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Definitions

• CRISPR-Cas systems of bacterial and archaeal adaptive immunity have been adapted for precise targeting of genomic DNA in eukaryotic cells.
• the CRISPR-Cas systems are easy to set up, scalable, and amenable to targeting multiple positions within the eukaryotic genome, thereby providing a major resource for new applications in genome engineering.
• Two distinct classes of CRISPR-Cas systems have been identified. Class 1 CRISPR- Cas systems utilize multi-protein effector complexes, whereas class 2 CRISPR-Cas systems utilize single-protein effectors (see, Makarova et al. (2017) CELL, 168: 328).
• type II and type V systems typically target DNA and type VI systems typically target RNA (id.).
• Naturally occurring type II effector complexes consist of Cas9, CRISPR RNA (crRNA), and trans-activating CRISPR RNA (tracrRNA), but the crRNA and tracrRNA can be fused as a single guide RNA in an engineered system for simplicity (see, Wang et al. (2016) ANNU. REV. BIOCHEM., 85: 227).
• type V systems such as type V-A, type V-C, and type V-D systems, do not require tracrRNA and use crRNA alone as the guide for cleavage of target DNA (see, Zetsche et al. (2015) CELL, 163: 759; Makarova et al. (2017) CELL, 168: 328).
• the CRISPR-Cas systems have been engineered for various purposes, such as genomic DNA cleavage, base editing, epigenome editing, and genomic imaging (see, e.g., Wang et al. (2016) ANNU. REV. BIOCHEM., 85: 227 and Rees et al. (2016) NAT. REV. GENET., 19: 770).
• Figures 2A-C show a series of schematic representation showing incorporation of a protecting group (e.g., a protective nucleotide sequence or a chemical modification) (Figure 2A), a donor template-recruiting sequence (Figure 2B), and an editing enhancer (Figure 2C) into a Type V-A CRISPR-Cas system.
• a protecting group e.g., a protective nucleotide sequence or a chemical modification
• Figure 2B e.g., a donor template-recruiting sequence
• an editing enhancer Figure 2C
• Figure 3 shows a schematic of a Type V-A nucleic acid guide nuclease comprising a dual guide nucleic acid.
• DETAILED DESCRIPTION Outline I. Engineered non-naturally-occurring dual guide CRISPR-cas systems A. Cas proteins B. Guide nucleic acids C. gNA modifications II. Composition and methods for targeting, editing, and/or modifying genomic DNA A. Ribonucleoprotein (RNP) delivery and “cas RNA” delivery B. CRISPR expression systems C. Donor templates D. Efficiency and specificity E. Multiplex F. Genomic safe harbors G. Guide nucleic acids III. Pharmaceutical compositions IV. Therapeutic uses A. Gene therapies V. Kits VI. Embodiments VI. Equivalents I.
• a CRISPR-Cas system generally comprises a Cas protein and one or more guide nucleic acids (gNAs).
• the Cas protein can be directed to a specific location in a double-stranded DNA target by recognizing a protospacer adjacent motif (PAM) in the non-target strand of the DNA, and the one or more guide nucleic acids can be directed to a specific location by hybridizing with a target nucleotide sequence, also referred to herein as a target sequence, in the target strand of the target polynucleotide.
• PAM protospacer adjacent motif
• a guide nucleic acid can be designed to comprise a nucleotide sequence called a spacer sequence that is at least partially complementary to and can hybridize with a target nucleotide sequence, where target nucleotide sequence is located adjacent to a PAM in an orientation operable with the Cas protein. It has been observed that not all CRISPR-Cas systems designed by these criteria are equally effective.
• the larger polynucleotide in which a target nucleotide sequence is located may be referred to as a target polynucleotide; e.g., a chromosome or other genomic DNA, or portion thereof, or any other suitable polynucleotide within which a target nucleotide sequence is located.
• the target polynucleotide in double stranded DNA comprises two strands.
• the strand of the DNA duplex to which the spacer sequence is complementary herein is called the “target strand,” while the strand to which the spacer sequence shares sequence identity herein is called the “non-target strand.”
• the cleavage site is distant from the PAM site (e.g., separated by at least 10, 11, 12, 13, 14, or 15 nucleotides downstream from the PAM on the non- target strand and/or separated by at least 15, 16, 17, 18, or 19 nucleotides upstream from the sequence complementary to PAM on the target strand).
• Elements in an exemplary single guide CRISPR Cas system e.g., a type V-A CRISPR-Cas system, are shown in Figure 1A.
• the single gNA can also be called a “crRNA” or “single gRNA” where it is present in the form of an RNA.
• the targeter nucleic acid further comprises a spacer sequence (305) at least partially complementary to a target nucleotide sequence (304), i.e., a protospacer, in a target polynucleotide (302) adjacent to a suitable PAM (303).
• a target nucleotide sequence i.e., a protospacer
• the nucleic acid-guided nuclease complex can generate one or more strand breaks (308) in the target polynucleotide at or near the target nucleotide sequence.
• the guide nucleic acid is capable of activating a Cas nuclease.
• a gNA capable of activating a particular Cas nuclease is said to be “compatible” with the Cas nuclease; a Cas nuclease capable of being activated by a particular gNA is said to be “compatible” with the gNA.
• CRISPR-Associated protein can refer to a naturally occurring Cas protein or an engineered Cas protein.
• Non-limiting examples of Cas protein engineering include but are not limited to mutations and modifications of the Cas protein that alter the activity of the Cas, alter the PAM specificity, broaden the range of recognized PAMs, and/or reduce the ability to modify one or more off-target loci as compared to a corresponding unmodified Cas.
• the altered activity of engineered Cas comprises altered ability (e.g., specificity or kinetics) to bind a naturally occurring gNA, e.g., gRNA or engineered gNA, e.g., gRNA, altered ability (e.g., specificity or kinetics) to bind a target nucleotide sequence, altered processivity of nucleic acid scanning, and/or altered effector (e.g., nuclease) activity.
• a Cas protein having nuclease activity can be referred to as a “CRISPR-Associated nuclease” or “Cas nuclease,” or simply “nuclease,” as used interchangeably herein.
• the Cas protein is a type V-A, type V-C, or type V-D Cas protein. In certain embodiments, the Cas protein is a type V-A Cas protein. In other embodiments, the Cas protein is a type II Cas protein, e.g., a Cas9 protein.
• a type V-A Cas nucleases comprises Cpf1. Cpf1 proteins are known in the art and are described, e.g., in U.S. Patent Nos.9,790,490 and 10,113,179. Cpf1 orthologs can be found in various bacterial and archaeal genomes.
• the Cpf1 protein is derived from Francisella novicida U112 (Fn), Acidaminococcus sp. BV3L6 (As), Lachnospiraceae bacterium ND2006 (Lb), Lachnospiraceae bacterium MA2020 (Lb2), Candidatus Methanoplasma termitum (CMt), Moraxella bovoculi 237 (Mb), Porphyromonas crevioricanis (Pc), Prevotella disiens (Pd), Francisella tularensis 1, Francisella tularensis subsp.
• a type V-A Cas nuclease comprises AsCpf1 or a variant thereof.
• a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 3 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 3 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A Cas nuclease comprises LbCpf1 or a variant thereof.
• a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 4 of International (PCT) Application Publication No. WO 2021158918.
• a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 4 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A Cas nuclease comprises FnCpf1 or a variant thereof.
• a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 5 of International (PCT) Application Publication No. WO 2021158918.
• a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 5 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A Cas nuclease comprises Prevotella bryantii Cpf1 (PbCpf1) or a variant thereof.
• a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 6 of International (PCT) Application Publication No.
• a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 6 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A Cas nuclease comprises Proteocatella sphenisci Cpf1 (PsCpf1) or a variant thereof.
• a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7 of International (PCT) Application Publication No. WO 2021158918.
• a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 7 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A Cas nuclease comprises Anaerovibrio sp.
• a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 8 of International (PCT) Application Publication No. WO 2021158918.
• a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 8 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A Cas nuclease comprises Moraxella caprae Cpf1 (McCpf1) or a variant thereof.
• a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 9 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 9 of International (PCT) Application Publication No.
• a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 11 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A Cas nuclease is not Cpf1. In certain embodiments, a type V-A Cas nuclease is not AsCpf1.
• MAD7 (SEQ ID NO: 37) MNNGTNNFQNFIGISSLQKTLRNALIPTETTQQFIVKNGIIKEDELRGENRQILKDIMDDYYRGF ISETLSSIDDIDWTSLFEKMEIQLKNGDNKDTLIKEQTEYRKAIHKKFANDDRFKNMFSAKLISD ILPEFVIHNNNYSASEKEEKTQVIKLFSRFATSFKDYFKNRANCFSADDISSSSCHRIVNDNAEI FFSNALVYRRIVKSLSNDDINKISGDMKDSLKEMSLEEIYSYEKYGEFITQEGISFYNDICGKVN SFMNLYCQKNKENKNLYKLQKLHKQILCIADTSYEVPYKFESDEEVYQSVNGFLDNISSKHIVER LRKIGDNYNGYNLDKIYIVSKFYESVSQKTYRDWETINTALEIHYNNILPGNGKSKADKVKKAVK N
• a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 38.
• MAD2 (SEQ ID NO: 38) MSSLTKFTNKYSKQLTIKNELIPVGKTLENIKENGLIDGDEQLNENYQKAKIIVDDFLRDFINKA LNNTQIGNWRELADALNKEDEDNIEKLQDKIRGIIVSKFETFDLFSSYSIKKDEKIIDDDNDVEE EELDLGKKTSSFKYIFKKNLFKLVLPSYLKTTNQDKLKIISSFDNFSTYFRGFFENRKNIFTKKP ISTSIAYRIVHDNFPKFLDNIRCFNVWQTECPQLIVKADNYLKSKNVIAKDKSLANYFTVGAYDY FLSQNGIDFYNNIIGGLPAFAGHEKIQGLNEFINQECQKDSELKSKLKNRHAFKMAVLFKQILSD REKSFVIDEFESDAQVIDAVKNFYAEQCKDNNVIFNLLNLIKNIAFLSDDELDGIFIEGKYLSSV SQKLYSDWSKLRNDIEDSANSKQ
• Csm1 proteins are known in the art and are described in U.S. Patent No.9,896,696. Csm1 orthologs can be found in various bacterial and archaeal genomes.
• a Csm1 protein is derived from Smithella sp. SCADC (Sm), Sulfuricurvum sp. (Ss), or Microgenomates (Roizmanbacteria) bacterium (Mb).
• a type V-A Cas nuclease comprises SmCsm1 or a variant thereof.
• a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 12 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 12 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A Cas nuclease comprises SsCsm1 or a variant thereof.
• the additional nucleotide sequence consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides. In certain embodiments, the additional nucleotide sequence consists of 2 nucleotides. In certain embodiments, the additional nucleotide sequence is reminiscent to the loop or a fragment thereof (e.g., one, two, three, or four nucleotides at the 3’ end of the loop) in a crRNA of a corresponding single guide CRISPR-Cas system. It is understood that an additional nucleotide sequence 5’ to the targeter stem sequence can be dispensable.
• the free energy change during the hairpin formation is greater than or equal to -5 kcal/mol, -6 kcal/mol, -7 kcal/mol, -8 kcal/mol, -9 kcal/mol, -10 kcal/mol, -11 kcal/mol, -12 kcal/mol, -13 kcal/mol, -14 kcal/mol, or -15 kcal/mol.
• the G is lower than or equal to -1 kcal/mol, e.g., lower than or equal to -2 kcal/mol, lower than or equal to -3 kcal/mol, lower than or equal to -4 kcal/mol, lower than or equal to -5 kcal/mol, lower than or equal to -6 kcal/mol, lower than or equal to -7 kcal/mol, lower than or equal to -7.5 kcal/mol, or lower than or equal to -8 kcal/mol.
• the G is greater than or equal to -10 kcal/mol, e.g., greater than or equal to -9 kcal/mol, greater than or equal to -8.5 kcal/mol, or greater than or equal to -8 kcal/mol. In certain embodiments, the G is in the range of -10 to -4 kcal/mol.
• the G is in the range of -8 to -4 kcal/mol, -7 to -4 kcal/mol, -6 to -4 kcal/mol, -5 to -4 kcal/mol, -8 to -4.5 kcal/mol, -7 to -4.5 kcal/mol, -6 to -4.5 kcal/mol, or -5 to - 4.5 kcal/mol.
• one or more base pairs may reduce the G, i.e., stabilize the nucleic acid complex.
• the nucleotide immediately 5’ to the targeter stem sequence comprises a uracil or is a uridine
• the nucleotide immediately 3’ to the modulator stem sequence comprises a uracil or is a uridine, thereby forming a nonconventional U-U base pair.
• the modulator nucleic acid or the single guide nucleic acid comprises a nucleotide sequence referred to herein as a “5’ tail” positioned 5’ to the modulator stem sequence.
• the 5’ tail is a nucleotide sequence positioned 5’ to the stem-loop structure of the crRNA.
• a 5’ tail in an engineered type V-A CRISPR-Cas system, whether single guide or dual guide, can be reminiscent to the 5’ tail in a corresponding naturally occurring type V-A CRISPR-Cas system.
• the second nucleotide in the 5’ tail, the position counted from the 3’ end comprises a uracil or is a uridine.
• the third nucleotide in the 5’ tail, the position counted from the 3’ end comprises an adenine or is an adenosine.
• This third nucleotide may form a base pair (e.g., a Watson-Crick base pair) with a nucleotide 5’ to the modulator stem sequence.
• the modulator nucleic acid comprises a uridine or a uracil-containing nucleotide 5’ to the modulator stem sequence.
• the 5’ tail comprises the nucleotide sequence of 5’- AUU-3’. In certain embodiments, the 5’ tail comprises the nucleotide sequence of 5’-AAUU-3’. In certain embodiments, the 5’ tail comprises the nucleotide sequence of 5’-UAAUU-3’. In certain embodiments, the 5’ tail is positioned immediately 5’ to the modulator stem sequence. [0086] In certain embodiments, the single guide nucleic acid, the targeter nucleic acid, and/or the modulator nucleic acid are designed to reduce the degree of secondary structure other than the hybridization between the targeter stem sequence and the modulator stem sequence.
• no more than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the single guide nucleic acid other than the targeter stem sequence and the modulator stem sequence participate in self-complementary base pairing when optimally folded.
• no more than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the targeter nucleic acid and/or the modulator nucleic acid participate in self-complementary base pairing when optimally folded.
• Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy.
• the single guide nucleic acid or the modulator nucleic acid further comprises a donor template-recruiting sequence capable of hybridizing with a donor template (see Figure 2B).
• Donor templates are described in the “Donor Templates” subsection of section II infra. The donor template and donor template-recruiting sequence can be designed such that they bear sequence complementarity.
• the donor template-recruiting sequence is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) complementary to at least a portion of the donor template. In certain embodiments, the donor template-recruiting sequence is 100% complementary to at least a portion of the donor template. In certain embodiments, where the donor template comprises an engineered sequence not homologous to the sequence to be repaired, the donor template-recruiting sequence is capable of hybridizing with the engineered sequence in the donor template.
• the single guide nucleic acid, the modulator nucleic acid, and/or the targeter nucleic acid can further comprise a protective nucleotide sequence that prevents or reduces nucleic acid degradation.
• the protective nucleotide sequence is at least 5 (e.g., at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50) nucleotides in length.
• a protective nucleotide sequence is typically located at the 5’ or 3’ end of the single guide nucleic acid, the modulator nucleic acid, and/or the targeter nucleic acid.
• nucleotide sequences can be present in the 5’ portion of a single nucleic acid or a modulator nucleic acid, including but not limited to a donor template- recruiting sequence, an editing enhancer sequence, a protective nucleotide sequence, and a linker connecting such sequence to the 5’ tail, if present, or to the modulator stem sequence. It is understood that the functions of donor template recruitment, editing enhancement, protection against degradation, and linkage are not exclusive to each other, and one nucleotide sequence can have one or more of such functions.
• the single guide nucleic acid or the modulator nucleic acid comprises a nucleotide sequence that is both a donor template-recruiting sequence and an editing enhancer sequence.
• the single guide nucleic acid or the modulator nucleic acid comprises a nucleotide sequence that is both a donor template-recruiting sequence and a protective sequence.
• the single guide nucleic acid or the modulator nucleic acid comprises a nucleotide sequence that is both an editing enhancer sequence and a protective sequence.
• the single guide nucleic acid or the modulator nucleic acid comprises a nucleotide sequence that is a donor template-recruiting sequence, an editing enhancer sequence, and a protective sequence.
• the nucleotide sequence 5’ to the 5’ tail, if present, or 5’ to the modulator stem sequence is 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-90, 30-80, 30- 70, 30-60, 30-50, 30-40, 40-90, 40-80, 40-70, 40-60, 40-50, 50-90, 50-80, 50-70, 50-60, 60-90, 60-80, 60-70, 70-90, 70-80, or 80-90 nucleotides in length.
• an engineered, non-naturally occurring system further comprises one or more compounds (e.g., small molecule compounds) that enhance HDR and/or inhibit NHEJ.
• compounds e.g., small molecule compounds
• Exemplary compounds having such functions are described in Maruyama et al. (2015) Nat Biotechnol.33(5): 538-42; Chu et al. (2015) Nat Biotechnol.33(5): 543-48; Yu et al. (2015) Cell Stem Cell 16(2): 142-47; Pinder et al. (2015) Nucleic Acids Res.43(19): 9379-92; and Yagiz et al. (2019) Commun. Biol.2: 198.
• an engineered, non- naturally occurring system further comprises one or more compounds selected from the group consisting of DNA ligase IV antagonists (e.g., SCR7 compound, Ad4 E1B55K protein, and Ad4 E4orf6 protein), RAD51 agonists (e.g., RS-1), DNA-dependent protein kinase (DNA-PK) antagonists (e.g., NU7441 and KU0060648), 3-adrenergic receptor agonists (e.g., L755507), inhibitors of intracellular protein transport from the ER to the Golgi apparatus (e.g., brefeldin A), and any combinations thereof.
• DNA ligase IV antagonists e.g., SCR7 compound, Ad4 E1B55K protein, and Ad4 E4orf6 protein
• RAD51 agonists e.g., RS-1
• DNA-PK DNA-dependent protein kinase
• 3-adrenergic receptor agonists e.g., L
• an engineered, non-naturally occurring system comprising a targeter nucleic acid and a modulator nucleic acid is tunable or inducible.
• the targeter nucleic acid, the modulator nucleic acid, and/or the Cas protein can be introduced to the target nucleotide sequence at different times, the system becoming active only when all components are present.
• the amounts of the targeter nucleic acid, the modulator nucleic acid, and/or the Cas protein can be titrated to achieve desired efficiency and specificity.
• Guide nucleic acids including a single guide nucleic acid, a targeter nucleic acid, and/or a modulator nucleic acid, may comprise a DNA (e.g., modified DNA), an RNA (e.g., modified RNA), or a combination thereof.
• the single guide nucleic acid comprises a DNA (e.g., modified DNA), an RNA (e.g., modified RNA), or a combination thereof.
• the targeter nucleic acid comprises a DNA (e.g., modified DNA), an RNA (e.g., modified RNA), or a combination thereof.
• the modulator nucleic acid comprises a DNA (e.g., modified DNA), an RNA (e.g., modified RNA), or a combination thereof.
• Spacer sequences can be presented as DNA sequences by including thymidines (T) rather than uridines (U). It is understood that corresponding RNA sequences and DNA/RNA chimeric sequences are also contemplated. For example, where the spacer sequence is an RNA, its sequence can be derived from a DNA sequence disclosed herein by replacing each T with U.
• engineered, non-naturally occurring systems comprising a targeter nucleic acid comprising: a spacer sequence designed to hybridize with a target nucleotide sequence and a targeter stem sequence; and a modulator nucleic acid comprising a modulator stem sequence complementary to the targeter stem sequence, and, optionally, a 5’ sequence, e.g., a tail sequence, wherein, in a single guide nucleic acid the targeter nucleic acid and the modulator nucleic acid are part of a single polynucleotide, and in a dual guide nucleic acid, the targeter nucleic acid and the modulator nucleic acid are separate nucleic acids; modifications can include one or more chemical modifications to one or more nucleotides or internucleotide linkages at or near the 3’ end of the targeter nucleic acid (dual and single and the targeter nucleic acid
• the Cas nuclease is a type V-A Cas nuclease.
• Modulator and/or targeter nucleic sequences can include further sequences, as detailed in the Guide Nucleic Acids section, and modifications can be in these further sequences, as appropriate and apparent to one of skill in the art.
• guide nucleic acid is oriented from 5’ at the modulator nucleic acid to 3’ at the modulator stem sequence, and 5’ at the targeter stem sequence to 3’ at the targeter sequence (see, e.g., Figure 1A and 1B); in certain embodiments, as appropriate, guide nucleic acid is oriented from 3’ at the modulator nucleic acid to 5’ at the modulator stem sequence, and 3’ at the targeter stem sequence to 5’ at the targeter sequence.
• the targeter nucleic acid may comprise a DNA (e.g., modified DNA), an RNA (e.g., modified RNA), or a combination thereof.
• the modulator nucleic acid may comprise a DNA (e.g., modified DNA), an RNA (e.g., modified RNA), or a combination thereof.
• the targeter nucleic acid is an RNA and the modulator nucleic acid is an RNA.
• a targeter nucleic acid in the form of an RNA is also called targeter RNA, and a modulator nucleic acid in the form of an RNA is also called modulator RNA.
• the nucleotide sequences disclosed herein are presented as DNA sequences by including thymidines (T) and/or RNA sequences including uridines (U). It is understood that corresponding DNA sequences, RNA sequences, and DNA/RNA chimeric sequences are also contemplated.
• 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, 30%-40%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%- 80%, 50%-70%, 50%-60%, 60%-80%, 60%-70%, or 70%-80% of gNA is RNA.
• 50% of the gNA is RNA.
• 70% of the gNA is RNA.
• 90% of the gNA is RNA.
• 100% of the gNA is RNA, e.g., a gRNA.
• the remaining portion of the gNA that is not RNA comprises a modified ribonucleotide, a deoxyribonucleotide, a modified deoxyribonucleotide, or a synthetic, e.g., unnatural nucleotide, for example, not intended to be limiting, threose nucleic acid, locked nucleic acid, peptide nucleic acid, arabinonucleic acid, hexose nucleic acid, among others.
• the targeter nucleic acid and/or the modulator nucleic acid are RNAs with one or more modifications in a ribose group, one or more modifications in a phosphate group, one or more modifications in a nucleobase, one or more terminal modifications, or a combination thereof.
• Exemplary modifications are disclosed in U.S. Patent Nos.10,900,034 and 10,767,175, U.S. Patent Application Publication No.2018/0119140, Watts et al. (2008) Drug Discov. Today 13: 842-55, and Hendel et al. (2015) NAT. BIOTECHNOL.33: 985.
• a targeter nucleic acid e.g., RNA
• the 3’ end of the targeter nucleic acid comprises the spacer sequence.
• the 3’ end of the targeter nucleic acid comprises the targeter stem sequence. Exemplary modifications are disclosed in Dang et al. (2015) Genome Biol.16: 280, Kocaz et al. (2019) Nature Biotech.37: 657-66, Liu et al.
• Modifications in a ribose group include but are not limited to modifications at the 2' position or modifications at the 4 position.
• the ribose comprises 2'-O-C1-4alkyl, such as 2'-O-methyl (2'-OMe, or M).
• the ribose comprises 2'-O-C1-3alkyl-O-C1-3alkyl, such as 2'-methoxyethoxy (2'-O—CH 2 CH 2 OCH 3 ) also known as 2'-O-(2-methoxyethyl) or 2'-MOE.
• the ribose comprises 2'-O-allyl.
• the ribose comprises 2'-O-2,4-Dinitrophenol (DNP).
• the ribose comprises 2'-halo, such as 2'-F, 2'-Br, 2'-Cl, or 2'-I.
• the ribose comprises 2'-NH 2 .
• the ribose comprises 2'-H (e.g., a deoxynucleotide). In certain embodiments, the ribose comprises 2'-arabino or 2'-F- arabino. In certain embodiments, the ribose comprises 2'-LNA or 2'-ULNA. In certain embodiments, the ribose comprises a 4'-thioribosyl. [0101] Modifications can also include a deoxy group, for example a 2'-deoxy-3'- phosphonoacetate (DP), a 2'-deoxy-3'-thiophosphonoacetate (DSP).
• DP 2'-deoxy-3'- phosphonoacetate
• DSP 2'-deoxy-3'-thiophosphonoacetate
• Internucleotide linkage modifications in a phosphate group include but are not limited to a phosphorothioate (S), a chiral phosphorothioate, a phosphorodithioate, a boranophosphonate, a C 1-4 alkyl phosphonate such as a methylphosphonate, a boranophosphonate, a phosphonocarboxylate such as a phosphonoacetate (P), a phosphonocarboxylate ester such as a phosphonoacetate ester, an amide, a thiophosphonocarboxylate such as a thiophosphonoacetate (SP), a thiophosphonocarboxylate ester such as a thiophosphonoacetate ester, and a 2' ,5 -linkage having a phosphodiester or any of the modified phosphates above.
• S phosphorothioate
• nucleobase examples include but are not limited to 2-thiouracil, 2- thiocytosine, 4-thiouracil, 6-thioguanine, 2-aminoadenine, 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5- methylcytosine, 5-methyluracil, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6- dehydrouracil, 5-propynylcytosine, 5-propynyluracil, 5-ethynylcytosine, 5-ethynyluracil, 5- allyluracil, 5-allylcytosine, 5-aminoallyluracil, 5-aminoallyl-cytosine, 5-bromouracil, 5- iodouraci
• Terminal modifications include but are not limited to polyethyleneglycol (PEG), hydrocarbon linkers (such as heteroatom (O,S,N)-substituted hydrocarbon spacers; halo- substituted hydrocarbon spacers; keto-, carboxyl-, amido-, thionyl-, carbamoyl-, thionocarbamaoyl-containing hydrocarbon spacers, propanediol), spermine linkers, dyes such as fluorescent dyes (for example, fluoresceins, rhodamines, cyanines), quenchers (for example, dabcyl, BHQ), and other labels (for example biotin, digoxigenin, acridine, streptavidin, avidin, peptid
• a terminal modification comprises a conjugation (or ligation) of the RNA to another molecule comprising an oligonucleotide (such as deoxyribonucleotides and/or ribonucleotides), a peptide, a protein, a sugar, an oligosaccharide, a steroid, a lipid, a folic acid, a vitamin and/or other molecule.
• an oligonucleotide such as deoxyribonucleotides and/or ribonucleotides
• a terminal modification incorporated into the RNA is located internally in the RNA sequence via a linker such as 2-(4-butylamidofluorescein)propane-1,3-diol bis(phosphodiester) linker, which is incorporated as a phosphodiester linkage and can be incorporated anywhere between two nucleotides in the RNA.
• a linker such as 2-(4-butylamidofluorescein)propane-1,3-diol bis(phosphodiester) linker, which is incorporated as a phosphodiester linkage and can be incorporated anywhere between two nucleotides in the RNA.
• the modification in the RNA is selected from the group consisting of incorporation of 2'-O-methyl- 3'phosphorothioate (MS), 2'-O-methyl-3'-phosphonoacetate (MP), 2'-O-methyl-3'- thiophosphonoacetate (MSP), 2'-halo-3'-phosphorothioate (e.g., 2'-fluoro-3'-phosphorothioate), 2'-halo-3'-phosphonoacetate (e.g., 2'-fluoro-3'-phosphonoacetate), and 2'-halo-3'- thiophosphonoacetate (e.g., 2'-fluoro-3'-thiophosphonoacetate).
• MS 2'-O-methyl- 3'phosphorothioate
• MP 2'-O-methyl-3'-phosphonoacetate
• MSP 2'-halo-3'-phosphorothioate
• 2'-halo-3'-phosphorothioate e
• modifications can include either a 5’ or a 3’ propanediol or C3 linker modification.
• the modification alters the stability of the RNA.
• the modification enhances the stability of the RNA, e.g., by increasing nuclease resistance of the RNA relative to a corresponding RNA without the modification.
• Stability- enhancing modifications include but are not limited to incorporation of 2'-O-methyl, a 2'-O-C 1- 4 alkyl, 2'-halo (e.g., 2'-F, 2'-Br, 2'-Cl, or 2'-I), 2' MOE, a 2'-O-C 1-3 alkyl-O-C 1-3 alkyl, 2'-NH 2 , 2'-H (or 2'-deoxy), 2'-arabino, 2'-F-arabino, 4 -thioribosyl sugar moiety, 3'-phosphorothioate, 3'- phosphonoacetate, 3'-thiophosphonoacetate, 3'-methylphosphonate, 3'-boranophosphate, 3'- phosphorodithioate, locked nucleic acid (“LNA”) nucleotide which comprises a methylene bridge between the 2' and 4' carbons of the ribose ring, and unlocked nucleic acid
• Specificity- enhancing modifications include but are not limited to 2-thiouracil, 2-thiocytosine, 4-thiouracil, 6-thioguanine, 2-aminoadenine, and pseudouracil. Within 10, 5, 4, 3, 2, or 1 nucleotide of the 3’ end, for example the 3’ end nucleotide, is modified.
• the modification alters the immunostimulatory effect of the RNA relative to a corresponding RNA without the modification. For example, in certain embodiments, the modification reduces the ability of the RNA to activate TLR7, TLR8, TLR9, TLR3, RIG-I, and/or MDA5.
• the targeter nucleic acid and/or the modulator nucleic acid comprise at least 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, or 40 modified nucleotides or internucleotide linkages.
• the modification can be made at one or more positions in the targeter nucleic acid and/or the modulator nucleic acid such that these nucleic acids retain functionality.
• the modified nucleic acids can still direct the Cas protein to the target nucleotide sequence and allow the Cas protein to exert its effector function.
• the particular modification(s) at a position may be selected based on the functionality of the nucleotide or internucleotide linkage at the position.
• a specificity-enhancing modification may be suitable for a nucleotide or internucleotide linkage in the spacer sequence, the targeter stem sequence, or the modulator stem sequence.
• a stability-enhancing modification may be suitable for one or more terminal nucleotides or internucleotide linkages in the targeter nucleic acid and/or the modulator nucleic acid.
• At least 1 e.g., at least 2, at least 3, at least 4, or at least 5 terminal nucleotides or internucleotide linkages at or near the 5’ end and/or at least 1 (e.g., at least 2, at least 3, at least 4, or at least 5) terminal nucleotides or internucleotide linkages at or near the 3’ end of the targeter nucleic acid are modified.
• At least 1 e.g., at least 2, at least 3, at least 4, or at least 5 terminal nucleotides or internucleotide linkages at or near the 5’ end and/or at least 1 (e.g., at least 2, at least 3, at least 4, or at least 5) terminal nucleotides or internucleotide linkages at or near the 3’ end of the modulator nucleic acid are modified.
• the targeter or modulator nucleic acid is a combination of DNA and RNA
• the nucleic acid as a whole is considered as an RNA
• the DNA nucleotide(s) are considered as modification(s) of the RNA, including a 2'-H modification of the ribose and optionally a modification of the nucleobase.
• composition and methods for targeting, editing, and/or modifying genomic DNA can be covalently conjugated to each other through one or more chemical modifications introduced into these nucleic acids, thereby increasing the stability of the double-stranded complex and/or improving other characteristics of the system.
• An engineered, non-naturally occurring system, such as disclosed herein, can be useful for targeting, editing, and/or modifying a target nucleic acid, such as a DNA (e.g., genomic DNA) in a cell or organism.
• the present invention provides a method of cleaving a target nucleic acid (e.g., DNA) comprising the sequence of a preselected target sequence or a portion thereof, the method comprising contacting the target DNA with an engineered, non-naturally occurring system disclosed herein, thereby resulting in cleavage of the target DNA.
• a target nucleic acid e.g., DNA
• the present invention provides a method of binding a target nucleic acid (e.g., DNA) comprising the sequence of a preselected target sequence or a portion thereof, the method comprising contacting the target DNA with an engineered, non-naturally occurring system disclosed herein, thereby resulting in binding of the system to the target DNA.
• This method can be useful, e.g., for detecting the presence and/or location of the a preselected target gene, for example, if a component of the system (e.g., the Cas protein) comprises a detectable marker.
• a component of the system e.g., the Cas protein
• a detectable marker e.g., a detectable marker associated with the target DNA.
• methods of modifying a target nucleic acid e.g., DNA
• a structure e.g., protein associated with the target DNA
• the method comprising contacting the target DNA with an engineered, non-naturally occurring system disclosed herein, wherein the Cas protein comprises an effector domain or is associated with an effector protein, thereby resulting in modification of the target DNA or the structure associated with the target DNA.
• the Cas protein is a type V-A Cas protein (e.g., Cas nuclease).
• a method of editing a human genomic sequence at one of a group of preselected target gene loci comprising delivering an engineered, non-naturally occurring system disclosed herein into a human cell, thereby resulting in editing of the genomic sequence at the target gene locus in the human cell.
• Exemplary methods of delivery are known in the art and described in, for example, U.S. Patent Nos.8,697,359, 10,113,167, 10,570,418, 10,829,787, 11,118,194, and 11,125,739 and U.S. Patent Application Publication Nos. 2015/0344912, 2018/0119140, and 2018/0282763.
• contacting a DNA e.g., genomic DNA
• a CRISPR- Cas complex does not require delivery of all components of the complex into the cell.
• one or more of the components may be pre-existing in the cell.
• the cell (or a parental/ancestral cell thereof) has been engineered to express the Cas protein, and the single guide nucleic acid (or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the single guide nucleic acid), the targeter nucleic acid (or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the targeter nucleic acid), and/or the modulator nucleic acid (or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the modulator nucleic acid) are delivered into the cell.
• the single guide nucleic acid or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the single guide nucleic acid
• the targeter nucleic acid or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the targeter nucleic
• the cell (or a parental/ancestral cell thereof) has been engineered to express the modulator nucleic acid, and the Cas protein (or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the Cas protein) and the targeter nucleic acid (or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the targeter nucleic acid) are delivered into the cell.
• the Cas protein or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the Cas protein
• the targeter nucleic acid or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the targeter nucleic acid
• the target cells can be mitotic or post-mitotic cells from any organism, such as a bacterial cell (e.g., E coli), an archaeal cell, a cell of a single-cell eukaryotic organism, a plant cell, an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, or the like, a fungal cell (e.g., a yeast cell, such as S. cervisiae), an animal cell, a cell from an invertebrate animal (e.g.
• a bacterial cell e.g., E coli
• an archaeal cell e.g., a cell of a single-cell eukaryotic organism
• a plant cell e.g., an algal cell, e.g., Botryococc
• target cells include but are not limited to a stem cell (e.g., an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell), a somatic cell (e.g., a fibroblast, a hematopoietic cell, a T lymphocyte (e.g., CD8+ T lymphocyte), an NK cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell), an in vitro or in vivo embryonic cell of an embryo at any stage (e.g., a 1-cell, 2-cell, 4-cell, 8-cell; stage zebrafish embryo).
• a stem cell e.g., an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell
• a somatic cell e.g., a fibroblast, a hematopoietic cell, a T lymphocyte (e.g., CD8
• Ribonucleoprotein (RNP) delivery and “cas RNA” delivery An engineered, non-naturally occurring system disclosed herein can be delivered into a cell by suitable methods known in the art, including but not limited to ribonucleoprotein (RNP) delivery and “Cas RNA” delivery described below.
• RNP ribonucleoprotein
• Cas RNA delivery described below.
• a CRISPR-Cas system including a single guide nucleic acid and a Cas protein, or a CRISPR-Cas system including a targeter nucleic acid, a modulator nucleic acid, and a Cas protein can be combined into a RNP complex and then delivered into the cell as a pre-formed complex.
• a “ribonucleoprotein” or “RNP,” as used herein, can refer to a complex comprising a nucleoprotein and a ribonucleic acid.
• nucleoprotein as provided herein can refer to a protein capable of binding a nucleic acid (e.g., RNA, DNA). Where the nucleoprotein binds a ribonucleic acid it can be referred to as “ribonucleoprotein.”
• the interaction between the ribonucleoprotein and the ribonucleic acid may be direct, e.g., by covalent bond, or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g.
• the ribonucleoprotein includes an RNA-binding motif non-covalently bound to the ribonucleic acid.
• positively charged aromatic amino acid residues e.g., lysine residues
• the RNA-binding motif may form electrostatic interactions with the negative nucleic acid phosphate backbones of the RNA.
• Patent No.11,118,194 nanoparticles, nanowires (see, Shalek et al. (2012) Nano Letters, 12: 6498), exosomes, and perturbation of cell membrane (e.g., by passing cells through a constriction in a microfluidic system, see, U.S. Patent No.11,125,739).
• the efficiency of RNP delivery can be enhanced by cell cycle synchronization (see, U.S. Patent No.10,570,418).
• an RNP is delivered into a cell by electroporation.
• a CRISPR-Cas system is delivered into a cell in a “approach, i.e., delivering (a) a single guide nucleic acid, or a combination of a targeter nucleic acid and a modulator nucleic acid, and (b) an RNA (e.g., messenger RNA (mRNA)) encoding a Cas protein.
• RNA e.g., messenger RNA (mRNA)
• the RNA encoding the Cas protein can be translated in the cell and form a complex with the single guide nucleic acid or combination of the targeter nucleic acid and the modulator nucleic acid intracellularly.
• the single guide nucleic acid, or the targeter nucleic acid and the modulator nucleic acid are generally provided in excess molar amount (e.g., at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 50 fold, or at least 100 fold) relative to the mRNA.
• the targeter nucleic acid and the modulator nucleic acid are annealed under suitable conditions prior to delivery into the cells.
• the targeter nucleic acid and the modulator nucleic acid are delivered into the cells without annealing in vitro.
• a variety of delivery systems can be used to introduce an “Cas RNA” system into a cell.
• Non-limiting examples of delivery methods or vehicles include microinjection, biolistic particles, liposomes (see, e.g., U.S. Patent No.10,829,787) such as molecular trojan horses liposomes that delivers molecules across the blood brain barrier (see, Pardridge et al. (2010) Cold Spring Harb. Protoc., doi:10.1101/pdb.prot5407), immunoliposomes, virosomes, polycations, lipid:nucleic acid conjugates, electroporation, nanoparticles, nanowires (see, Shalek et al.
• the CRISPR-Cas system is delivered into a cell in the form of (a) a single guide nucleic acid or a combination of a targeter nucleic acid and a modulator nucleic acid, and (b) a DNA comprising a regulatory element operably linked to a Cas coding sequence.
• the DNA can be provided in a plasmid, viral vector, or any other form described in the “CRISPR Expression Systems” subsection.
• Such delivery method may result in constitutive expression of Cas protein in the target cell (e.g., if the DNA is maintained in the cell in an episomal vector or is integrated into the genome), and may increase the risk of off-targeting which is undesirable when the Cas protein has nuclease activity.
• this approach is useful when the Cas protein comprises a non-nuclease effector (e.g., a transcriptional activator or repressor). It is also useful for research purposes and for genome editing of plants.
• a non-nuclease effector e.g., a transcriptional activator or repressor
• the present invention provides a CRISPR expression system comprising: (a) a nucleic acid comprising a first regulatory element operably linked to a nucleotide sequence encoding a targeter nucleic acid and (b) a nucleic acid comprising a second regulatory element operably linked to a nucleotide sequence encoding a modulator nucleic acid.
• a CRISPR expression system further comprises a nucleic acid comprising a third regulatory element operably linked to a nucleotide sequence encoding a Cas protein, such as a Cas protein disclosed herein.
• the Cas protein is a type V-A, type V-C, or type V-D Cas protein (e.g., Cas nuclease). In certain embodiments, the Cas protein is a type V-A Cas protein (e.g., Cas nuclease).
• the term “operably linked” can mean that the nucleotide sequence of interest is linked to the regulatory element in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
• the nucleic acids of a CRISPR expression system described above may be independently selected from various nucleic acids such as DNA (e.g., modified DNA) and RNA (e.g., modified RNA).
• the nucleic acids comprising a regulatory element operably linked to one or more nucleotide sequences encoding the guide nucleic acids are in the form of DNA.
• the nucleic acid comprising a third regulatory element operably linked to a nucleotide sequence encoding the Cas protein is in the form of DNA.
• the third regulatory element can be a constitutive or inducible promoter that drives the expression of the Cas protein.
• Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
• Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors and replication defective viral vectors) do not autonomously replicate in the host cell. Certain vectors, however, may be integrated into the genome of the host cell and thereby are replicated along with the host genome. A skilled person in the art will appreciate that different vectors may be suitable for different delivery methods and have different host tropism, and will be able to select one or more vectors suitable for the use.
• regulatory element can refer to a transcriptional and/or translational control sequence, such as a promoter, enhancer, transcription termination signal (e.g., polyadenylation signal), internal ribosomal entry sites (IRES), protein degradation signal, or the like, that provide for and/or regulate transcription of a non-coding sequence (e.g., a targeter nucleic acid or a modulator nucleic acid) or a coding sequence (e.g., a Cas protein) and/or regulate translation of an encoded polypeptide.
• a transcriptional and/or translational control sequence such as a promoter, enhancer, transcription termination signal (e.g., polyadenylation signal), internal ribosomal entry sites (IRES), protein degradation signal, or the like, that provide for and/or regulate transcription of a non-coding sequence (e.g., a targeter nucleic acid or a modulator nucleic acid) or a coding sequence (e.g., a Cas protein) and/or regulate translation
• Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
• tissue-specific regulatory sequences may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or particular cell types (e.g., lymphocytes).
• a vector comprises one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof.
• pol III promoters include, but are not limited to, U6 and H1 promoters.
• a vector can be introduced into host cells to produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., CRISPR transcripts, proteins, enzymes, mutant forms thereof, or fusion proteins thereof).
• the nucleotide sequence encoding the Cas protein is codon optimized for expression in a prokaryotic cell, e.g., E coli, eukaryotic host cell, e.g., a yeast cell (e.g., S. cerevisiae), a mammalian cell (e.g., a mouse cell, a rat cell, or a human cell), or a plant cell.
• a prokaryotic cell e.g., E coli
• eukaryotic host cell e.g., a yeast cell (e.g., S. cerevisiae)
• a mammalian cell e.g., a mouse cell, a rat cell, or a human cell
• Various species exhibit particular bias for certain codons of a particular amino acid.
• Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules.
• mRNA messenger RNA
• tRNA transfer RNA
• the predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at kazusa.or.jp/codon/ and these tables can be adapted in a number of ways (see, Nakamura et al.
• C. Donor templates Cleavage of a target nucleotide sequence in the genome of a cell by a CRISPR-Cas system or complex can activate DNA damage pathways, which may rejoin the cleaved DNA fragments by NHEJ or HDR. HDR requires a repair template, either endogenous or exogenous, to transfer the sequence information from the repair template to the target.
• an engineered, non-naturally occurring system or CRISPR expression system further comprises a donor template.
• the term “donor template” can refer to a nucleic acid designed to serve as a repair template at or near the target nucleotide sequence upon introduction into a cell or organism.
• the donor template is complementary to a polynucleotide comprising the target nucleotide sequence or a portion thereof.
• a donor template may overlap with one or more nucleotides of a target nucleotide sequences (e.g. about or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, or more nucleotides).
• the nucleotide sequence of the donor template is typically not identical to the genomic sequence that it replaces. Rather, the donor template may contain one or more substitutions, insertions, deletions, inversions or rearrangements with respect to the genomic sequence, so long as sufficient homology is present to support homology-directed repair.
• the donor template comprises a non-homologous sequence flanked by two regions of homology (i.e., homology arms), such that homology-directed repair between the target DNA region and the two flanking sequences results in insertion of the non-homologous sequence at the target region.
• the donor template comprises a non- homologous sequence 10-100 nucleotides, 50-500 nucleotides, 100-1,000 nucleotides, 200-2,000 nucleotides, or 500-5,000 nucleotides in length positioned between two homology arms.
• the homologous region(s) of a donor template has at least 50% sequence identity to a genomic sequence with which recombination is desired.
• the homology arms are designed or selected such that they are capable of recombining with the nucleotide sequences flanking the target nucleotide sequence under intracellular conditions.
• the donor template comprises a first homology arm homologous to a sequence 5’ to the target nucleotide sequence and a second homology arm homologous to a sequence 3’ to the target nucleotide sequence.
• the first homology arm is at least 50% (e.g., at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a sequence 5’ to the target nucleotide sequence.
• the second homology arm is at least 50% (e.g., at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a sequence 3’ to the target nucleotide sequence.
• the nearest nucleotide of the donor template is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, or more nucleotides from the target nucleotide sequence.
• the donor template further comprises an engineered sequence not homologous to the sequence to be repaired. Such engineered sequence can harbor a barcode and/or a sequence capable of hybridizing with a donor template-recruiting sequence disclosed herein.
• the donor template further comprises one or more mutations relative to the genomic sequence, wherein the one or more mutations reduce or prevent cleavage, by the same CRISPR-Cas system, of the donor template or of a modified genomic sequence with at least a portion of the donor template sequence incorporated.
• the PAM adjacent to the target nucleotide sequence and recognized by the Cas nuclease is mutated to a sequence not recognized by the same Cas nuclease.
• the target nucleotide sequence e.g., the seed region
• the one or more mutations are silent with respect to the reading frame of a protein-coding sequence encompassing the mutated sites.
• the donor template can be provided to the cell as single-stranded DNA, single- stranded RNA, double-stranded DNA, or double-stranded RNA. It is understood that a CRISPR- Cas system, such as a system disclosed herein, may possess nuclease activity to cleave the target strand, the non-target strand, or both. When HDR of the target strand is desired, a donor template having a nucleic acid sequence complementary to the target strand is also contemplated. [0146] The donor template can be introduced into a cell in linear or circular form.
• the ends of the donor template may be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art.
• one or more dideoxynucleotide residues are added to the 3 terminus of a linear molecule and/or self- complementary oligonucleotides are ligated to one or both ends (see, for example, Chang et al. (1987) PROC. NATL. ACAD SCI USA, 84: 4959; Nehls et al. (1996) SCIENCE, 272: 886; see also the chemical modifications for increasing stability and/or specificity of RNA disclosed supra).
• Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.
• additional lengths of sequence may be included outside of the regions of homology that can be degraded without impacting recombination.
• a donor template can be a component of a vector as described herein, contained in a separate vector, or provided as a separate polynucleotide, such as an oligonucleotide, linear polynucleotide, or synthetic polynucleotide.
• the donor template is a DNA.
• a donor template is in the same nucleic acid as a sequence encoding the single guide nucleic acid, a sequence encoding the targeter nucleic acid, a sequence encoding the modulator nucleic acid, and/or a sequence encoding the Cas protein, where applicable.
• a donor template is provided in a separate nucleic acid.
• the donor template is introduced as an AAV, e.g., a pseudotyped AAV.
• the capsid proteins of the AAV can be selected by a person skilled in the art based upon the tropism of the AAV and the target cell type.
• the donor template is introduced into a hepatocyte as AAV8 or AAV9.
• the donor template is introduced into a hematopoietic stem cell, a hematopoietic progenitor cell, or a T lymphocyte (e.g., CD8 + T lymphocyte) as AAV6 or an AAVHSC (see, U.S. Patent No.9,890,396).
• sequence of a capsid protein may be modified from a wild-type AAV capsid protein, for example, having at least 50% (e.g., at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to a wild-type AAV capsid sequence.
• the donor template can be delivered to a cell (e.g., a primary cell) by various delivery methods, such as a viral or non-viral method disclosed herein.
• a non- viral donor template is introduced into the target cell as a naked nucleic acid or in complex with a liposome or poloxamer.
• a non-viral donor template is introduced into the target cell by electroporation.
• a viral donor template is introduced into the target cell by infection.
• the engineered, non-naturally occurring system can be delivered before, after, or simultaneously with the donor template (see, International (PCT) Application Publication No. WO 2017/053729). A skilled person in the art will be able to choose proper timing based upon the form of delivery (consider, for example, the time needed for transcription and translation of RNA and protein components) and the half-life of the molecule(s) in the cell.
• the donor template e.g., as an AAV
• the donor template is introduced into the cell within 4 hours (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 90, 120, 150, 180, 210, or 240 minutes) after the introduction of the engineered, non-naturally occurring system.
• the donor template is conjugated covalently to a modulator nucleic acid. Covalent linkages suitable for this conjugation are known in the art and are described, for example, in U.S.
• the donor template is covalently linked to a modulator nucleic acid (e.g., the 5’ end of the modulator nucleic acid) through an internucleotide bond.
• the donor template is covalently linked to a modulator nucleic acid (e.g., the 5’ end of the modulator nucleic acid) through a linker.
• the donor template can comprise any nucleic acid chemistry.
• the donor template can comprise DNA and/or RNA nucleotides.
• the donor template can comprise single-stranded DNA, linear single- stranded RNA, linear double-stranded DNA, linear double-stranded RNA, circular single- stranded DNA, circular single-stranded RNA, circular double-stranded DNA, or circular double- stranded RNA.
• the donor template comprises a mutation in a PAM sequence to partially or completely abolish binding of the RNP to the DNA.
• the donor template is present at a concentration of at least 0.05, 0.01, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 3, or 4, and/or no more than 0.01, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 3, 4, or 5 ⁇ g ⁇ L -1 , for example 0.01-5 ⁇ g ⁇ L -1 .
• the donor template comprises one or more promoters.
• the donor template comprises a promoter that shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99.5% sequence identity with any one of SEQ ID NOs: 78-85 of Table 4. TABLE 4: Promoter sequences
• An engineered, non-naturally occurring system can be evaluated in terms of efficiency and/or specificity in nucleic acid targeting, cleavage, or modification.
• an engineered, non-naturally occurring system has high efficiency. For example, in certain embodiments, at least 1, 1.5, 2, 2.5, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% of a population of nucleic acids having the target nucleotide sequence and a cognate PAM, when contacted with the engineered, non-naturally occurring system, is targeted, cleaved, or modified.
• the genomes of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% of a population of cells, when the engineered, non-naturally occurring system is delivered into the cells, are targeted, cleaved, or modified.
• the frequency of off-target events e.g., targeting, cleavage, or modification, depending on the function of the CRISPR-Cas system
• off-target events were summarized in Lazzarotto et al. (2016) Nat Protoc.13(11): 2615-42, and include discovery of in situ Cas off-targets and verification by sequencing (DISCOVER-seq) as disclosed in Wienert et al.
• the off-target events include targeting, cleavage, or modification at a given off-target locus (e.g., the locus with the highest occurrence of off-target events detected).
• the off-target events include targeting, cleavage, or modification at all the loci with detectable off-target events, collectively.
• genomic mutations are detected in no more than 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, or 5% of the cells at any off-target loci (in aggregate).
• the ratio of the percentage of cells having an on-target event to the percentage of cells having any off-target event is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000. It is understood that genetic variation may be present in a population of cells, for example, by spontaneous mutations, and such mutations are not included as off-target events. E.
• the method of targeting, editing, and/or modifying a genomic DNA disclosed herein can be conducted in multiplicity.
• a library of targeter nucleic acids can be used to target multiple genomic loci; a library of donor templates can also be used to generate multiple insertions, deletions, and/or substitutions.
• the multiplex assay can be conducted in a screening method wherein each separate cell culture (e.g., in a well of a 96-well plate or a 384-well plate) is exposed to a different guide nucleic acid having a different targeter stem sequence and/or a different donor template.
• the multiplex assay can also be conducted in a selection method wherein a cell culture is exposed to a mixed population of different guide nucleic acids and/or donor templates, and the cells with desired characteristics (e.g., functionality) are enriched or selected by advantageous survival or growth, resistance to a certain agent, expression of a detectable protein (e.g., a fluorescent protein that is detectable by flow cytometry), etc.
• the plurality of guide nucleic acids and/or the plurality of donor templates are designed for saturation editing. For example, in certain embodiments, each nucleotide position in a sequence of interest is systematically modified with each of all four traditional bases, A, T, G and C.
• each sequence in each gene from a pool of genes of interest is modified, for example, according to a CRISPR design algorithm.
• each sequence from a pool of exogenous elements of interest e.g., protein coding sequences, non-protein coding genes, regulatory elements
• each sequence from a pool of exogenous elements of interest is inserted into one or more given loci of the genome.
• the multiplex methods suitable for the purpose of carrying out a screening or selection method which is typically conducted for research purposes, may be different from the methods suitable for therapeutic purposes.
• constitutive expression of certain elements e.g., a Cas nuclease and/or a guide nucleic acid
• constitutive expression of a Cas nuclease and/or a guide nucleic acid may be desirable.
• the constitutive expression provides a large window during which other elements can be introduced.
• constitutive expression of certain elements can increase the efficiency and reduce the complexity of a screening or selection process.
• Inducible expression of certain elements of the system disclosed herein may also be used for research purposes given similar advantages.
• Expression may be induced by an exogenous agent (e.g., a small molecule) or by an endogenous molecule or complex present in a particular cell type (e.g., at a particular stage of differentiation).
• exogenous agent e.g., a small molecule
• endogenous molecule or complex present in a particular cell type (e.g., at a particular stage of differentiation).
• Methods known in the art, such as those described herein, can be used for constitutively or inducibly expressing one or more elements.
• the specificity of CRISPR nucleases is at least partially dictated by the uniqueness of the spacer (in combination with spacer sequence’s proximity to a requisite PAM) and its off-target score can be calculated with algorithms, such as crispr.mit.edu (Hsu et al. (2013) Nat. Biotech.31: 827-832).
• the method disclosed herein further comprises a step of identifying a guide nucleic acid, a Cas protein, a donor template, or a combination of two or more of these elements from the screening or selection process.
• a set of barcodes may be used, for example, in the donor template between two homology arms, to facilitate the identification.
• the method further comprises harvesting the population of cells; selectively amplifying a genomic DNA or RNA sample including the target nucleotide sequence(s) and/or the barcodes; and/or sequencing the genomic DNA or RNA sample and/or the barcodes that has been selectively amplified.
• the present invention provides a library comprising a plurality of guide nucleic acids, such as a plurality of guide nucleic acids disclosed herein.
• the present invention provides a library comprising a plurality of nucleic acids each comprising a regulatory element operably linked to a different guide nucleic acid such as a different guide nucleic acid disclosed herein.
• These libraries can be used in combination with one or more Cas proteins or Cas-coding nucleic acids, such as disclosed herein, and/or one or more donor templates, such as disclosed herein, for a screening or selection method.
• Exogenous genes e.g., transgenes
• inserted into the genome of a target human cell either randomly, e.g., through retroviral vectors, or in a targeted manner, e.g., through the action of a nucleic acid-guided nuclease, such as Cas, may interact with other genomic elements in unpredictable ways.
• transgenes due to the complex transcriptional regulation of genes in mammalian cells through networks of cis and trans regulatory elements, such as proximal and distal enhancers, and multiple transcription factors, attempts to alter the default genomic architecture by integration of exogenous DNA, e.g., transgenes, or synthetic sequences can affect the expression of the transgene itself leading to complete attenuation or complete silencing, and/or the expression of both nearby and distant endogenous genes that can, e.g., compromise the safety checkpoints that healthy cells have including dysregulation of expression of key genes, such as oncogenes and tumor suppressor genes, that can alter cellular behavior in dramatic ways, i.e., promoting clonal expansion or malignant transformation of the host.
• exogenous DNA e.g., transgenes, or synthetic sequences
• key genes such as oncogenes and tumor suppressor genes
• Expression of exogenous genes, e.g., transgenes, in desired cell types and/or developmental/differentiation stages relies on integration into suitable target polynucleotide comprising a target nucleotide sequence that results in sufficient expression, to a degree sufficient for the intended purpose, from the candidate locus.
• suitable target polynucleotide comprising a target nucleotide sequence that results in sufficient expression, to a degree sufficient for the intended purpose, from the candidate locus.
• Expression from a specific genomic site can be affected by many factors including but not limited to cell type and differentiation stage, as one or more components of the target polynucleotide get activated during differentiation while others get silenced, and changes in chromatin architecture.
• gNAs novel guide nucleic acids
• a target polynucleotide includes a polynucleotide in which a target nucleotide sequence is located.
• a “target nucleotide sequence” includes a sequence to which a guide sequence can bind, e.g., has complementarity to, where binding between a target nucleotide sequence and a guide sequence may allow the activity of a nucleic acid-guided nuclease complex.
• Certain embodiments disclosed herein concern novel nucleic acid-guided nuclease complexes, e.g., RNPs, such as Cas bound to a gNA, that are complementary to a target nucleotide sequence within a target polynucleotide and hydrolyze the phosphodiester back bone (also referred as cleave or cut) in at least one position on at least one strand of the target polynucleotide.
• Certain embodiments disclosed herein concern methods for selecting and using gNAs, e.g., gRNAs, for genome engineering.
• Certain embodiments concern methods for using gNAs that are complementary to a target nucleotide sequence within a target polynucleotide, synthesizing the gNA and nucleic-acid-guided nuclease, and/or combining the nucleic guided nuclease with the gNA to form a nucleic acid-guided nuclease complex, e.g., RNP.
• Certain embodiments disclosed herein concern methods.
• Certain embodiments disclosed herein concern methods for engineering genomes.
• exogenous DNA or a “transgene” includes any gene, natural or synthetic, which is introduced into the genome of an organism or cell to which it is not endogenous.
• the transgene may or may not retain the ability to be expressed and/or produce RNA or protein in the human target cell.
• the transgene may or may not alter the resulting phenotype of the human target cell.
• Certain embodiments include human target cells, e.g., a eukaryotic cell, e.g., a mammalian cell, such as a human cell, for example a stem cell or an immune cell, generated through a method where the nucleic acid-guided nuclease complex, e.g., RNP, is introduced, e.g., transfected, into a human target cell along with a donor template, e.g., as an exogenous DNA or a transgene, such as a chimeric antigen receptor (CAR), in which the nucleic-acid guided nuclease cleaves at or near a targets sequence in a target polynucleotide and the donor template is used to repair the cleaved target polynucleotide introducing at least a portion of the donor template into the target polynucleotide.
• a eukaryotic cell e.g., a mammalian cell, such as a human cell
• a “site of cleavage” includes the location or locations at which a nucleic acid-guided nuclease complex will hydrolyze the phosphodiester backbone of a single- stranded or double-stranded target polynucleotide, after binding at a target nucleotide sequence in the target polynucleotide.
• binding of the nucleic acid-guided nuclease complex to a target nucleotide sequence within the target polynucleotide can result in hydrolysis of one of the strands of the target polynucleotide at or near the target nucleotide sequence, resulting in strand cleavage.
• the nucleic acid-guided nuclease complex can cleave either strand of the target polynucleotide.
• binding of the nucleic acid-guided nuclease complex to a target nucleotide sequence within a target polynucleotide can result in hydrolysis of both strands of the target polynucleotide at or near the target nucleotide sequence, resulting in cleavage of both strands.
• the sites of cleavage can be the same for both strands, resulting in a blunt end, or the sites of cleavage for each strand can be offset resulting in single strand overhangs, e.g., sticky ends.
• mismatches at or near the site of cleavage may or may not affect the cleavage efficiency of the nucleic acid-guided nuclease complex.
• Exemplary characteristics of a target nucleotide sequence that can demonstrate predictable function without potentially harmful alterations in human target cell genomic activity include one or more of (1) >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene, (2) >150 kb, for example, >200, such as >250, and in some cases >300 kb away from any miRNA/other functional small RNA, (3) >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end, (4) >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any replication origin, (5) >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any ultra-conserved element, (6) demonstrating low transcriptional activity, (7) outside of a copy number variable region, (8) located in open chromatin, and (9) unique
• compositions In certain embodiments, provided herein are compositions. In certain embodiments, provided herein are compositions for engineering a human target cell at suitable target nucleotide sequences within a target polynucleotide of the human target cell. [0173] In certain embodiments, a suitable target polynucleotide that comprises a target nucleotide sequence has at least one of the exemplary characteristics. In certain embodiments, a suitable target polynucleotide that comprises a target nucleotide sequence has at least two of the exemplary characteristics. In certain embodiments, a suitable target polynucleotide that comprises a target nucleotide sequence has at least three of the exemplary characteristics.
• a suitable target polynucleotide that comprises a target nucleotide sequence has at least four of the exemplary characteristics. In certain embodiments, a suitable target polynucleotide that comprises a target nucleotide sequence has at least five of the exemplary characteristics. In certain embodiments, a suitable target polynucleotide that comprises a target nucleotide sequence has at least six of the exemplary characteristics. In certain embodiments, a suitable target polynucleotide that comprises a target nucleotide sequence has at least seven of the exemplary characteristics. In certain embodiments, a suitable target polynucleotide that comprises a target nucleotide sequence has at least eight of the exemplary characteristics.
• a suitable target polynucleotide that comprises a target nucleotide sequence has all the exemplary characteristics.
• a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end.
• a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end and further comprises at one additional exemplary characteristic.
• a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end and further comprises at least two additional exemplary characteristics.
• a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end and further comprises at least three additional exemplary characteristics.
• a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end and further comprises at least four additional exemplary characteristics.
• a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end and further comprises at least five additional exemplary characteristics.
• a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end and further comprises at least six additional exemplary characteristics.
• a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end and further comprises at least seven additional exemplary characteristics.
• a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end and further comprises all eight additional exemplary characteristics.
• a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene.
• a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene and further comprises at one additional exemplary characteristic.
• a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene and further comprises at least two additional exemplary characteristics.
• a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene and further comprises at least three additional exemplary characteristics.
• a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene and further comprises at least four additional exemplary characteristics.
• a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene and further comprises at least five additional exemplary characteristics.
• a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene and further comprises at least six additional exemplary characteristics.
• a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene and further comprises at least seven additional exemplary characteristics.
• a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene and further comprises all eight additional exemplary characteristics.
• a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene, and >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end.
• a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene, >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end, and further comprises at least one additional exemplary characteristic.
• a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene, >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end, and further comprises at least two additional exemplary characteristics.
• a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene, >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end, and further comprises at least three additional exemplary characteristics.
• a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene, >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end, and further comprises at least four additional exemplary characteristics.
• a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene, >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end, and further comprises at least five additional exemplary characteristics.
• a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene, >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end, and further comprises at least six additional exemplary characteristics.
• a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene, >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end, and further comprises all seven additional exemplary characteristics.
• a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end and >150, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene.
• a suitable target polynucleotide comprising a target nucleotide sequence may comprise any one of SEQ ID NOs: 2020- 2043 of Table 5.
• a suitable target polynucleotide comprising a target nucleotide sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or completely identical to any one of SEQ ID NOs: 2020-2043.
• a suitable target polynucleotide comprising a target nucleotide sequence is at least 98% identical to any one of SEQ ID NOs: 2020-2043. In a more preferred embodiment, a suitable target polynucleotide comprising a target nucleotide sequence is at least 99% identical to any one of SEQ ID NOs: 2020-2043. [0179] In certain embodiments, a suitable target polynucleotide comprising a target nucleotide sequence, e.g., for transgene insertion, may comprise any one of SEQ ID NOs: 2020- 2042 of Table 5.
• a suitable target polynucleotide comprising a target nucleotide sequence is at least 98% identical to any one of SEQ ID NOs: 2020-2042. In a more preferred embodiment, a suitable target polynucleotide comprising a target nucleotide sequence is at least 99% identical to any one of SEQ ID NOs: 2020-2042. [0180] In certain embodiments, a suitable target polynucleotide comprising a target nucleotide sequence, e.g., for transgene insertion, may comprise any one of SEQ ID NOs: 2020- 2041 and 2043 of Table 5.
• a suitable target polynucleotide comprising a target nucleotide sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or completely identical to any one of SEQ ID NOs: 2020-2041 and 2043.
• a suitable target polynucleotide comprising a target nucleotide sequence is at least 98% identical to any one of SEQ ID NOs: 2020-2041 and 2043. In a more preferred embodiment, a suitable target polynucleotide comprising a target nucleotide sequence is at least 99% identical to any one of SEQ ID NOs: 2020-2041 and 2043. [0181] In certain embodiments, a suitable target polynucleotide comprising a target nucleotide sequence, e.g., for transgene insertion, may comprise any one of SEQ ID NOs: 2020- 2041 of Table 5.
• a suitable target polynucleotide comprising a target nucleotide sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or completely identical to any one of SEQ ID NOs: 2020-2041.
• a suitable target polynucleotide comprising a target nucleotide sequence is at least 98% identical to any one of SEQ ID NOs: 2020-2041. In a more preferred embodiment, a suitable target polynucleotide comprising a target nucleotide sequence is at least 99% identical to any one of SEQ ID NOs: 2020-2041.
• the guide nucleic acid comprises: (i) a targeter nucleic acid comprising: (a) a spacer sequence configured to hybridize with a target nucleotide sequence, and (b) a targeter stem sequence; and (ii) a modulator nucleic acid comprising: (a) a modulator stem sequence complementary to the target stem sequence, and (b) a 5’ sequence.
• the modulator nucleic acid comprises a sequence at least 50, 60, 70, 80, 90, 95, 99, 99.5, or 100% identical to any one of the sequences listed in Table 6.
• the composition comprises an RNP comprising the targeter nucleic acid, the modulator nucleic acid, and a Cas protein (e.g., Cas nuclease).
• a Cas protein e.g., Cas nuclease.
• the method comprising incubating a single guide nucleic acid, such as a single guide nucleic acid disclosed herein, with a Cas protein, thereby producing a complex of the single guide nucleic acid and the Cas protein (e.g., an RNP).
• the method further comprises purifying the complex (e.g., the RNP).
• a method of producing a composition comprising incubating a targeter nucleic acid and a modulator nucleic acid, such as a targeter nucleic acid and a modulator nucleic acid disclosed herein, under suitable conditions, thereby producing a composition (e.g., pharmaceutical composition) comprising a complex of the targeter nucleic acid and the modulator nucleic acid.
• a modulator nucleic acid such as a targeter nucleic acid and a modulator nucleic acid disclosed herein
• a guide nucleic acid, an engineered, non-naturally occurring system, a CRISPR expression system, or a cell comprising such system or modified by such system disclosed herein is combined with a pharmaceutically acceptable carrier.
• pharmaceutically acceptable can refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit-to-risk ratio.
• a subject composition comprises a subject DNA-targeting RNA, e.g., gRNA, and a buffer for stabilizing nucleic acids.
• a pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
• suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta- cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents
• amino acids
• a pharmaceutical composition may contain nanoparticles, e.g., polymeric nanoparticles, liposomes, or micelles (See Anselmo et al. (2016) Bioeng. Transl. Med.1: 10-29).
• the pharmaceutical composition comprises an inorganic nanoparticle.
• Exemplary inorganic nanoparticles include, e.g., magnetic nanoparticles (e.g., Fe3MnO2) or silica.
• the outer surface of the nanoparticle can be conjugated with a positively charged polymer (e.g., polyethylenimine, polylysine, polyserine) which allows for attachment (e.g., conjugation or entrapment) of payload.
• a positively charged polymer e.g., polyethylenimine, polylysine, polyserine
• the pharmaceutical composition comprises an organic nanoparticle (e.g., entrapment of the payload inside the nanoparticle).
• Exemplary organic nanoparticles include, e.g., SNALP liposomes that contain cationic lipids together with neutral helper lipids which are coated with polyethylene glycol (PEG) and protamine and nucleic acid complex coated with lipid coating.
• the pharmaceutical composition comprises a liposome, for example, a liposome disclosed in International (PCT) Application Publication No. WO 2015/148863.
• the pharmaceutical composition comprises a targeting moiety to increase target cell binding or update of nanoparticles and liposomes.
• targeting moieties include cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars, and cell penetrating peptides.
• the pharmaceutical composition comprises a fusogenic or endosome-destabilizing peptide or polymer.
• a pharmaceutical composition may contain a sustained- or controlled-delivery formulation.
• sustained- or controlled-delivery means such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art.
• Sustained-release preparations may include, e.g., porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules.
• Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly (2- hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D( _ )-3-hydroxybutyric acid.
• Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art.
• a pharmaceutical composition of the invention can be administered by a variety of methods known in the art. The route and/or mode of administration vary depending upon the desired results. Administration can be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target.
• the pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
• the active compound e.g., the guide nucleic acid, engineered, non-naturally occurring system, or CRISPR expression system disclosed herein
• the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
• Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents
• antibacterial agents such as benzyl alcohol or methyl parabens
• antioxidants such as ascorbic acid or sodium bisulfite
• chelating agents such as EDTA
• buffers such as acetates, citrates or phosphates
• suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
• the carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof.
• Pharmaceutical formulations preferably are sterile. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes.
• compositions of the invention can be prepared in accordance with methods well known and routinely practiced in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably manufactured under GMP conditions.
• compositions of the invention typically employ a therapeutically effective dose or efficacious dose of the guide nucleic acid, engineered, non- naturally occurring system, or CRISPR expression system disclosed herein is employed in the pharmaceutical compositions of the invention.
• the compositions disclosed herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
• Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. [0202] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
• the selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions disclosed herein employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.
• Guide nucleic acids, engineered, non-naturally occurring systems, and the CRISPR expression systems, e.g., as disclosed herein, are useful for targeting, editing, and/or modifying the genomic DNA in a cell or organism.
• treatment can refer to obtaining a desired pharmacologic and/or physiologic effect.
• the effect may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease or delaying the disease progression.
• Treatment covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease, i.e., causing regression of the disease.
• a disease or disorder may be identified by genetic methods and treated prior to manifestation of any medical symptom.
• it can be important to control the concentration of the CRISPR-Cas system delivered.
• Optimal concentrations can be determined by testing different concentrations in a cellular, tissue, or non-human eukaryote animal model and using deep sequencing to analyze the extent of modification at potential off-target genomic loci. The concentration that gives the highest level of on-target modification while minimizing the level of off-target modification is generally selected for ex vivo or in vivo delivery.
• the guide nucleic acid, the engineered, non-naturally occurring system, and the CRISPR expression system disclosed herein can be used to treat any suitable disease or disorder that can be improved by the system in a cell.
• certain methods disclosed herein is particularly suitable for editing or modifying a proliferating cell, such as a stem cell (e.g., a hematopoietic stem cell), a progenitor cell (e.g., a hematopoietic progenitor cell or a lymphoid progenitor cell), or a memory cell (e.g., a memory T cell).
• a stem cell e.g., a hematopoietic stem cell
• a progenitor cell e.g., a hematopoietic progenitor cell or a lymphoid progenitor cell
• a memory cell e.g., a memory T cell
• the cells can include autologous cells derived from a subject to be treated, or alternatively allogenic cells derived from a donor.
• the immune cell is a T cell, which can be, for example, a cultured T cell, a primary T cell, a T cell from a cultured T cell line (e.g., Jurkat, SupTi), or a T cell obtained from a mammal, for example, from a subject to be treated. If obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched or purified.
• the T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4 + /CD8 + double positive T cells, CD4 + helper T cells (e.g., Th1 and Th2 cells), CD8 + T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), regulatory T cells, naive T cells, or the like.
• an immune cell e.g., a T cell, is engineered to express an exogenous gene.
• an engineered CRISPR system disclosed herein may catalyze DNA cleavage at the gene locus, allowing for site-specific integration of the exogenous gene at the gene locus by HDR.
• an immune cell e.g., a T cell
• a chimeric antigen receptor i.e., the T cell comprises an exogenous nucleotide sequence encoding a CAR.
• the term “chimeric antigen receptor” or “CAR” includes any artificial receptor including an antigen-specific binding moiety and one or more signaling chains derived from an immune receptor.
• CARs can comprise a single chain fragment variable (scFv) of an antibody specific for an antigen coupled via hinge and transmembrane regions to cytoplasmic domains of T cell signaling molecules, e.g. a T cell costimulatory domain (e.g., from CD28, CD137, OX40, ICOS, or CD27) in tandem with a T cell triggering domain (e.g. from CD3 ).
• T cell costimulatory domain e.g., from CD28, CD137, OX40, ICOS, or CD27
• a T cell expressing a chimeric antigen receptor is referred to as a CAR T cell.
• Exemplary CAR T cells include CD19 targeted CTL019 cells (see, Grupp et al. (2015) BLOOD, 126: 4983), 19-28z cells (see, Park et al. (2015) J. CLIN.
• an immune cell e.g., a T cell
• binds an antigen e.g., a cancer antigen
• an endogenous T cell receptor TCR
• an immune cell e.g., a T cell
• is engineered to express an exogenous TCR e.g., an exogenous naturally occurring TCR or an exogenous engineered TCR.
• T cell receptors comprise two chains referred to as the ⁇ - and ⁇ - chains, that combine on the surface of a T cell to form a heterodimeric receptor that can recognize MHC-restricted antigens.
• Each of ⁇ - and ⁇ -chain comprises a constant region and a variable region.
• Each variable region of the ⁇ - and ⁇ -chains defines three loops, referred to as complementary determining regions (CDRs) known as CDR 1 , CDR 2 , and CDR 3 that confer the T cell receptor with antigen binding activity and binding specificity.
• CDRs complementary determining regions
• a CAR or TCR binds a cancer antigen selected from B-cell maturation antigen (BCMA), mesothelin, prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD70, CD74, CD123, CD133, CD138, epithelial glycoprotein2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor-type tyrosine- protein kinase (FLT3), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a and (FRa and ), Ganglioside G2 (GD2), Ganglioside G2 (GD2), Gan
• TCR subunit loci e.g., the TCR constant (TRAC) locus, the TCR constant 1 (TRBC1) locus, and the TCR constant 2 (TRBC2) locus. It is understood that insertion in the TRAC locus reduces tonic CAR signaling and enhances T cell potency (see, Eyquem et al. (2017) NATURE, 543: 113).
• T cells also express major histocompatibility complex (MHC) or human leukocyte antigen (HLA) genes, and inactivation of these endogenous gene may reduce an immune response, thereby allowing use of allogeneic T cells as starting materials for preparation of CAR T cells.
• MHC major histocompatibility complex
• HLA human leukocyte antigen
• the cell may be modified to have partially reduced or no expression of the immune checkpoint protein.
• the immune cell e.g., a T cell
• the immune cell is engineered to have less than 80% (e.g., less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%) of the expression of the immune checkpoint protein relative to a corresponding unmodified or parental cell.
• the immune cell e.g., a T cell
• Exemplary approaches to reduce expression of immune checkpoint proteins using CRISPR systems are described in International (PCT) Publication No. WO 2017/017184, Cooper et al.
• embodiment 20 provided herein is the method of any one of embodiments 17-19, wherein the method comprises delivering at least two gNAs, or polynucleotides encoding the gNAs, wherein each gNA comprises a different spacer sequence such that when complexed with a nucleic acid-guided nuclease, the nucleic acid-guided nuclease complexes form strand breaks in the genome at or near each of the target nucleotide sequences.
• embodiment 29 provided herein is the composition of embodiment 21-28, wherein the modulator nucleic acid comprises at least one modified nucleotide and at least two modified internucleotide linkages within the first five nucleotides from the 5’ end.
• embodiment 30 provided herein is the composition of embodiment 21-29, further comprising a Type V nucleic acid-guided nuclease.
• embodiment 31 provided herein is the composition of embodiment 30, wherein the Type V nucleic acid-guided nuclease is at least 80% identical to an ABW, ART, or MAD nuclease.
• embodiment 32 provided herein is the composition of any one of embodiments 21- 31, wherein the modulator nucleic acid comprises a sequence at least 50, 60, 70, 80, 90, 95, 99, 99.5, or 100% identical to any one of the sequences listed in Table 6.
• embodiment 33 provided herein is the composition of any one of embodiments 21- 32, wherein the targeter nucleic acid comprises a sequence at least 50, 60, 70, 80, 90, 95, 99, 99.5, or 100% identical to any one of the sequences listed in Table 7.
• a method of editing a genome of a eukaryotic cell comprising (I) delivering to the eukaryotic cell (A) one or more synthetic guide nucleic acids (gNA), or polynucleotides encoding the one or more gNAs, comprising (i) a targeter nucleic acid comprising: (a) a spacer sequence configured to hybridize with a target nucleotide sequence, and (b) a targeter stem sequence; and (ii) a modulator nucleic acid comprising: (a) a modulator stem sequence complementary to the target stem sequence, and (b) a 5’ sequence; wherein (1) the targeter nucleic acid and modulator nucleic acids are separate polynucleotides, (2) the predicted minimum free energy of the targeter stem sequence and the modulator stem sequence as determined by the RNAcofold WebServer is between -10 and -4 kcal/mol, and (3) the gNA is capable of binding to and forming
• compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.
• an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

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