrRNA, wherein the crRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 19, and the tracrRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 20. The composition of claim 32, comprising a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is at least 95% or 100% identical to SEQ ID NO: 19, and the nucleotide sequence of the tracrRNA is at least 95% or 100% identical to SEQ ID NO: 20. The composition of any one of claims 1-26, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 2, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of sequence selected from SEQ ID NO: 76 - 79. The composition of claim 35, wherein the PAM is of SEQ ID NO: 11. The composition of claim 35 or 36, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 51. The composition of claim 37, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 52 or 53. The composition of claim 35 or 36, wherein the guide nucleic acid is sgRNA, and wherein the sgRNA is at least 90% identical to any one of SEQ ID NO: 54-56. The composition of claim 36, wherein the guide nucleic acid comprises at least one sequence that is at least 90% identical to a sequence selected from SEQ ID NOS: 23-28. The composition of claim 40, comprising a crRNA and a tracrRNA, wherein the crRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 23, and the tracrRNA comprises a nucleotide sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 24 and SEQ ID NO: 25 The composition of claim 40, comprising a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is at least 95% or 100% identical to SEQ ID NO: 23, and the nucleotide sequence of the tracrRNA is at least 95% or 100% identical to a sequence selected from SEQ ID NO: 24 and SEQ ID NO: 25 The composition of any one of claims 1-26, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 3, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 80. The composition of claim 43, wherein the PAM is of SEQ ID NO: 12. The composition of claim 43 or 44, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 57. The composition of claim 45, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 58. The composition of claim 44, wherein the guide nucleic acid comprises at least one sequence that is at least 90% identical to a sequence selected from SEQ ID NOS: 29 and 30. The composition of claim 47, comprising a crRNA and a tracrRNA, wherein the crRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 29, and the tracrRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 30. The composition of claim 47, comprising a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is at least 95% or 100% identical to SEQ ID NO: 29, and the nucleotide sequence of the tracrRNA is at least 95% or 100% identical to SEQ ID NO: 30. The composition of any one of claims 1-26, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 4, and wherein a portion of the guide nucleic acid

205 that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 81. The composition of claim 50, wherein the PAM is of SEQ ID NO: 13. The composition of claim 50 or 51, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 59. The composition of claim 52, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNAis at least 90% identical to SEQ ID NO: 60. The composition of claim 51, wherein the guide nucleic acid comprises at least one sequence that is at least 90% identical to a sequence selected from SEQ ID NOS: 31 and 32. The composition of claim 54, comprising a crRNA and a tracrRNA, wherein the crRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 31, and the tracrRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 32. The composition of claim 54, comprising a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is at least 95% or 100% identical to SEQ ID NO: 31, and the nucleotide sequence of the tracrRNA is at least 95% or 100% identical to SEQ ID NO: 32. The composition of any one of claims 1-26, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 5, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 82. The composition of claim 57, wherein the PAM is of SEQ ID NO: 14. The composition of claim 57 or 58, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 61. The composition of claim 59, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 62 or 63. The composition of claim 58, wherein the guide nucleic acid comprises at least one sequence that is at least 90% identical to a sequence selected from SEQ ID NOS: 33-35. The composition of claim 61, comprising a crRNA and a tracrRNA, wherein the crRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 33, and the tracrRNA comprises a nucleotide sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 34 and SEQ ID NO: 35. The composition of claim 61, comprising a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is at least 95% or 100% identical to SEQ ID NO: 33, and the nucleotide sequence of the tracrRNA is at least 95% or 100% identical to a sequence selected from SEQ ID NO: 34 and SEQ ID NO: 35

206 The composition of any one of claims 1-26, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 6, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 83. The composition of claim 64, wherein the PAM is of SEQ ID NO: 15. The composition of claim 64 or 65, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 64. The composition of claim 66, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 65 or 66. The composition of claim 64 or 65, wherein the guide nucleic acid is sgRNA, and wherein the sgRNA is at least 90% identical to any one of SEQ ID NO: 67-68. The composition of claim 65, wherein the guide nucleic acid comprises at least one sequence that is at least 90% identical to a sequence selected from SEQ ID NOS: 36-40. The composition of claim 69, comprising a crRNA and a tracrRNA, wherein the crRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 36, and the tracrRNA comprises a nucleotide sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 37 and SEQ ID NO: 38 The composition of claim 69, comprising a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is at least 95% or 100% identical to SEQ ID NO: 36, and the nucleotide sequence of the tracrRNA is at least 95% or 100% identical to a sequence selected from SEQ ID NO: 37 and SEQ ID NO: 38. The composition of any one of claims 1-26, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 7, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 84. The composition of claim 72, wherein the PAM is of SEQ ID NO: 16. The composition of claim 72 or 73, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 69. The composition of claim 74, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 70. The composition of claim 72, wherein the guide nucleic acid comprises at least one sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 41 and 42. The composition of claim 76, comprising a crRNA and a tracrRNA, wherein the crRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 41, and the tracrRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 42.

207 The composition of claim 76, comprising a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is at least 95% or 100% identical to SEQ ID NO: 41, and the nucleotide sequence of the tracrRNA is at least 95% or 100% identical to SEQ ID NO: 42. The composition of any one of claims 1-26, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 8, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 85. The composition of claim 79, wherein the PAM is of SEQ ID NO: 17. The composition of claim 79 or 80, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 71. The composition of claim 81, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 72. The composition of claim 80, wherein the guide nucleic acid comprises at least one sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 43 and 44. The composition of claim 83, comprising a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is at least 95% or 100% identical to SEQ ID NO: 43, and the nucleotide sequence of the tracrRNA is at least 95% or 100% identical to SEQ ID NO: 44. The composition of any one of claims 1-26, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 9, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 86. The composition of claim 85, wherein the PAM is of SEQ ID NO: 18. The composition of claim 85 or 86, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 73. The composition of claim 87, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 74. The composition of claim 86, wherein the guide nucleic acid comprises at least one sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 45 and 46. The composition of claim 89, comprising a crRNA and a tracrRNA, wherein the nucleotide sequence of the crRNA is at least 95% or 100% identical to SEQ ID NO: 45, and the nucleotide sequence of the tracrRNA is at least 95% or 100% identical to SEQ ID NO: 46. The composition of any one of claims 1-23, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 87, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 118.

208 The composition of claim 91, wherein the PAM is of SEQ ID NO: 98. The composition of claim 91 or 92, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 109. The composition of claim 93, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 127. The composition of any one of claims 1-23, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 88, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 119. The composition of claim 95, wherein the PAM is of SEQ ID NO: 98 or 99. The composition of claim 95 or 96, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 110. The composition of claim 97, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 128 or 129. The composition of any one of claims 1-23, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 89, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 120. The composition of claim 99, wherein the PAM is of SEQ ID NO: 98 or 99. The composition of claim 99 or 100, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 111 The composition of claim 101, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 130 or 131. The composition of any one of claims 1-23, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 90, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 121. The composition of claim 103, wherein the PAM is of SEQ ID NO: 98 or 100. The composition of claim 103 or 104, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 112. The composition of claim 105, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 132.

209 The composition of any one of claims 1-23, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 91, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 122. The composition of claim 107, wherein the PAM is of SEQ ID NO: 98 or 101. The composition of claim 107 or 108, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 113. The composition of claim 109, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 133. The composition of any one of claims 1-23, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 92, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 123. The composition of claim 111, wherein the PAM is of SEQ ID NO: 98 or 102. The composition of claim 111 or 112, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 114. The composition of claim 113, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 134 or 135. The composition of any one of claims 1-23, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 93, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 124. The composition of claim 114, wherein the PAM is of SEQ ID NO: 98 or 102. The composition of claim 114 or 115, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 115. The composition of claim 116, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 136 or 137. The composition of any one of claims 1-23, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 94, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 123. The composition of claim 119, wherein the PAM is of SEQ ID NO: 103 or 104. The composition of claim 119 or 120, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 114.

210 The composition of claim 121, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 138. The composition of any one of claims 1-23, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 95, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 125. The composition of claim 123, wherein the PAM is of SEQ ID NO: 105 or 106. The composition of claim 123 or 124, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 116. The composition of claim 125, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 139. The composition of any one of claims 1-23, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 96, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 123. The composition of claim 127, wherein the PAM is of SEQ ID NO: SEQ ID NO: 98. The composition of claim 127 or 128, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 114. The composition of claim 129, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 140. The composition of any one of claims 1-23, wherein the effector protein comprises a sequence that is at least 90% identical to SEQ ID NO: 97, and wherein a portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80% identical to an equal length portion of SEQ ID NO: 126. The composition of claim 131, wherein the PAM is of SEQ ID NO: SEQ ID NO: 107 or 108. The composition of claim 131 or 132, wherein the guide nucleic acid comprises a crRNA, and wherein the crRNA is at least 90% identical to SEQ ID NO: 117. The composition of claim 133, wherein the guide nucleic acid further comprises an intermediary RNA, wherein the guide nucleic acid is not transactivating or transactivated, wherein the intermediary RNA is at least 90% identical to SEQ ID NO: 141. The composition of any one of claims 1-134, wherein the effector protein comprises a nuclear localization signal.

211 The composition of any one of claims 1-135, wherein the length of the effector protein is at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, or at least 600 linked amino acid residues. The composition of any one of claims 1-136, wherein the length of the effector protein is less than about 700 linked amino acids. The composition of any one of claims 1-137, wherein the length of the effector protein is about 300 to about 400, about 350 to about 450, about 400 to about 500, about 450 to about 550, about 500 to about 600, or about 550 to about 650 linked amino acids. The composition of any one of claims 1-138, comprising a donor nucleic acid. The composition of any one of claims 1-139, comprising a fusion partner protein linked to the effector protein. The composition of claim 140, wherein the fusion partner protein is directly fused to the N terminus or C terminus of the effector protein via an amide bond. The composition of claim 141, wherein the fusion partner protein is directly fused to the N terminus or C terminus of the effector protein via a peptide linker. The composition of any one of claims 140-142, wherein the fusion partner protein comprises a polypeptide selected from a deaminase, a transcriptional activator, a transcriptional repressor, or a functional domain thereof. The composition of any one of claims 1-143, wherein the effector protein comprises at least one mutation that reduces its nuclease activity relative to the effector protein without the mutation as measured in a cleavage assay, optionally wherein the effector protein is a catalytically inactive nuclease. A composition comprising a nucleic acid expression vector, wherein the nucleic acid vector encodes at least one of the effector proteins and the guide nucleic acid of the composition of any one of claims 1-144. The composition of claim 145, comprising a donor nucleic acid, optionally wherein the donor nucleic acid is encoded by the nucleic acid expression vector or an additional nucleic acid expression vector. The composition of claim 145 or 146, wherein the nucleic acid expression vector is a viral vector. The composition of claim 147, wherein the viral vector is an adeno-associated viral (AAV) vector. A composition comprising a virus, wherein the virus comprises the composition of any one of claims 145-148. A pharmaceutical composition, comprising the composition of any one of claims 1-149, and a pharmaceutically acceptable excipient.

212 A system comprising the composition of any one of claims 1-149, and at least one detection reagent for detecting a target nucleic acid. The system of claim 151, wherein the at least one detection reagent is selected from a reporter nucleic acid, a detection moiety, an additional effector protein, or a combination thereof, optionally wherein the reporter nucleic acid comprises a fluorophore, a quencher, or a combination thereof. The system of claim 151 or 152, comprising at least one amplification reagent for amplifying a target nucleic acid. The system of claim 153, wherein the at least one amplification reagent is selected from the group consisting of a primer, a polymerase, a deoxynucleoside triphosphate (dNTP), a ribonucleoside triphosphate (rNTP), and combinations thereof. The system of any one of claims 151 to 154, wherein the system comprises a device with a chamber or solid support for containing the composition, target nucleic acid, detection reagent or combination thereof. A method of detecting a target nucleic acid in a sample, comprising the steps of:

(a) contacting the sample with:

(i) the composition of any one of claims 1-150 or the system of any one of claims 151- 155, and

(ii) a reporter nucleic acid comprising a detectable moiety that produces a detectable signal in the presence of the target nucleic acid and the composition or system; and

(b) detecting the detectable signal. The method of claim 156, wherein the reporter nucleic acid comprises a fluorophore, a quencher, or a combination thereof, and wherein the detecting comprises detecting a fluorescent signal. The method of claim 156 or 157, comprising reverse transcribing the target nucleic acid, amplifying the target nucleic acid, in vitro transcribing the target nucleic acid, or any combination thereof. The method of any one of claims 156-158, comprising reverse transcribing the target nucleic acid and/or amplifying the target nucleic acid before contacting the sample with the composition. The method of any one of claims 156-158, comprising reverse transcribing the target nucleic acid and/or amplifying the target nucleic acid after contacting the sample with the composition. The method of any one of claims 158-160, wherein amplifying comprises isothermal amplification. The method of any one of claims 156-161, wherein the target nucleic acid is from a pathogen. The method of claim 162, wherein the pathogen is a vims. The method of any one of claims 156-163, wherein the target nucleic acid comprises RNA.

213 The method of any one of claims 156-163, wherein the target nucleic acid comprises DNA. A method of modifying a target nucleic acid, the method comprising contacting the target nucleic acid with the composition of any one of claims 1-150, or the system of any one of claims 151-155, thereby modifying the target nucleic acid. The method of claim 166, wherein modifying the target nucleic acid comprises cleaving the target nucleic acid, deleting a nucleotide of the target nucleic acid, inserting a nucleotide into the target nucleic acid, substituting a nucleotide of the target nucleic acid with an alternative nucleotide or an additional nucleotide, or any combination thereof. The method of claim 166 or 167, comprising contacting the target nucleic acid with a donor nucleic acid. The method of any one of claims 166-168, wherein the target nucleic acid comprises a mutation associated with a disease. The method of claim 169, wherein the disease is selected from an autoimmune disease, a cancer, an inherited disorder, an ophthalmological disorder, a metabolic disorder, or a combination thereof. The method of claim 169, wherein the disease is any disease set forth in TABLE 9. The method of any one of claims 166-171, wherein contacting the target nucleic acid comprises contacting a cell, wherein the target nucleic acid is located in the cell. The method of claim 172, wherein the contacting occurs in vitro. The method of claim 172, wherein the contacting occurs in vivo. The method of claim 172, wherein the contacting occurs ex vivo. A cell comprising the composition of any one of claims 1-150. A cell modified by the composition of any one of claims 1-150. A cell modified by the system of any one of claims 151-155. A cell comprising a modified target nucleic acid, wherein the modified target nucleic acid is a target nucleic acid modified according to any one of the methods of claims 166-175. The cell of any one of claims 176-179, wherein the cell is a eukaryotic cell. The cell of any one of claims 176-179, wherein the cell is a mammalian cell. The cell of any one of claims 176-179, wherein the cell is a prokaryotic cell. The cell of any one of claims 176-179, wherein the cell is a plant cell. The cell of any one of claims 176-179, wherein the cell is an animal cell. The cell of claim 184, wherein the cell is a T cell, optionally wherein the T cell is a natural killer T cell (NKT). The cell of claim 185, wherein the cell is a chimeric antigen receptor T cell (CAR T-cell). The cell of claim 184, wherein the cell is an induced pluripotent stem cell (iPSC). A population of cells according to any one of claims 176-187.

214 A method of producing a protein, the method comprising,

(i) contacting a cell comprising a target nucleic acid to the composition of any one of claims 1-150, thereby editing the target nucleic acid to produce a modified cell comprising a modified target nucleic acid; and

(ii) producing a protein from the cell that is encoded, transcriptionally affected, or translationally affected by the modified nucleic acid. A method of treating a disease comprising administering to a subject in need thereof a composition according to any one of claims 1-150, or a cell according to any one of claims 176- 187. A system for modifying or detecting a target nucleic acid, comprising at least two components each individually comprising one of the following: i) an effector protein, or a nucleic acid encoding an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of sequences recited in TABLE 1; and ii) a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid, wherein the guide nucleic acid at least partially binds to the target nucleic acid. A system for modifying or detecting a target nucleic acid, comprising at least two components each individually comprising one of the following: i) an effector protein, or a nucleic acid encoding an effector protein, wherein the amino acid sequence of the effector protein is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of sequences recited in TABLE 1; and ii) a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid, wherein the guide nucleic acid at least partially binds to the target nucleic acid. A system for modifying or detecting a target nucleic acid, comprising at least two components each individually comprising one of the following: i) an effector protein, or a nucleic acid encoding an effector protein, wherein the effector protein comprises about 100, about 120, about 140, about 160, about 180, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 340, about 360, about 380, about 400, about 420, about 440, about 460, about 480, about 500, about 520, about 540, about 560, about 580, about 600, or about 620 contiguous amino acids of any one of sequences recited in TABLE 1; and ii) a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid, wherein the guide nucleic acid at least partially binds to the target nucleic acid. A system for modifying or detecting a target nucleic acid, comprising at least two components each individually comprising one of the following:

215 i) an effector protein, or a nucleic acid encoding an effector protein, wherein the effector protein comprises the amino acid sequence located at positions 1-100, 150-250, 101-200, 250-350, 201-300, 350-450, 301-400, 350-450, 401-500, 450-550, 501-600, or 550-615 of any one of sequences recited in TABLE 1; and ii) a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid, wherein the guide nucleic acid at least partially binds to the target nucleic acid. A system for modifying or detecting a target nucleic acid, comprising at least two components each individually comprising one of the following: i) an effector protein, or a nucleic acid encoding an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to an equal length portion of any one of sequences recited in TABLE 1, wherein the portion of the sequence is about 30%, about 40% about 50%, about 60%, about 70%, about 80%, or about 90% of any one of sequences recited in TABLE 1; and ii) a guide nucleic acid, or a nucleic acid encoding a guide nucleic acid, wherein the guide nucleic acid at least partially binds to the target nucleic acid. The system of any one of claims 191-195, wherein at least a portion of the guide nucleic acid binds the effector protein. The system of any one of claims 191-196, wherein the portion of the guide nucleic acid that is bound by the effector protein comprises at least 10, at least 15, at least 20, at least 25, or at least 30 contiguous nucleotides of any one of sequences recited in TABLE 4. The system of any one of claims 191-196, wherein the portion of the guide nucleic acid that is bound by the effector protein comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to an equal length portion of any one of sequences recited in TABLE 4. The system of any one of claims 191-198 wherein the effector protein comprises a nuclear localization signal comprising any one of the amino acid sequences recited in TABLE 2. The system of any one of claims 191-199, wherein the system further comprises a component comprising a donor nucleic acid. The system of any one of claims 191-200, wherein the system further comprises a component comprising a fusion partner protein. The system of claim 201, wherein the fusion partner protein is fused to the effector protein. The system of claim 201, wherein the fusion partner protein is not fused to the effector protein. The system of any one of claims 191-203, wherein the effector protein comprises a catalytic activity that is 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less, relative to a naturally occurring counterpart effector protein.

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