WO2022104381A1

WO2022104381A1 - A MINIMAL CRISPRi/a SYSTEM FOR TARGETED GENOME REGULATION

Info

Publication number: WO2022104381A1
Application number: PCT/US2021/072393
Authority: WO
Inventors: Lei S. QI; Xiaoshu Xu
Original assignee: The Board Of Trustees Of The Leland Stanford Junior University
Priority date: 2020-11-13
Filing date: 2021-11-12
Publication date: 2022-05-19

Abstract

The present disclosure relates to systems, compositions, and methods for modulating the expression of a target nucleic acid employing a nuclease-deficient variant of a Cas14 CRISPR-Cas effector protein.

Description

A Minimal CRISPRi/a System for Targeted Genome Regulation

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 63/113,217 filed November 13, 2020, the full disclosure of which is incorporated by reference in its entirety for all purposes.

FIELD

[0002] The present disclosure generally relates to systems, compositions, and methods for modulating the expression of a target nucleic acid in a cell. The systems include a nuclease- deficient variant of a Casl4 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) effector protein (dCasl4) and a guide RNA complementary to a target sequence in the target nucleic acid, wherein the dCasl4 protein interacts with the guide RNA and binds to the target nucleic acid sequence in a site specific manner, thereby modulating expression of the target nucleic acid.

BACKGROUND

[0003] Gene regulation is core to manipulating cell function, cell fate, and implementing gene- or cell-based therapies. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) has shown tremendous utility for research, as well as development of novel gene and cell therapies. Many current CRISPRi/a systems and methods are based on Type II CRISPR Cas9 systems using nuclease-deactivated Cas9 (dCas9) and corresponding single guide RNAs (sgRNAs). However, these Cas9-based systems suffer from various limitations. The relatively large size of Cas9 proteins, which are usually 1,000-1,500 amino acids in length, makes them challenging to encode on a single viral vector for delivery. This large size also impairs delivery efficiency into many primary human cell types both tn vitro and in vivo when using various delivery modalities such as ribonucleoproteins (RNPs), nanoparticles, and electroporation, in DNA, RNA, or protein forms. Efforts to reduce the size of Cas9 proteins have encountered substantial challenges, such as preventing significantly decreased activity, as the protein evolved with inter-connected domains and disrupting one domain interferes with the function of the whole protein. Therefore, there is currently a need for the discovery and/or engineering of new, smaller Cas molecules that can be used for DNA- or RNA-targeted gene regulation. SUMMARY

[0004] This section provides a general summary of the disclosure, and is not comprehensive of its full scope or all of its features.

[0005] In one aspect, provided herein is a system comprising: a Casl4 protein or variant thereof, or nucleic acid encoding the Casl4 protein or variant; and a guide RNA (gRNA) or nucleic acid encoding the gRNA, wherein the gRNA comprises a spacer having sufficient complementary to a target nucleotide sequence in a target nucleic acid such that it can hybridize to the target nucleic acid. In some embodiments, the gRNA is a single-guide RNA (sgRNA).

[0006] In some embodiments, according to any of the systems described above, the Casl4 protein or variant thereof recognizes a protospacer adjacent motif (PAM) having the sequence 5'-TTTG-3' or 5'-TTTA-3' located immediately 5' (upstream) of the protospacer (see, e.g., FIG. 2E showing exemplary Casl4 system gRNA spacers and corresponding nucleic acid target sites). In some embodiments, the Casl4 protein or variant is a dCasl4 protein having reduced or eliminated nuclease activity as compared to the parental Casl4 protein from which it is derived. In some embodiments, the parental Casl4 protein comprises the amino acid sequence of SEQ ID NO: 58. In some embodiments, the dCasl4 protein comprises one or more amino acids that have been altered or otherwise removed to reduce or eliminate its nuclease activity. In some embodiments, the one or more amino acids include D326 and D510 with respect to SEQ ID NO: 58. In some embodiments, one or both of D326 and D510 are substituted with an amino acid that reduces, substantially eliminates, or eliminates nuclease activity. In some embodiments, one or both of D326 and D510 are substituted with alanine. In some embodiments, the dCas14 protein comprises the amino acid sequence of SEQ ID NO: 60.

[0007] In some embodiments, according to any of the systems described above, the target nucleic acid is a target dsDNA. In some embodiments, a protospacer in the target dsDNA targeted by the gRNA spacer has a PAM selected from 5'-TTTG-3' and 5'-TTTA-3'. In some embodiments, the target dsDNA is a genomic DNA and the target nucleotide sequence is present in the sense strand of the genomic DNA corresponding to an mRNA transcribed from the genomic DNA. In some embodiments, the target dsDNA is a genomic DNA and the target nucleotide sequence is present in the anti-sense strand of the genomic DNA complementary to an mRNA transcribed from the genomic DNA. In some embodiments, targeting of the genomic DNA by the Casl4 systems is capable of modulating transcription of the mRNA. In some embodiments, targeting of the genomic DNA by the Casl4 system is capable of repressing transcription of the mRNA. In some embodiments, targeting of the genomic DNA by the Casl4 system is capable of enhancing transcription of the mRNA. In some embodiments, targeting of the mRNA by the Gas 14 system is capable of modulating translation of the mRNA. In some embodiments, targeting of the mRNA by the Cas14 system is capable of repressing translation of the mRNA. In some embodiments, targeting of the mRNA by the Casl4 system is capable of enhancing translation of the mRNA.

[0008] In some embodiments, according to any of the systems described above, the target nucleic acid is a target RNA. In some embodiments, the target RNA is an mRNA transcribed from a genomic DNA, and the target nucleotide sequence is present in the sense strand of the genomic DNA corresponding to the mRNA. In some embodiments, any protospacers in the genomic DNA targeted by the gRNA spacer do not have a PAM selected from 5'-TTTG-3' and 5'-TTTA-3'. In some embodiments, targeting of the mRNA by the Casl4 system is capable of modulating translation of the mRNA. In some embodiments, targeting of the mRNA by the Casl4 system is capable of repressing translation of the mRNA. In some embodiments, targeting of the mRNA by the Casl4 system is capable of enhancing translation of the mRNA. [0009] In some embodiments, according to any of the systems described above, the target nucleotide sequence is present in a gene sequence. In some embodiments, the target nucleotide sequence is present in a promoter region of the gene. In some embodiments, the target nucleotide sequence is present in a 5’ UTR region of the gene. In some embodiments, the target nucleotide sequence is present in a 5’ UTR/RBS region of the gene. In some embodiments, the target nucleotide sequence is present in a coding region of the gene.

[0010] In another aspect, provided herein is a method for modulating the expression of a target gene in a cell to produce an engineered cell, the method comprising providing to the cell a system according to any of the embodiments described above, wherein the target nucleotide sequence is present in the target gene, thereby producing an engineered cell having modulated expression of the target gene.

[0011] In another aspect, provided herein is an engineered cell produced by a method according to any of the embodiments described above.

[0012] In another aspect, provided herein is an engineered cell comprising a system according to any of the embodiments described above.

[0013] In another aspect, provided herein is a method for treating a disease or condition associated with a target gene in a subject in need thereof, the method comprising providing to the subject an engineered cell according to any of the embodiments described above, wherein the expression of the target gene in the engineered cell is modulated such that the disease or condition is treated.

[0014] In another aspect, provided herein is a method for treating a disease or condition associated with a target gene in a subject in need thereof, the method comprising modifying a cell in the subject to produce an engineered cell according to any of the embodiments described above, wherein the expression of the target gene in the engineered cell is modulated such that the disease or condition is treated.

[0015] In another aspect, provided herein is a kit comprising one or more components of a system according to any of the embodiments described above.

[0016] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1A shows a schematic of an exemplary' dCasl4al-mediated CRISPRi system consisting of a catalytic dead mutant Casl4al/D326A/D510A (dCasl4al-l) and a designed sgRNA chimera. The protein dCasl4al-l is expressed under the control of an anhydrotetracycline (aTc)-inducible promoter Ptet. The sgRNA is expressed from a minimal constitute promoter Pj23119.

[0018] FIG. IB shows a model for a hypothesis of gene repression in Escherichia coli (E. coli) mediated by the dCasl4al CRISPRi system of FIG. 1A.

[0019] FIG. 1C shows a schematic of an exemplary' synthetic fluorescence-based reporter system in E. coli.

[0020] FIGS. 2A and 2C show results for mRFP (FIG. 2A) and GFP (FIG. 2C) expression in cells with the reporter system of FIG. 1C and engineered to express dCas9 and sgRR2, an sgRNA targeting the coding sequence of mRFP on the non-template strand.

[0021] FIGS. 2B and 2D show results for mRFP (FIG. 2B) and GFP (FIG. 2D) expression in cells with the reporter system of FIG. 1C and engineered to express dCasl4al-l and a series of sgRNAs (alGRRl, alGRR2, alGRR3, alGRR4, alGRR5, and alGRR6) targeting the promoter regions of sfGRP and mRFP. sgRNAs alNT2 and alNT3 were included as control non-target sgRNAs.

[0022] FIG. 2E shows the binding sites on the promoter regions of sfGRP and mRFP for sgRNAs alGRRl, alGRR2, alGRR3, alGRR4, alGRR5, and alGRR6.

[0023] FIGS. 3A-3F show results for GFP (FIGS. 3A, 3C, and 3E) and RFP (FIGS. 3B, 3D, and 3F) expression in the reporter system shown in FIG. 1C following treatment with a dCasl4al CRISPRi system with sgRNAs targeting seven different sites in the GFP coding sequence as indicated (site G-2: alGR4-alGR6; site G-3: alGR7-alGR9; site G-4: alGRIO and alGR12; site G-5: alGR13-alGR15; site G-6: alGR16-alGR18; site G-7: alGR19 and alGR21; and site G-8: alGR22-alGR24) (FIGS. 3C, 3D, 3E, and 3F) or a dCas9 CRISPRi system with sgRR2 (FIGS. 3A and 3B). sgRNAs alNT2 and alNT3 were included as control non-target sgRNAs.

[0024] FIG. 3G shows the binding sites in the sfGFP expression cassette for sgRNAs alGR4- alGR24.

[0025] FIGS. 4A-4E show results for GFP (FIGS. 4A, 4C, and 4E) and RFP (FIGS. 4B, 4D, and 4E) expression in the reporter system shown in FIG. 1C following treatment with a dCasl4al CRISPRi system with sgRNAs targeting seven different sites in the RFP coding sequence as indicated (site R-l: alRRl-alRR15; site R-2: alRR16-alRR18; site R-3: alRR19- alRR21; site R-4: alRR22-alRR23; site R-5: alRR25-alRR27; site R-6: alRR28; and site R- 7: alRR29) (FIGS. 4C, 4D, and 4E) or a dCas9 CRISPRi system with sgRR2 (FIGS. 4A and 4B). FIG. 4E shows results for GFP and RFP expression at 6 hours, 7 hours, 8 hours, 9 hours, and 10.5 hours after the induction of dCasl4al-l for alRRl-alRR4, alRR28, and alRR29. sgRNAs alNTl, alNT2, or alNT3 were included as control non-target sgRNAs.

[0026] FIG. 4F shows the binding sites in the mRFP expression cassette for sgRNAs alRRl- alRR29.

[0027] FIGS. 5A and 5B show results for GFP (FIG. 5A) and RFP (FIG. 5B) expression in the reporter system shown in FIG. 1C following treatment with a dCasl4al CRISPRi system with sgRNAs targeting the 5’ UTR region on the sense strand of the GFP expression cassette and partially matching a region of the 5’ UTR of RFP (alGRl, alGR2, and alGR3). sgRNAs alNT2 and alNT3 were included as control non-target sgRNAs.

[0028] FIG. 5C shows the binding sites in the sfGFP and mRFP expression cassettes for sgRNAs alGRl -alGR3.

[0029] FIGS. 6A-6C show illustrative schematics for a dCasl4al CRISPR system targeting both DNA and RNA (FIG. 6A), DNA only (FIG. 6B), and RNA only (FIG. 6C).

[0030] FIG. 7 shows a photograph of a gel used in an electrophoretic mobility shift assay (EMSA) study that demonstrated strong binding of dCasl4 to ssDNA. DETAILED DESCRIPTION OF THE DISCLOSURE

[0031] The present invention relates to engineered or isolated CRISPR-Cas effector protein Casl4, such as nuclease-deficient Casl4 (dCasl4). The invention further relates to CRISPR- Cas systems comprising such Casl4 proteins, as well as polynucleotide sequences encoding such Casl4 proteins or systems and vectors or vector systems comprising such and delivery systems comprising such. The invention further relates to cells or cell lines or organisms comprising such Casl4 proteins, CRISPR-Cas systems, polynucleotide sequences, vectors, vector systems, and/or delivery systems. The invention further relates to medical and nonmedical uses of such Casl4 proteins, CRISPR-Cas systems, polynucleotide sequences, vectors, vector systems, delivery systems, cells, cell lines, and/or organisms, such as for targeted genome regulation. The Casl4 protein may be a wild type Casl4 protein or a mutated (such as comprising point mutaiion(s) and/or truncations) Casl4 protein. In one aspect, embodiments disclosed herein are directed to engineered Gas 14 proteins that comprise at least one modification compared to an unmodified Casl4 protein that reduces or eliminates its endonuclease activity.

[0032] Further aspects of the present disclosure are directed to methods of modulating expression of a target nucleic acid in a cell. The methods include providing to the cell a guide RNA complementary' to the target nucleic acid sequence and a dCasl4 protein. According to another aspect, the present disclosure provides nucleic acids encoding the guide RNA and the dCasl4 protein. In some embodiments, the nucleic acids encoding the guide RNA and the dCasl4 protein are present on a single vector or on separate vectors, such as engineered DNA plasmid vectors or viral vectors. The vector is then used to deliver the nucleic acids encoding the guide RNA and the dCasl4 protein into the desired cells or tissues. Once delivered into the cell, the dCasl4 protein and the guide RNA are expressed. The guide RNA comprises a portion that is complementary to a sequence of a target site and guides the dCasl4 protein to the target site. In this manner, expression of the target nucleic acid sequence is modulated depending on the location of the target site.

[0033] Using a dCas14 al -mediated CRISPRi system based on a catalyticly dead variant of Cas 14al , targeted repression of two different genes at different target sites throughout the genes was successfully achieved. Notably, the dCasl4 system was able to target both dsDNA (when an appropriate PAM was present) and RNA (requiring only a matching spacer sequence in the gRNA). [0034] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols generally identify similar components, unless context dictates otherwise. The illustrative alternatives described in the detailed description, drawings, and claims are not meant to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this application.

[0035] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2nd edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4th edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (MJ. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboraotry Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboraotry Manual, 2nd edition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al . (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology- and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hoflcer and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011). Definitions

[0036] As used herein, the singular forms “a,” “an,” and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0037] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0038] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0039] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes, such as variations of +/- 10% or less, +/- 1-5% or less, +/- 1% or less, and +/- 0.1% or less from the specified value. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0040] The terms “subject” and “individual” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. In some cases, a subject is a patient. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0041] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub- combination was individually and explicitly disclosed herein.

[0042] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.

CAS14 NUCLEIC ACID-TARGETING SYSTEMS

[0043] Class 2 CRISPR-Cas systems endow microbes with diverse mechanisms for adaptive immunity. CRISPR systems are generally divided into two classes, with class 1 systems using a complex of multiple Cas proteins to degrade foreign nucleic acids, and class 2 systems using a single, generally larger, Cas protein for the same purpose. Class 1 is divided into types I, HI, and IV, and class 2 is divided into types II, V, and VI. Provided herein is an analysis of RNA- guided, nucleic acid-targeting CRISPR systems classified as Type V-F employing Cas 14 or an engineered variant thereof as the effector protein.

[0044] Embodiments of the present disclosure are directed to Cas 14-based regulation of gene expression, such as at the transcriptional and/or translational level. RNA-guided nucleic acidbinding proteins are readily known to those of skill in the art to bind to DNA or RNA for various purposes, including those having nuclease activity, such as Type II RNA-guided DNA- binding Cas9 endonucleases. Cas 14 proteins are Type V subtype F RNA-guided nucleic acidbinding proteins that can be targeted to DNA and/or RNA, and are much smaller than typical CRISPR effectors, ranging in size from about 400 amino acids to about 700 amino acids (Y an, W. X., et al. (2019). Science, 363(6422), 88-91; Harrington, L. B., et al. (2018). Science, 362(6416), 839-842). At least 24 different Casl4 variants have been identified that cluster into three subgroups, Cas 14a, Cas 14b, and Cas 14c, based on sequence comparison, all of which share a predicted RuvC nuclease domain characteristic of type V CRISPR-Cas DNA -targeting enzymes. The small size of Cas 14 proteins allows Cas 14 proteins and effector domain fusions thereof to be paired with a CRISPR array encoding multiple guide RNAs while remaining under the packaging size limit of the versatile adeno-associated virus (AAV) delivery vehicle for primary cell and in vivo delivery. Targeted AAV delivery of dCasl4 systems to cells can allow for long-term expression of a corrective payload that avoids permanent genetic modifications or frequent re-administration (Chiriboga et al., 2016), complementing other nucleic acid-targeting technologies such as DNA nuclease editing or antisense oligonucleotides. RNA mis-splicing diseases have been estimated to account for up to 15% of genetic diseases (Hammond and Wood, 2011), highlighting the potential for engineered splice effectors capable of multiplexed targeting. CRISPR-Casl4 and engineered variants such as dCasl4 allow for flexible nucleic acid engineering, regulation of gene expression, and therapeutics, expanding the genome editing and regulation toolbox. dCasl4

[0045] Modifications to Casl4 proteins are contemplated by the present disclosure. In some embodiments, a Casl4 protein is altered or otherwise modified to inactivate its nuclease activity. Such alteration or modification includes altering one or more amino acids to inactivate the nuclease activity or the nuclease domain, as well as removing the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e., the nuclease domain, such that the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e. nuclease domain, are absent from the Casl4 protein. Thus, in some embodiments, a nuclease-null Gas 14 protein (dCasl4) includes polypeptide sequences modified to inactivate nuclease activity or removal of a polypeptide sequence or sequences to inactivate nuclease activity. The dCasl4 protein retains the ability to bind to target nucleic acid even though the nuclease activity has been inactivated. Accordingly, the dCasl4 protein includes the polypeptide sequence or sequences required for nucleic acid binding.

[0046] In some embodiments, provide herein is a dCas14 protein where one or more amino acids of the parental Casl4 protein from which it is derived have been altered or otherwise removed to reduce or eliminate its nuclease activity. In some embodiments, the amino acids include D326 and D510 with respect to SEQ ID NO: 58. In some embodiments, one or both of D326 and D510 are substituted with an amino acid that reduces, substantially eliminates, or eliminates nuclease activity'. In some embodiments, one or both of D326 and D510 are substituted with alanine (i.e., D326A and/or D510A). In some embodiments, the dCasl4 exhibits reduced or eliminated nuclease activity', or nuclease activity is absent or substantially absent within levels of detection. According to this embodiment, nuclease activity for a dCas 14 may be undetectable using known assays, i.e. below the level of detection of known assays.

[0047] In some embodiments, the dCas14 protein includes homologs and orthologs thereof which retain the ability of the protein to be guided by RNA to bind to target nucleic acid. In some embodiments, the dCasl4 protein comprises the amino acid sequence of SEQ ID NO: 60 or a variant thereof having at least 80% (such as at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) sequence identity to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the dCasl4 protein comprises the amino acid sequence of SEQ ID NO: 62 or a variant thereof having at least 80% (such as at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) sequence identity to the amino acid sequence of SEQ ID NO: 62. In some embodiments, the dCasl4 protein comprises the amino acid sequence of SEQ ID NO: 63 or a variant thereof having at least 80% (such as at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) sequence identity to the amino acid sequence of SEQ ID NO: 63.

[0048] In some embodiments, the dCasl4 protein is attached to, bound to, or fused with an effector domain, such as a transcriptional regulatory domain or an epigenetic modifying domain.

[0049] In some embodiments, provided herein is a dCas 14 fusion protein comprising a dCas 14 protein fused to an effector domain. In some embodiments, the effector domain is fused to the C-terminus of the dCas14 protein. In some embodiments, the effector domain is fused to the N-terminus of the dCas14 protein. In some embodiments, the effector domain comprises a subcellular localization signal. In some embodiments, the subcellular localization signals is an organelle localization signal, such as a nuclear localization signal (NLS), nuclear export signal (NES), or mitochondrial localization signal. In some embodiments, the effector domain comprises a polypeptide that can (i) cleave a nucleic acid (e.g., DNA and/or RNA), (ii) affect RNA stability, (iii) edit a nucleotide, (iv) activate transcription, (v) repress transcription, (iv) activate translation, (v) repress translation, (vi) methylate a nucleic acid (e.g., DNA and/or RNA), (vii) demethylate a nucleic acid (e.g., DNA and/or RNA), (viii) affect RNA splicing, (ix) enable affinity purification or immunoprecipitation (e.g., FLAG, HA, biotin, or HALO tags), and/or (x) enable proximity-based protein labeling and identification.

[0050] Also provided herein is an isolated nucleic acid molecule encoding a dCasl4 protein according to any of the embodiments described herein. In some embodiments, the isolated nucleic acid molecule is part of a vector (such as a plasmid or viral vector), and can be operably linked to a promoter. Nucleotide sequences encoding dCasl4 can be generated based on the amino acid sequences provided herein. In some examples, the isolated nucleic acid molecule encoding a dCasl4 protein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 61. In some embodiments, the isolated nucleic acid molecule encoding a dCasl4 protein comprises a dCasl4 protein coding sequence that is codon optimized for expression in a eukaryotic cell (e.g., a mammalian cell, such as a human cell). For example, nucleic acid molecules encoding the dCasl4 proteins disclosed herein can be designed to have codons that are preferentially used by a particular organism of interest. In some embodiments, the nucleotide sequence encoding dCasl4 is optimized for expression in human cells.

Guide RNA

[0051] In general, bacterial and archaeal CRISPR-Cas systems rely on short guide RNAs in complex with Cas proteins to direct degradation of complementary sequences present within invading foreign nucleic acid. See Deltcheva, E. et al. Nature 471, 602-607 (2011); Gasiunas, G., et al. Proceedings of the National Academy of Sciences of the United States of America 109, E2579-2586 (2012); Jinek, M. et al. Science 337, 816-821 (2012); Sapranauskas, R. et al. Nucleic acids research 39, 9275- 9282 (2011); and Bhaya, D., Davison, M. & Barrangou, R. Anmial review of genetics 45, 273-297 (2011).

[0052] Embodiments of the present disclosure are directed to the use of a CRISPR Cas 14 system and, in particular, a guide RNA which may include one or more of a spacer sequence, a tracr mate sequence and a tracr sequence. The term spacer sequence is understood by those of skill in the art and may include any polynucleotide having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. The guide RNA may be formed from a spacer sequence covalently connected to a tracr mate sequence (which may be referred to as a crRNA) and a separate tracr sequence, wherein the tracr mate sequence is hybridized to a portion of the tracr sequence. According to certain aspects, the tracr mate sequence and the tracr sequence are connected or linked such as by covalent bonds by a linker, which construct may be referred to as a fusion of the tracr mate sequence and the tracr sequence. In some embodiments, the linker is a polynucleotide linker. Accordingly, a guide RNA may be a two component species (i.e., separate crRNA and tracr RNA which hybridize together) or a unimolecular species (i.e., a crRNA-tracr RNA fusion, often termed an “sgRNA”). An exemplary sgRNA sequence is provided in SEQ ID NO: 57, with the sequence from position 1-205 corresponding to a fusion of tracr and tracr mate sequences (scaffold) and the sequence from position 206-225 corresponding to the spacer.

[0053] In some embodiments, the guide RNA is between about 10 to about 500 nucleotides. In some embodiments, the guide RNA is between about 20 to about 100 nucleotides. In some embodiments, the spacer sequence is between about 10 and about 500 nucleotides in length. In some embodiments, the tracr mate sequence is between about 10 and about 500 nucleotides in length. In some embodiments, the tracr sequence is between about 10 and about 100 nucleotides in length. In some embodiments, the linker nucleic acid sequence is between about 10 and about 100 nucleotides in length.

[0054] In some embodiments, methods of making a guide RNA as described herein are employed, such as by expressing constructs encoding the guide RNA using promoters and terminators and optionally other genetic elements known in the art for such purposes.

[0055] In some embodiments, the guide RNA may be delivered directly to a cell as a native species by methods known to those of skill in the art, including injection or lipofection, or delivered indirectly to a cell as transcribed from a cognate DNA, with the cognate DNA introduced into the cell through electroporation, transient or stable transfection (including lipofection), or viral transduction.

Systems

[0056] In some embodiments, the compositions herein comprise, or the methods herein comprise delivering, one or more components of a Casl4 nuclei acid-targeting system (also referred to herein as a “Casl4 system”). In general, “Casl4 system” as used in the present application refers collectively to elements involved in the activity of a Casl4 protein in association with a compatible guide RNA (gRNA) to be targeted to a particular nucleic acid sequence (also referred to herein as a “target sequence,” or “protospacer-like sequence” in the context of an endogenous CRISPR-Cas system) as guided by the spacer sequence of the gRNA, and can include nucleic acid encoding the Casl4 protein and/or the gRNA. In some embodiments, the Casl4 protein and/or gRNA are derived from a particular organism comprising an endogenous Casl4 system. In general, a Casl4 system is characterized by elements that promote the formation of a nucleic acid-targeting complex including a Casl4 protein and a gRNA at the site of a target sequence, which can be present in a DNA molecule or an RNA molecule. In the context of a Casl4 system, “target sequence” refers to a sequence to which a guide sequence (also referred to herein as a “spacer” or “spacer sequence”) in a gRNA of the system is designed to have complementarity, where hybridization between the target sequence and the gRNA allows for localization of the Cas 14 protein to the target sequence. Full complementarity is not necessarily required, provided there is sufficient complementarity to allow for hybridization between the target sequence and the gRNA. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, a mitochondrion or a chloroplast.

[0057] In some embodiments, provided herein is a CRISPR-Cas system including (a) a Cas 14 protein or variant thereof, or nucleic acid encoding the Cas 14 protein or variant (such as an mRNA or a vector encoding the Cas 14 protein or variant); and (b) a CRISPR-Cas system guide RNA (gRNA), or nucleic acid encoding the gRNA, wherein the gRNA comprises a spacer having sufficient complementary to a target nucleotide sequence in a target nucleic acid (e.g., RNA, ssDNA, or dsDNA) such that it can hybridize to the target nucleic acid. The Cas 14 protein or variant is capable of forming a complex with the gRNA, and the gRNA can direct the complex to one or more target nucleic acid molecules comprising the target nucleotide sequence. This targeting can allow the Casl4/gRNA complex to bind to, modify, or detect the one or more target nucleic acid molecules. In some embodiments, the Casl4 protein or variant is a dCasl4 protein according to any of the embodiments described herein, e.g., a dCasl4 protein having reduced or eliminated nuclease activity as compared to the parental Cas 14 protein from which it is derived. In some embodiments, the parental Cas 14 protein comprises or consists of the amino acid sequence of SEQ ID NO: 58 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the dCasl4 protein comprises one or more amino acids that have been altered or otherwise removed to reduce or eliminate its nuclease activity. In some embodiments, the amino acids include D326 and D510 with respect to SEQ ID NO: 58. In some embodiments, one or both of D326 and D510 are substituted with an amino acid that reduces, substantially eliminates, or eliminates nuclease activity. In some embodiments, one or both of D326 and D510 are substituted with alanine (i.e., D326A and/or D510A). In some embodiments, the dCasl4 exhibits reduced or eliminated nuclease activity, or nuclease activity' is absent or substantially absent within levels of detection. In some embodiments, the dCasl4 protein comprises or consists of the amino acid sequence of SEQ ID NO: 60, 62, or 63, or a variant thereof having at least about 80% sequence identity' to the amino acid sequence of SEQ ID NO: 60, 62, or 63. In some embodiments, the dCasl4 protein comprises or consists of the amino acid sequence of SEQ ID NO: 60 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 60.

[0058] In some embodiments, according to any of the Casl4 systems described herein, the target nucleic acid is dsDNA. In such embodiments, dsDNA-targeting specificity is determined, at least in part, by two parameters: the gRNA spacer targeting a protospacer in the target dsDNA (the sequence in the target dsDNA corresponding to the gRNA spacer on the non-complementary DNA strand) and a short sequence, the protospacer-adjacent motif (PAM), located immediately 5' (upstream) of the protospacer on the non-complementary DNA strand. In some embodiments, the PAM is 5'-lllG-3' or 5'-TTTA-3'. In some embodiments, the PAM is 5'-TTTG-3'. In some embodiments, the PAM is 5'-TTTA-3'.

[0059] In some embodiments, according to any of the Casl4 systems described herein, the target nucleic acid is RNA. In such embodiments, RNA-targeting specificity is determined, at least in part, by the gRNA spacer targeting a protospacer-like sequence in the target RNA (the sequence in the target RNA complementary to the gRNA spacer), and is independent of the sequence located immediately 5' (upstream) of the protospacer-like sequence. In some embodiments, the Cas14 system is also capable of targeting a dsDNA molecule, wherein the gRNA spacer is selected such that it targets a protospacer in the target dsDNA molecule having a PAM selected from 5'-TTTG-3' and 5'-TH A-3'. In other embodiments, the Casl4 system is incapable of targeting a dsDNA molecule, wherein the gRNA spacer is selected such that any protospacers in the dsDNA molecule targeted by the gRNA spacer do not have a PAM selected from 5'-TTTG-3' and 5'-TTTA-3'.

[0060] In some embodiments, according to any of the Casl4 systems described herein, the target nucleotide sequence is present in a target dsDNA and a target RNA, and the Casl4 system is capable of targeting both the target dsDNA and the target RNA. In some embodiments, a protospacer in the target dsDNA targeted by the gRNA spacer has a PAM selected from 5'-TTTG-3' and 5'-TTTA-3'. In some embodiments, the target dsDNA is a genomic DNA and the target RNA is an mRNA transcribed from the genomic DNA, wherein the target nucleotide sequence is present in the sense strand (non-template strand) of the genomic DNA (see, e.g., FIG. 6A), wherein targeting of the genomic DNA by the Casl4 systems is capable of modulating transcription of the mRNA, and wherein targeting of the mRNA by the Casl4 system is capable of modulating translation of the mRNA. In some embodiments, targeting of the genomic DNA by the Casl4 system is capable of repressing transcription of the mRNA, and targeting of the mRNA by the Casl4 system is capable of repressing translation of the mRNA. In some embodiments, targeting of the genomic DNA by the Casl4 system is capable of enhancing transcription of the mRNA, and targeting of the mRNA by the Casl4 system is capable of enhancing translation of the mRNA. In some embodiments, the Casl4 protein or variant is a dCasl4 protein or a dCasl4 fusion protein, e.g., a dCasl4 fusion protein comprising one or more effector domains capable of modulating transcription and/or translation.

[0061] In some embodiments, according to any of the Casl4 systems described herein, the target nucleotide sequence is present in a target dsDNA and a non-target RNA, and the Cas 14 system is capable of targeting the target dsDNA but not the non-target RNA. In some embodiments, a protospacer in the target dsDNA targeted by the gRNA spacer has a PAM selected from 5'-TTTG-3' and 5'-TTTA-3'. In some embodiments, the target dsDNA is a genomic DNA and the non-target RNA is an mRNA transcribed from the genomic DNA, wherein the target nucleotide sequence is present in the anti-sense strand (template strand) of the genomic DNA (see, e.g., FIG. 6B), and wherein targeting of the genomic DNA by the Casl4 systems is capable of modulating transcription of the mRNA. In some embodiments, targeting of the genomic DNA by the Cas 14 system is capable of repressing transcription of the mRNA. In some embodiments, targeting of the genomic DNA by the Casl4 system is capable of enhancing transcription of the mRNA. In some embodiments, the Cas 14 protein or variant is a dCasl4 protein or a dCasl4 fusion protein, e.g., adCasl4 fusion protein comprising one or more effector domains capable of modulating transcription.

[0062] In some embodiments, according to any of the Casl4 systems described herein, the target nucleotide sequence is present in a target RNA and a non-target dsDNA, and the Cas 14 system is capable of targeting the target RNA but not the non-target dsDNA. In some embodiments, any protospacers in the non-target dsDNA targeted by the gRNA spacer do not have a PAM selected from 5'-THG-3' and 5'-THA-3'. In some embodiments, the non-target dsDNA is a genomic DNA and the target RNA is an mRNA transcribed from the genomic DNA, wherein the target nucleotide sequence is present in the sense strand of the genomic DNA (see, e.g., FIG. 6C), and wherein targeting of the mRNA by the Cas 14 system is capable of modulating translation of the mRNA. In some embodiments, targeting of the mRNA by the Cas 14 system is capable of repressing translation of the mRNA. In some embodiments, targeting of the mRNA by the Cas 14 system is capable of enhancing translation of the mRNA. In some embodiments, the Cas 14 protein or variant is a d Cas 14 protein or a dCasl4 fusion protein, e.g., a dCasl4 fusion protein comprising one or more effector domains capable of modulating translation. In some embodiments, the Casl4 protein or variant is an endonuclease- competent Cas 14 protein capable of cleaving the target RNA.

[0063] In some embodiments, according to any of the Casl4 systems described herein, the target nucleotide sequence is present in a gene sequence. In some embodiments, the target nucleotide sequence is present in a promoter region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a 5 ’ UTR region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a 5’ UTR/RBS region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a coding region of the gene, and can be present in the sense strand or anti-sense strand of the gene.

[0064] In some embodiments, according to any of the Cas 14 systems described herein, one or more vectors driving expression of one or more elements of a Casl4 system are introduced into a host cell such that expression of the elements of the Cas 14 system directs formation of a nucleic acid-targeting complex at one or more target sequence sites in the host cell. For example, a Cas 14 protein and a guide RNA could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the Cas 14 system not included in the first vector. Cas 14 system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to (“upstream” of) or 3' with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding a Cas 14 protein and a guide RNA. In some embodiments, the Cas 14 protein and guide RNA are operably linked to and expressed from the same promoter.

Methods of the Disclosure

Methods of targeting nucleic acid molecules

[0065] Provided herein are methods of targeting (e.g., binding to, modifying, detecting, etc.) one or more target nucleic acid molecules (e.g., RNA, ssDNA, or dsDNA) using a Cas 14 system as described herein. Such methods can include contacting one or more target nucleic acid molecules with a non-naturally occurring or engineered (e.g., does not naturally occur in the cell or system into which it is introduced) CRISPR-Casl4 system comprising a Cas 14 protein or variant thereof and a gRNA. In some embodiments, the spacer sequence within the gRNA molecule is not naturally occurring, and has been modified to be complementary' to a target nucleic acid molecule. In some embodiments, the target nucleic acid molecule is a DNA molecule, such as a genomic DNA. In some embodiments, the target nucleic acid molecule is an RNA molecule, such as an mRNA.

[0066] In some embodiments, provided herein is a method of targeting (e.g., binding to, modifying, detecting, etc.) one or more target nucleic acid molecules (e.g., RNA, ssDNA, and/or dsDNA) comprising contacting the one or more target nucleic acid molecules with a Casl4 system comprising (a) a Casl4 protein or variant thereof, or nucleic acid encoding the Casl4 protein or variant (such as an mRNA or a vector encoding the Casl4 protein or variant); and (b) a gRNA, or nucleic acid encoding the gRNA, wherein the gRNA comprises a spacer having sufficient complementary to a target nucleotide sequence in the one or more target nucleic acid molecules such that it can hybridize to them. In some embodiments, the Casl4 protein or variant is a dCasl4 protein according to any of the embodiments described herein, e.g., a dCasl4 protein having reduced or eliminated nuclease activity as compared to the parental Casl4 protein from which it is derived. In some embodiments, the parental Casl4 protein comprises or consists of the amino acid sequence of SEQ ID NO: 58 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the dCasl4 protein comprises or consists of the amino acid sequence of SEQ ID NO: 60, 62, or 63, or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 60, 62, or 63. In some embodiments, the dCasl4 protein comprises or consists of the amino acid sequence of SEQ ID NO: 60 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the one or more target nucleic acid molecules are present in a cell, and the contacting comprises introducing into the cell the Casl 4 system, for example using endocytosis (e.g., receptor-mediated endocytosis, micropinocytosis), a liposome, a particle, an exosome, a microvesicle, a gene gun, electroporation, a virus, an RNP-antibody fusion (e.g., Casl4/gRNA RNP tethered to an antibody, antibody fragment, or other targeting moiety), or combinations thereof. Thus, cells can be transformed, transduced, transfected, or otherwise contacted with appropriate components of the Casl4 system, resulting in recombinant cells. In some embodiments, the one or more target nucleic acid molecules are present in a cell-free system (such as a biological or environmental sample, e.g., a cell lysate), and the contacting comprises introducing into the cell-free system the Casl 4 system (for example in a diagnostic method to detect a target nucleic acid molecule).

[0067] Targeting a nucleic acid molecule can include one or more of cutting or nicking the target nucleic acid molecule; modulating the expression of a gene present in the target nucleic acid molecule (such as by regulating transcription of the gene from a target DNA and/or regulating translation of the gene from a target RNA, e.g., to downregulate or upregulate expression of the gene); visualizing, labeling, or detecting the target nucleic acid molecule; binding the target nucleic acid molecule, editing the target nucleic acid molecule, trafficking the target nucleic acid molecule, and masking the target nucleic acid molecule. In some embodiments, modifying the target nucleic acid molecule includes introducing one or more of a nucleobase substitution, a nucleobase deletion, a nucleobase insertion, a break in the target nucleic acid molecule, methylation of the target nucleic acid molecule, and demethylation of the nucleic acid molecule. In some embodiments, such methods are used to treat a disease, such as a disease in a human. In such embodiments, one or more target nucleic acid molecules are associated with the disease.

Methods of modulating transcription

[0068] In some embodiments, provided herein are methods of modulating transcription of a target nucleic acid in a cell. The methods generally involve contacting the target nucleic acid with an enzymatically inactive Casl4 protein and a gRNA. The methods are useful in a variety of applications.

[0069] In some embodiments, provided herein is a method of selectively modulating transcription of a target DNA in a cell, e.g., a human cell. The method generally involves: a) introducing into the cell: i) a gRNA, or a nucleic acid comprising a nucleotide sequence encoding the gRNA; and ii) a Casl4 protein or variant thereof, or a nucleic acid comprising a nucleotide sequence encoding the Casl4 protein or variant, where the Casl4 protein or variant exhibits reduced endodeoxyribonuclease activity. The gRNA and the Casl4 protein or variant form a complex in the cell; the complex selectively modulates transcription of a target DNA in the cell.

[0070] In one aspect, provided herein is a method of selectively modulating transcription of a target DNA in a cell, the method comprising providing to the cell (a) a Casl4 protein or variant thereof, or nucleic acid encoding the Casl4 protein or variant; and (b) a gRNA or nucleic acid encoding the gRNA, wherein the gRNA is capable of targeting the Casl4 protein or variant to a target sequence in the target DNA. In some embodiments, the Casl4 protein or variant and the gRNA are configured such that a complex formed by association of the Cas 14 protein or variant with the gRNA is capable of binding the target sequence. In some embodiments, the method comprises providing to the cell the Cas 14 protein or variant. In some embodiments, the method comprises providing to the cell nucleic acid encoding the Cas 14 protein or variant. In some embodiments, the method comprises providing to the cell the gRNA. In some embodiments, the method comprises providing to the cell nucleic acid encoding the gRNA. In some embodiments, the gRNA is an sgRNA. In some embodiments, the method further comprises providing to the cell one or more additional gRNAs or nucleic acid encoding the one or more additional gRNAs.

[0071] In some embodiments, according to any of the methods of selectively modulating transcription of a target DNA in a cell described herein, the Cas 14 protein or variant is a dCas 14 protein according to any of the embodiments described herein, e.g., a dCasl4 protein having reduced or eliminated nuclease activity as compared to the parental Cas 14 protein from which it is derived. In some embodiments, the parental Casl4 protein comprises or consists of the amino acid sequence of SEQ ID NO: 58 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the dCas14 protein comprises one or more amino acids that have been altered or otherwise removed to reduce or eliminate its nuclease activity. In some embodiments, the amino acids include D326 and D510 with respect to SEQ ID NO: 58. In some embodiments, one or both of D326 and D510 are substituted with an amino acid that reduces, substantially eliminates, or eliminates nuclease activity. In some embodiments, one or both of D326 and D510 are substituted with alanine (i.e., D326A and/or D510A). In some embodiments, the dCasl4 exhibits reduced or eliminated nuclease activity, or nuclease activity is absent or substantially absent within levels of detection. In some embodiments, the dCasl4 protein comprises or consists of the amino acid sequence of SEQ ID NO: 60, 62, or 63, or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 60, 62, or 63. In some embodiments, the dCas14 protein comprises or consists of the amino acid sequence of SEQ ID NO: 60 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 60.

[0072] In some embodiments, according to any of the methods of selectively modulating transcription of a target DNA in a cell described herein, the target nucleic acid is dsDNA. In some embodiments, according to any of the methods of selectively modulating transcription of a target DNA in a cell described herein, the target nucleotide sequence is present in a target dsDNA and a target RNA, and the Casl4 system is capable of targeting both the target dsDNA and the target RNA. In some embodiments, according to any of the methods of selectively- modulating transcription of a target DNA in a cell described herein, the target nucleotide sequence is present in a target dsDNA and a non-target RNA, and the Cas 14 system is capable of targeting the target dsDNA but not the non-target RNA.

[0073] In some embodiments, according to any of the methods of selectively modulating transcription of a target DNA in a cell described herein, the target nucleotide sequence is present in a gene sequence. In some embodiments, the target nucleotide sequence is present in a promoter region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a 5’ UTR region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a 5’ UTR/RBS region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a coding region of the gene, and can be present in the sense strand or anti-sense strand of the gene.

[0074] In some embodiments, a transcription modulation method described herein allows for selective modulation (e.g., reduction or increase) of transcription of a target DNA in a cell. For example, in some embodiments, “selective” reduction of transcription of a target DNA reduces transcription of the target DNA by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or greater than 90%, compared to the level of transcription of the target DNA in the absence of a gRNA/Casl4 protein or variant complex. In some embodiments, selective reduction of transcription of a target DNA reduces transcription of the target DNA, but does not substantially reduce transcription of a non-target DNA, e.g., transcription of a non-target DNA is reduced, if at all, by less than 10% compared to the level of transcription of the non-target DNA in the absence of the gRNA/Cas 14 protein or variant complex. In some embodiments, “selective” increase of transcription of a target DNA increases transcription of the target DNA by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or greater than 90%, compared to the level of transcription of the target DNA in the absence of a gRNA/Cas 14 protein or variant complex. In some embodiments, selective increase of transcription of a target DNA increases transcription of the target DNA, but does not substantially increase transcription of a non-target DNA, e.g., transcription of a non-target DNA is increased, if at all, by less than 10% compared to the level of transcription of the non- target DNA in the absence of the gRNA/Casl4 protein or variant complex.

[0075] In some embodiments, the Casl4 protein or variant has activity that modulates the transcription of target DNA (e.g., in the case of a Casl4 fusion protein or variant thereof, etc.). In some embodiments, a Casl4 fusion protein or variant thereof comprising a heterologous polypeptide that exhibits the ability to increase or decrease transcription (e.g., transcriptional activator or transcription repressor polypeptides) is used to increase or decrease the transcription of target DNA at a specific location in a target DNA, which is guided by the spacer of the gRNA. Examples of source polypeptides for providing a Casl4 fusion protein or variant thereof with transcription modulatory activity include, but are not limited to lightinducible transcription regulators, small molecule/drug-responsive transcription regulators, transcription factors, transcription repressors, etc. In some embodiments, the method is used to control the transcription of a targeted gene-coding RNA (protein-encoding mRNA) and/or a targeted non-coding RNA (e.g., tRNA, rRNA, snoRNA, siRNA, miRNA, long ncRNA. etc.). In some embodiments, the Casl4 protein or variant has enzymatic activity' that modifies a polypeptide associated with DNA (e.g., histone). In some embodiments, the enzymatic activity is methyltransferase activity-, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity (e.g., ubiquitination activity), deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity-, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity glycosylation activity (e.g., from GlcNAc transferase) or deglycosylation activity. The enzymatic activities listed herein catalyze covalent modifications to proteins. Such modifications are known in the art to alter the stability or activity of the target protein (e.g., phosphorylation due to kinase activity can stimulate or silence protein activity depending on the target protein). Of particular interest as protein targets are histones. Histone proteins are known in the art to bind DNA and form complexes known as nucleosomes. Histones can be modified (e.g., by methylation, acetylation, ubiquitination, phosphorylation) to elicit structural changes in the surrounding DNA, thus controlling the accessibility of potentially large portions of DNA to interacting factors such as transcription factors, polymerases and the like. A single histone can be modified in many different ways and in many different combinations (e.g., trimethylation of lysine 27 of histone 3, H3K27, is associated with DNA regions of repressed transcription while trimethylation of lysine 4 of histone 3, H3K4, is associated with DNA regions of active transcription). Thus, a Casl4 fusion protein or variant thereof with histone-modifying activity finds use in the site-specific control of chromosomal structure and can be used to alter the histone modification pattern in a selected region of target DNA. Such methods find use in both research and clinical applications.

Methods of modulating translation

[0076] In some embodiments, provided herein are methods of modulating translation of a target RNA in a cell. The methods generally involve contacting the target RNA with an enzymatically inactive Casl4 protein variant and a gRNA. The methods are usefill in a variety of applications.

[0077] In some embodiments, provided herein is a method of selectively modulating translation of a target RNA in a cell, e.g., a human cell. The method generally involves: a) introducing into the cell: i) a gRNA, or a nucleic acid comprising a nucleotide sequence encoding the gRNA; and ii) a Casl4 protein or variant thereof, or a nucleic acid comprising a nucleotide sequence encoding the Casl4 protein or variant, where the Casl4 protein or variant exhibits reduced endodeoxyribonuclease activity. The gRNA and the Casl4 protein or variant form a complex in the cell; the complex selectively modulates translation of a target RNA in the cell.

[0078] In one aspect, provided herein is a method of selectively modulating translation of a target RNA in a cell, the method comprising providing to the cell (a) a Cas14 protein or variant thereof, or nucleic acid encoding the Casl4 protein or variant; and (b) a gRNA or nucleic acid encoding the gRNA, wherein the gRNA is capable of targeting the Casl4 protein or variant to a target sequence in the target RNA. In some embodiments, the Casl4 protein or variant and the gRNA are configured such that a complex formed by association of the Casl4 protein or variant with the gRNA is capable of binding the target sequence. In some embodiments, the method comprises providing to the cell the Casl4 protein or variant. In some embodiments, the method comprises providing to the cell nucleic acid encoding the Casl4 protein or variant. In some embodiments, the method comprises providing to the cell the gRNA. In some embodiments, the method comprises providing to the cell nucleic acid encoding the gRNA. In some embodiments, the gRNA is an sgRNA. In some embodiments, the method further comprises providing to the cell one or more additional gRNAs or nucleic acid encoding the one or more additional gRNAs.

[0079] In some embodiments, according to any of the methods of selectively modulating translation of a target RNA in a cell described herein, the Casl4 protein or variant is a dCasl4 protein according to any of the embodiments described herein, e.g., a dCasl4 protein having reduced or eliminated nuclease activity as compared to the parental Cas 14 protein from which it is derived. In some embodiments, the parental Casl4 protein comprises or consists of the amino acid sequence of SEQ ID NO: 58 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the dCasl4 protein comprises one or more amino acids that have been altered or otherwise removed to reduce or eliminate its nuclease activity. In some embodiments, the amino acids include D326 and D510 with respect to SEQ ID NO: 58. In some embodiments, one or both of D326 and D510 are substituted with an amino acid that reduces, substantially eliminates, or eliminates nuclease activity. In some embodiments, one or both of D326 and D510 are substituted with alanine (i.e., D326A and/or D510A). In some embodiments, the dCasl4 exhibits reduced or eliminated nuclease activity, or nuclease activity is absent or substantially absent within levels of detection. In some embodiments, the dCasl4 protein comprises or consists of the amino acid sequence of SEQ ID NO: 60, 62, or 63, or a variant thereof having at least about 80% sequence identity' to the amino acid sequence of SEQ ID NO: 60, 62, or 63. In some embodiments, the dCasl4protein comprises or consists of the amino acid sequence of SEQ ID NO: 60 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 60.

[0080] In some embodiments, according to any of the methods of selectively modulating translation of a target RNA in a cell described herein, the target nucleic acid is RNA. In some embodiments, according to any of the methods of selectively modulating translation of a target RNA in a cell described herein, the target nucleotide sequence is present in a target dsDNA and a target RNA, and the Cas 14 system is capable of targeting both the target dsDNA and the target RNA. In some embodiments, according to any of the methods of selectively modulating translation of a target RNA in a cell described herein, the target nucleotide sequence is present in a target RNA and a non-target dsDNA, and the Cas 14 system is capable of targeting the target RNA but not the non-target dsDNA .

[0081] In some embodiments, according to any of the methods of selectively modulating translation of a target RNA in a cell described herein, the target nucleotide sequence is present in a gene sequence. In some embodiments, the target nucleotide sequence is present in a promoter region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a 5’ UTR region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a 5’ UTR/RBS region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a coding region of the gene, and can be present in the sense strand or anti-sense strand of the gene.

[0082] In some embodiments, a translation modulation method described herein allows for selective modulation (e.g., reduction or increase) of a target RNA in a cell. For example, in some embodiments, “selective” reduction of translation of a target RNA reduces translation of the target RNA by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or greater than 90%, compared to the level of translation of the target RNA in the absence of a gRNA/Casl4 protein or variant complex. In some embodiments, selective reduction of translation of a target RNA reduces translation of the target RNA, but does not substantially reduce translation of a non-target RNA, e.g., translation of a non-target RNA is reduced, if at all, by less than 10% compared to the level of translation of the non-target RNA in the absence of the gRNA/Casl4 protein or variant complex. In some embodiments, “selective” increase of translation of a target RNA increases translation of the target RNA by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or greater than 90%, compared to the level of translation of the target RNA in the absence of a gRNA/Casl4 protein or variant complex. In some embodiments, selective increase of translation of a target RNA increases translation of the target RNA, but does not substantially increase translation of a non-target RNA, e.g., translation of a non-target RNA is increased, if at all, by less than 10% compared to the level of translation of the non-target RNA in the absence of the gRNA/Casl4 protein or variant complex.

[0083] In some embodiments, the Casl4 protein or variant has activity that modulates the translation of target RNA (e.g., in the case of a Casl4 fusion protein or variant thereof, etc.). In some embodiments, a Casl4 fusion protein or variant thereof comprising a heterologous polypeptide that exhibits the ability to increase or decrease translation (e.g., translational activator or translation repressor polypeptides) is used to increase or decrease the translation of target RNA, which is guided by the spacer of the gRNA.

Methods of treating a disease or condition

[0084] In some aspects of the disclosure, one or more components of a Casl4 system are employed to modify target gene expression from cellular DNA in vivo or ex vivo for purposes of treating a disease or condition in a subject. In some of these embodiments, components of a Casl4 system include (i) a gRNA or nucleic acid encoding the gRNA; and (ii) a Casl4 protein or variant thereof, or nucleic acid encoding the Casl4 protein or variant. The Casl4 system components can be incorporated into a variety of formulations. More particularly, the Casl4 system components of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers or diluents.

[0085] In some embodiments, provided herein are pharmaceutical preparations or compositions comprising components of a Casl4 system including (i) a gRNA or nucleic acid encoding the gRNA; and (ii) a Gas 14 protein or variant thereof, or nucleic acid encoding the Casl4 protein or variant, present in a pharmaceutically acceptable vehicle. “Pharmaceutically acceptable vehicles” may be vehicles approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopeia or other generally recognized pharmacopeia for use in mammals, such as humans. The term “vehicle” refers to a diluent, adjuvant, excipient, or carrier with which a compound of the disclosure is formulated for administration to a mammal. Such pharmaceutical vehicles can be lipids, e.g., liposomes, e.g., liposome dendrimers; liquids, such as water and oils, including those of petroleinn, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, saline; gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. Pharmaceutical compositions may be formulated into preparations in solid, semisolid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. As such, administration of the Casl4 system components can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intra-tracheal, intraocular, etc., administration. The active agent may be systemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation. The active agent may be formulated for immediate activity or it may be formulated for sustained release.

[0086] Pharmaceutical compositions can include, depending on the formulation desired, pharmaceutically acceptable, non-toxic carriers of diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity' of the combination. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation can include other carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients and the like. The compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents.

[0087] The composition can also include any of a variety of stabilizing agents, such as an antioxidant for example. When the pharmaceutical composition includes a polypeptide, the polypeptide can be complexed with various well-known compounds that enhance the in vivo stability of the polypeptide, or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, and/or enhance solubility or uptake). Examples of such modifications or complexing agents include sulfate, gluconate, citrate and phosphate. The nucleic acids or polypeptides of a composition can also be complexed with molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[0088] In some embodiments, provided herein is a method of treating a disease or condition in a subject in need thereof, the method comprising: 1) modulating the expression of a target gene in input cells according to any of the methods described herein, thereby producing engineered cells and administering the engineered cells to the subject; or 2) modulating the expression of a target gene in input cells in the subject according to any of the methods described herein, thereby producing engineered cells in the subject. In some embodiments, the input cells of 1) are autologous to the subject. In some embodiments, the input cells of 1) are allogenic to the subject. In some embodiments, the subject is human.

[0089] In some embodiments, provided herein is a method of treating a disease or condition in a subject in need thereof, the method comprising providing to input cells (a) a Cas 14 protein or variant thereof, or nucleic acid encoding the Cas14 protein or variant; and (b) a gRNA or nucleic acid encoding the gRNA, wherein the gRNA is capable of targeting the Cas 14 protein or variant to a target sequence in the genomes of the input cells to modulate the expression of a target gene, thereby producing engineered cells, and administering the engineered cells to the subject. In some embodiments, the Cas 14 protein or variant and the gRNA are configured such that a complex formed by association of the Cas 14 protein or variant with the gRNA is capable of binding the target sequence. In some embodiments, the method comprises providing to the input cells the Cas 14 protein or variant. In some embodiments, the method comprises providing to the input cells nucleic acid encoding the Cas 14 protein or variant. In some embodiments, the method comprises providing to the input cells the gRNA. In some embodiments, the method comprises providing to the input cells nucleic acid encoding the gRNA. In some embodiments, the gRNA is an sgRNA. In some embodiments, the method further comprises providing to the input cells one or more additional gRNAs or nucleic acid encoding the one or more additional gRNAs. In some embodiments, the input cells are autologous to the subject. In some embodiments, the input cells are allogenic to the subject. In some embodiments, the subject is human.

[0090] The number of administrations of treatment to a subject may vary. In some embodiments, introducing the engineered cells into the subject is a one-time event. In some embodiments, the method further comprises one or more additional administrations of engineered cells.

[0091] In some embodiments, provided herein is a method of treating a disease or condition in a subject in need thereof, the method comprising providing to input cells in the subject (a) a Cas 14 protein or variant thereof, or nucleic acid encoding the Cas 14 protein or variant; and (b) a gRNA or nucleic acid encoding the gRNA, wherein the gRNA is capable of targeting the Cas 14 protein or variant to a target sequence in the genomes of the input cells to modulate the expression of a target gene, thereby producing engineered cells in the subject. In some embodiments, the Cas 14 protein or variant and the gRNA are configured such that a complex formed by association of the Cas 14 protein or variant with the gRNA is capable of binding the target sequence. In some embodiments, the method comprises providing to the input cells the Cas 14 protein or variant. In some embodiments, the method comprises providing to the input cells nucleic acid encoding the Cas 14 protein or variant. In some embodiments, the method comprises providing to the input cells the gRNA. In some embodiments, the method comprises providing to the input cells nucleic acid encoding the gRNA. In some embodiments, the gRNA is an sgRNA. In some embodiments, the method further comprises providing to the input cells one or more additional gRNAs or nucleic acid encoding the one or more additional gRNAs. In some embodiments, the subject is human.

[0092] In some embodiments, according to any of the methods of treating a disease or condition described herein, the Cas 14 protein or variant is a dCasl4 protein according to any of the embodiments described herein, e.g., a dCasl4 protein having reduced or eliminated nuclease activity as compared to the parental Cas 14 protein from which it is derived. In some embodiments, the parental Cas 14 protein comprises or consists of the amino acid sequence of SEQ ID NO: 58 or a variant thereof having at least about 80% sequence identity' to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the dCasl4 protein comprises one or more amino acids that have been altered or otherwise removed to reduce or eliminate its nuclease activity. In some embodiments, the amino acids include D326 and D510 with respect to SEQ ID NO: 58. In some embodiments, one or both of D326 and D510 are substituted with an amino acid that reduces, substantially eliminates, or eliminates nuclease activity. In some embodiments, one or both of D326 and D510 are substituted with alanine (i.e., D326A and/or D510A). In some embodiments, the dCasl4 exhibits reduced or eliminated nuclease activity, or nuclease activity is absent or substantially absent within levels of detection. In some embodiments, the dCasl4 protein comprises or consists of the amino acid sequence of SEQ ID NO: 60, 62, or 63, or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 60, 62, or 63. In some embodiments, the dCasl4 protein comprises or consists of the amino acid sequence of SEQ ID NO : 60 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 60.

[0093] In some embodiments, according to any of the methods of treating a disease or condition described herein, the target nucleic acid is dsDNA and/or RNA. In some embodiments, according to any of the methods of treating a disease or condition described herein, the target nucleotide sequence is present in a target dsDNA and a target RNA, and the Casl4 system is capable of targeting both the target dsDNA and the target RNA. In some embodiments, according to any of the methods of treating a disease or condition described herein, the target nucleotide sequence is present in a target dsDNA and a non-target RNA, and the Cas 14 system is capable of targeting the target dsDNA but not the non-target RNA. In some embodiments, according to any of the methods of treating a disease or condition described herein, the target nucleotide sequence is present in a target RNA and a non-target dsDNA, and the Casl4 system is capable of targeting the target RNA but not the non-target dsDNA.

[0094] In some embodiments, according to any of the methods of treating a disease or condition described herein, the target nucleotide sequence is present in a gene sequence. In some embodiments, the target nucleotide sequence is present in a promoter region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a 5’ UTR region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a 5’ UTR/RBS region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a coding region of the gene, and can be present in the sense strand or anti-sense strand of the gene.

Engineered Cells

[0095] In some embodiments, the Cas 14 systems described herein are used in eukaryotic cells, such as mammalian cells, for example, human cells, to produce engineered cells with modulated expression of a target gene. Any human cell is contemplated for use with the Cas 14 systems disclosed herein.

[0096] In some embodiments, an engineered cell ex vivo or in vitro includes: (a) a Cas 14 protein or variant thereof, or nucleic acid encoding the Cas 14 protein or variant; and (b) a gRNA or nucleic acid encoding the gRNA, wherein the gRNA is capable of targeting the Cas 14 protein or variant to a target sequence in a target nucleic acid in the cell. In some embodiments, the Casl4 protein or variant and the gRNA are configured such that a complex formed by association of the Cas 14 protein or variant with the gRNA is capable of binding the target sequence in the target nucleic acid. In some embodiments, the engineered cell comprises the Cas 14 protein or variant. In some embodiments, the engineered cell comprises nucleic acid encoding the Cas 14 protein or variant. In some embodiments, the engineered cell comprises the gRNA. In some embodiments, the engineered cell comprises nucleic acid encoding the gRNA. In some embodiments, the gRNA is an sgRNA. In some embodiments, the engineered cell further comprises one or more additional gRNAs or nucleic acid encoding the one or more additional gRNAs.

[0097] In one aspect, some embodiments disclosed herein relate to a method of engineering an input cell that includes introducing into the input cell, such as an animal cell, one or more components of a Cas 14 system as described herein, and selecting or screening for an engineered cell transformed by the one or more system components. The term “engineered cell” refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in feet, be identical to the parent cell, but are still included within the scope of the term as used herein. Techniques for transforming a wide variety of cell are known in the art.

[0098] In a related aspect, some embodiments relate to engineered cells, for example, engineered animal cells that include a heterologous nucleic acid and/or polypeptide as described herein. The nucleic acid can be stably integrated in the host genome, or can be episomally replicating, or present in the engineered cell as a mini-circle expression vector for stable or transient expression.

[0099] In some embodiments, provided herein is an engineered cell, e.g., an isolated engineered cell, prepared by modulating the expression of a target gene in an input cell according to any of the methods described herein, thereby producing the engineered cell.

[0100] In some embodiments, provided herein is an engineered cell prepared by a method comprising providing to an input cell (a) a Casl4 protein or variant thereof, or nucleic acid encoding the Casl4 protein or variant; and (b) a gRNA or nucleic acid encoding the gRNA, wherein the gRNA is capable of targeting the Casl4 protein or variant to a target sequence in the genome of the input cell to modulate the expression of a target gene. In some embodiments, the Casl4 protein or variant and the gRNA are configured such that a complex formed by association of the Casl4 protein or variant with the gRNA is capable of binding the target sequence. In some embodiments, the method comprises providing to the input cell the Casl4 protein or variant. In some embodiments, the method comprises providing to the input cell nucleic acid encoding the Gas 14 protein or variant. In some embodiments, the method comprises providing to the input cell the gRNA. In some embodiments, the method comprises providing to the input cell nucleic acid encoding the gRNA. In some embodiments, the gRNA is an sgRNA. In some embodiments, the method further comprises providing to the input cell one or more additional gRNAs or nucleic acid encoding the one or more additional gRNAs.

[0101] In some embodiments, according to any of the engineered cells described herein, the Casl4 protein or variant is a dCasl4 protein according to any of the embodiments described herein, e.g., a dCasl4 protein having reduced or eliminated nuclease activity as compared to the parental Casl4 protein from which it is derived. In some embodiments, the parental Casl4 protein comprises or consists of the amino acid sequence of SEQ ID NO: 58 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the dCasl4 protein comprises one or more amino acids that have been altered or otherwise removed to reduce or eliminate its nuclease activity. In some embodiments, the amino acids include D326 and D510 with respect to SEQ ID NO: 58. In some embodiments, one or both of D326 and D510 are substituted with an amino acid that reduces, substantially eliminates, or eliminates nuclease activity. In some embodiments, one or both of D326 and D510 are substituted with alanine (i.e., D326A and/or D510A). In some embodiments, the dCasl4 exhibits reduced or eliminated nuclease activity, or nuclease activity is absent or substantially absent within levels of detection. In some embodiments, the dCasl4 protein comprises or consists of the amino acid sequence of SEQ ID NO: 60, 62, or 63, or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 60, 62, or 63. In some embodiments, the dCas14 protein comprises or consists of the amino acid sequence of SEQ ID NO: 60 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 60.

[0102] In some embodiments, according to any of the engineered cells described herein, the target nucleic acid is dsDNA and/or RNA. In some embodiments, according to any of the engineered cells described herein, the target nucleotide sequence is present in a target dsDNA and a target RNA, and the Cas 14 system is capable of targeting both the target dsDNA and the target RNA. In some embodiments, according to any of the engineered cells described herein, the target nucleotide sequence is present in a target dsDNA and a non-target RNA, and the Cas 14 system is capable of targeting the target dsDNA but not the non-target RNA. In some embodiments, according to any of the engineered cells described herein, the target nucleotide sequence is present in a target RNA and a non-target dsDNA, and the Cas 14 system is capable of targeting the target RNA but not the non-target dsDNA.

[0103] In some embodiments, according to any of the engineered cells described herein, the target nucleotide sequence is present in a gene sequence. In some embodiments, the target nucleotide sequence is present in a promoter region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a 5 ’ UTR region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a 5’ UTR/RBS region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a coding region of the gene, and can be present in the sense strand or anti-sense strand of the gene.

[0104] All cells suitable to be a target cell as discussed above are also suitable to be an engineered cell. For example, an engineered cell can be prepared from an input cell from any organism, e.g., a bacterial cell, 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, Chlorela pyrenoidosa, Sargassum patens C. Agardh, and the like), a fungal cell (e.g., a yeast cell), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.). In some embodiments, the engineered cell can be prepared from any input cell from a human.

[0105] In some embodiments, an engineered cell is in vitro. In some embodiments, an engineered cell is in vivo. In some embodiments, an engineered cell is a prokaryotic cell or is derived from a prokaryotic cell. In some embodiments, an engineered cell is a bacterial cell or is derived from a bacterial cell. In some embodiments, an engineered cell is an archaeal cell or is derived from an archaeal cell. In some embodiments, an engineered cell is a eukaryotic cell or is derived from a eukaryotic cell. In some embodiments, an engineered cell is a plant cell or is derived from a plant cell. In some embodiments, an engineered cell is an animal cell or is derived from an animal cell. In some embodiments, an engineered cell is an invertebrate cell or is derived from an invertebrate cell. In some embodiments, an engineered cell is a vertebrate cell or is derived from a vertebrate cell. In some embodiments, an engineered cell is a mammalian cell or is derived from a mammalian cell. In some embodiments, an engineered cell is a rodent cell or is derived from a rodent cell. In some embodiments, an engineered cell is a human cell or is derived from a human cell. In some embodiments, the engineered cell is a human cell or is derived from a human cell.

[0106] The present disclosure further provides progeny of an engineered cell, where the progeny can include the same exogenous nucleic acid or polypeptide as the engineered cell fiom which it was derived. The present disclosure further provides, in some embodiments, a composition comprising an engineered cell.

Compositions

[0107] In one aspect, some embodiments disclosed herein relate to a composition that includes (i) a Cas 14 protein or variant thereof, or nucleic acid encoding the Cas 14 protein or variant; and/or (ii) a gRNA or nucleic acid encoding the gRNA. In some embodiments, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable excipient and/or carrier.

[0108] In some embodiments, provided herein is a composition comprising (i) a Cas 14 protein or variant thereof, or nucleic acid encoding the Cas 14 protein or variant; and/or (ii) a gRNA or nucleic acid encoding the gRNA. In some embodiments, the composition is used for carrying out a method of the present disclosure, e.g., a method for site-specific modification of a target DNA; etc.

[0109] In some embodiments, the composition includes: (a) a Cas 14 protein or variant thereof having at least about 90% (such as at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 58, 60, 62, or 63, or nucleic acid encoding the Cas 14 protein or variant; and/or (b) a gRNA or nucleic acid encoding the gRNA, wherein the gRNA is capable of targeting the Cas 14 protein or variant to a target sequence. In some embodiments, the composition comprises the Cas 14 protein or variant. In some embodiments, the composition comprises nucleic acid encoding the Cas 14 protein or variant. In some embodiments, the composition comprises the gRNA. In some embodiments, the composition comprises nucleic acid encoding the gRNA. In some embodiments, the gRNA is an sgRNA. In some embodiments, the composition comprises one or more additional gRNAs or nucleic acid encoding the one or more additional gRNAs.

[0110] In some embodiments, according to any of the compositions described herein, the Casl4 protein or variant is a dCasl4 protein according to any of the embodiments described herein, e.g., a dCasl4 protein having reduced or eliminated nuclease activity as compared to the parental Cas 14 protein from which it is derived. In some embodiments, the parental Cas 14 protein comprises or consists of the amino acid sequence of SEQ ID NO: 58 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the dCasl4 protein comprises one or more amino acids that have been altered or otherwise removed to reduce or eliminate its nuclease activity. In some embodiments, the amino acids include D326 and D510 with respect to SEQ ID NO: 58. In some embodiments, one or both of D326 and D510 are substituted with an amino acid that reduces, substantially eliminates, or eliminates nuclease activity. In some embodiments, one or both of D326 and D510 are substituted with alanine (i.e., D326A and/or D510A). In some embodiments, the dCasl4 exhibits reduced or eliminated nuclease activity, or nuclease activity is absent or substantially absent within levels of detection. In some embodiments, the dCasl4 protein comprises or consists of the amino acid sequence of SEQ ID NO: 60, 62, or 63, or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 60, 62, or 63. In some embodiments, the dCasl4 protein comprises or consists of the amino acid sequence of SEQ ID NO: 60 or a variant thereof having at least about 80% sequence identity' to the amino acid sequence of SEQ ID NO: 60.

[0111] In some embodiments, according to any of the compositions described herein, the target nucleic acid is dsDNA and/or RNA. In some embodiments, according to any of the compositions described herein, the target nucleotide sequence is present in a target dsDNA and a target RNA, and the Casl4 system is capable of targeting both the target dsDNA and the target RNA. In some embodiments, according to any of the compositions described herein, the target nucleotide sequence is present in a target dsDNA and a non-target RNA, and the Cas 14 system is capable of targeting the target dsDNA but not the non-target RNA. In some embodiments, according to any of the compositions described herein, the target nucleotide sequence is present in a target RNA and a non-target dsDNA, and the Cas 14 system is capable of targeting the target RNA but not the non-target dsDNA.

[0112] In some embodiments, according to any of the compositions described herein, the target nucleotide sequence is present in a gene sequence. In some embodiments, the target nucleotide sequence is present in a promoter region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a 5’ UTR region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a 5’ UTR/RBS region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a coding region of the gene, and can be present in the sense strand or anti-sense strand of the gene.

Kits

[0113] In some embodiments, provided herein are kits for carrying out a method described herein. A kit can include one or more of: a Cas 14 protein or variant thereof; nucleic acid encoding a Cas 14 protein or variant thereof; a gRNA; and nucleic acid encoding a gRNA. A kit may include a complex that includes two or more of: a Casl4 protein or variant thereof; a nucleic acid comprising a nucleotide encoding a Cas 14 protein or variant thereof; a gRNA; and a nucleic acid encoding a gRNA.

[0114] In some embodiments, a kit includes: (a) a Cas 14 protein or variant thereof having at least about 90% (such as at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 58, 60, 62, or 63, or nucleic acid encoding the Cas 14 protein or variant; and (b) a gRNA or nucleic acid encoding the gRNA, wherein the gRNA is capable of targeting the Casl4 protein or variant to a target sequence. In some embodiments, the kit comprises the Cas 14 protein or variant. In some embodiments, the kit comprises nucleic acid encoding the Cas 14 protein or variant. In some embodiments, the kit comprises the gRNA. In some embodiments, the kit comprises nucleic acid encoding the gRNA. In some embodiments, the gRNA is an sgRNA. In some embodiments, the kit comprises one or more additional gRNAs or nucleic acid encoding the one or more additional gRNAs. In some embodiments, the kit further comprises a reagent for reconstituting and/or diluting one or more of the kits components.

[0115] In some embodiments, according to any of the kits described herein, the Casl4 protein or variant is a dCas14 protein according to any of the embodiments described herein, e.g., a dCas14 protein having reduced or eliminated nuclease activity as compared to the parental Casl4 protein from which it is derived. In some embodiments, the parental Casl4 protein comprises or consists of the amino acid sequence of SEQ ID NO: 58 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the dCasl4 protein comprises one or more amino acids that have been altered or otherwise removed to reduce or eliminate its nuclease activity. In some embodiments, the amino acids include D326 and D510 with respect to SEQ ID NO: 58. In some embodiments, one or both of D326 and D510 are substituted with an amino acid that reduces, substantially eliminates, or eliminates nuclease activity. In some embodiments, one or both of D326 and D510 are substituted with alanine (i.e., D326A and/or D510A). In some embodiments, the dCasl4 exhibits reduced or eliminated nuclease activity, or nuclease activity' is absent or substantially absent within levels of detection. In some embodiments, the dCasl4 protein comprises or consists of the amino acid sequence of SEQ ID NO: 60, 62, or 63, or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 60, 62, or 63. In some embodiments, the dCasl4 protein comprises or consists of the amino acid sequence of SEQ ID NO: 60 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 60.

[0116] In some embodiments, according to any of the kits described herein, the target nucleic acid is dsDNA and/or RNA. In some embodiments, according to any of the kits described herein, the target nucleotide sequence is present in a target dsDNA and a target RNA, and the Casl4 system is capable of targeting both the target dsDNA and the target RNA. In some embodiments, according to any of the kits described herein, the target nucleotide sequence is present in a target dsDNA and a non-target RNA, and the Casl4 system is capable of targeting the target dsDNA but not the non-target RNA. In some embodiments, according to any of the kits described herein, the target nucleotide sequence is present in a target RNA and a non-target dsDNA, and the Cas14 system is capable of targeting the target RNA but not the non-target dsDNA.

[0117] In some embodiments, according to any of the kits described herein, the target nucleotide sequence is present in a gene sequence. In some embodiments, the target nucleotide sequence is present in a promoter region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a 5’ UTR region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a 5’ UTR/RBS region of the gene, and can be present in the sense strand or anti-sense strand of the gene. In some embodiments, the target nucleotide sequence is present in a coding region of the gene, and can be present in the sense strand or anti-sense strand of the gene.

[0118] A kit as described herein can further include one or more additional reagents, where such additional reagents can be selected from: a buffer for introducing the Casl4 protein or variant into a cell; a dilution buffer; a reconstitution solution; a wash buffer; a control reagent; a control expression vector or polyribonucleotide; a reagent for in vitro production of the Cas 14 protein or variant from DNA, and the like.

[0119] In some embodiments of any of the kits described herein, the kit includes a gRNA (e.g., an sgRNA). In some embodiments, the kit includes two or more gRNAs.

[0120] Components of a kit can be in separate containers; or can be combined in a single container.

[0121] In addition to the above-mentioned components, a kit can further include instructions for using the components of the kit to practice the methods. Hie instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging or sub-packaging) etc. In some embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

EMBODIMENTS

[0122] The following embodiments are contemplated. All combinations of features and embodiment are contemplated.

[0123] Embodiment 1: A system comprising: a Casl4 protein or variant thereof, or nucleic acid encoding the Casl4 protein or variant, wherein the Casl4 protein or variant is a dCasl4 protein having reduced or eliminated nuclease activity as compared to a parental Cas 14 protein from which it is derived; and a guide RNA (gRNA) or nucleic acid encoding the gRNA, wherein the gRNA comprises a spacer having sufficient complementary to a target nucleotide sequence in a target nucleic acid such that it can hybridize to the target nucleic acid.

[0124] Embodiment 2: An embodiment of embodiment 1, wherein the gRNA is a single-guide RNA (sgRNA).

[0125] Embodiment 3: An embodiment of embodiment 1 or 2, wherein the gRNA comprises a spacer having a length ranging from 20 nucleotides to 34 nucleotides.

[0126] Embodiment 4: An embodiment of any of the embodiments of embodiment 1 to 3, wherein the Cas 14 protein or variant thereof recognizes a protospacer adjacent motif (PAM) having the sequence 5'-THG-3' or 5'-lllA-3'.

[0127] Embodiment 5: An embodiment of any of the embodiments of embodiment 1 to 4, wherein the parental Cas 14 protein comprises the amino acid sequence of SEQ ID NO: 58.

[0128] Embodiment 6: An embodiment of any of the embodiments of embodiment 1 to 5, wherein the dCasl4 protein comprises one or more amino acids that have been altered or otherwise removed to reduce or eliminate its nuclease activity.

[0129] Embodiment 7: An embodiment of embodiment 6, wherein the one or more amino acids include D326 and D510 with respect to SEQ ID NO: 58.

[0130] Embodiment 8: An embodiment of embodiment 7, wherein one or both of D326 and D510 are substituted with an amino acid that reduces, substantially eliminates, or eliminates nuclease activity.

[0131] Embodiment 9: An embodiment of embodiment 8, wherein one or both of D326 and D510 are substituted with alanine.

[0132] Embodiment 10: An embodiment of any of the embodiments of embodiment 1 to 9, wherein the dCasl4 protein comprises the amino acid sequence of SEQ ID NO: 60.

[0133] Embodiment 11: An embodiment of any of the embodiments of embodiment 1 to 10, wherein the target nucleic acid is a target dsDNA.

[0134] Embodiment 12: An embodiment of embodiment 11, wherein a protospacer in the target dsDNA targeted by the gRNA spacer has a PAM selected from 5'-TTTG-3' and 5'-lll A-3'.

[0135] Embodiment 13: An embodiment of embodiment 11 or 12, wherein the target dsDNA is a genomic DNA and the target nucleotide sequence is present in the sense strand of the genomic DNA corresponding to an mRNA transcribed from the genomic DNA. [0136] Embodiment 14: An embodiment of embodiment 11 or 12, wherein the target dsDNA is a genomic DNA and the target nucleotide sequence is present in the anti-sense strand of the genomic DNA complementary to an mRNA transcribed from the genomic DNA.

[0137] Embodiment 15: An embodiment of any of the embodiments of embodiment 1 to 10, wherein the target nucleic acid is a target RNA.

[0138] Embodiment 16: An embodiment of embodiment 15, wherein the target RNA is an mRNA transcribed from a genomic DNA, and the target nucleotide sequence is present in the sense strand of the genomic DNA corresponding to the mRNA.

[0139] Embodiment 17: An embodiment of embodiment 16, wherein any protospacers in the genomic DNA targeted by the gRNA spacer do not have a PAM selected from 5'-TH"G-3' and 5'-TTTA-3'.

[0140] Embodiment 18: An embodiment of any of the embodiments of embodiment 1 to 10, wherein the target nucleic acid is a target ssDNA.

[0141] Embodiment 19: An embodiment of any of the embodiments of embodiment 13, 14, and 16, wherein targeting of the genomic DNA by the Casl4 systems is capable of modulating transcription of the mRNA.

[0142] Embodiment 20: An embodiment of embodiment 19, wherein targeting of the genomic DNA by the Casl4 system is capable of repressing transcription of the mRNA.

[0143] Embodiment 21 : An embodiment of embodiment 19, wherein targeting of the genomic DNA by the Casl4 system is capable of enhancing transcription of the mRNA.

[0144] Embodiment 22: An embodiment of any of the embodiments of embodiment 13, 16, and 17, wherein targeting of the mRNA by the Casl4 system is capable of modulating translation of the mRNA.

[0145] Embodiment 23: An embodiment of embodiment 22, wherein targeting of the mRNA by the Casl4 system is capable of repressing translation of the mRNA.

[0146] Embodiment 24: An embodiment of embodiment 22, wherein targeting of the mRNA by the Casl4 system is capable of enhancing translation of the mRNA.

[0147] Embodiment 25: An embodiment of any of the embodiments of embodiment 1 to 24, wherein the target nucleotide sequence is present in a gene sequence.

[0148] Embodiment 26: An embodiment of embodiment 25, wherein the target nucleotide sequence is present in a promoter region of the gene.

[0149] Embodiment 27: An embodiment of embodiment 25, wherein the target nucleotide sequence is present in a 5’ UTR region of the gene. [0150] Embodiment 28: An embodiment of embodiment 25, wherein the target nucleotide sequence is present in a 5’ UTR/RBS region of the gene.

[0151] Embodiment 29: An embodiment of embodiment 25, wherein the target nucleotide sequence is present in a coding region of the gene.

[0152] Embodiment 30: A method for modulating the expression of a target gene in a cell to produce an engineered cell, the method comprising providing to the cell the system of an embodiment of any of the embodiments of embodiment 1 to 29, wherein the target nucleotide sequence is present in the target gene, thereby producing an engineered cell having modulated expression of the target gene.

[0153] Embodiment 31: An embodiment of embodiment 30, wherein the engineered cell is a prokaryotic cell.

[0154] Embodiment 32: An embodiment of embodiment 31, wherein the prokaryotic cell is a bacterial cell.

[0155] Embodiment 33: An engineered cell produced by the method of embodiment 30.

[0156] Embodiment 34: An embodiment of embodiment 33, wherein the engineered cell is a prokaryotic cell.

[0157] Embodiment 35: An embodiment of embodiment 34, wherein the prokaryotic cell is a bacterial cell.

[0158] Embodiment 36: An engineered cell comprising the system of an embodiment of any of the embodiments of embodiment 1 to 29.

[0159] Embodiment 37: An embodiment of embodiment 36, wherein the engineered cell is a prokaryotic cell.

[0160] Embodiment 38: An embodiment of embodiment 37, wherein the prokaryotic cell is a bacterial cell.

[0161] Embodiment 39: A kit comprising one or more components of the system of an embodiment of any of the embodiments of embodiment 1 to 29.

EXAMPLES

[0162] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry', and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory- and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY : Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference.

[0163] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.

Example 1: Design and characterization of a dCas14al-mediated CRISPRi/a system

[0164] This example demonstrates the design and characterization of a CRISPRi/a system based on a nuclease-deficient variant of Cas 14al .

[0165] The dCasl4al-mediated CRISPRi/a system used in this example was based on a catalytically dead variant of Cas 14al having D326 A and D510A amino acid substitutions (also referred to herein as dCasl4al-l) and sgRNA chimera (e.g., sgRNA designed to be compatible or incompatible with the PAM requirements for Casl4al, such as 5'-TTTG-3' or 5'-TTTA-3' PAM located immediately 5' of the target protospacer). In this system, the dCasl4al-l protein was placed under the control of an anhydrotetracycline (aTc)-inducible promoter, Ptet, and the sgRNAs were placed under the control of a minimal constitute promoter, PJ23119. See FIG. 1A.

[0166] Binding of a complex of dCasl4al-l with a compatible sgRNA to a target protein coding region was predicted to be able to repress expression of the target gene, e.g., by blocking the binding of elongation of RNA polymerase (RNAP). See FIG. IB.

[0167] In order to test this model, a synthetic fluorescence-based reporter system in E. colt was used as previously described in Qi, L. S., et al. (2013). Cell, 152(5), 1173-1183. Sequences encoding sfGFP and mRFP, both individually under the control of promoter PJ233119, were inserted into the nsfA locus of E. coll strain MG1655 to generate the reporter strain EC001 (FIG. 1C).

[0168] In one study, reporter strain EC001 was transformed with plasmids encoding dCas9 and sgRNA sgRR2 (spacer sequence of SEQ ID NO: 64) targeting mRFP but not GFP, or plasmids encoding dCasl4al-l and one of the following sgRNAs targeting the 5’ UTR common to both the mRFP and GFP expression cassettes: alGRRl (spacer sequence of SEQ ID NO: 23), alGRR2 (spacer sequence of SEQ ID NO: 24), alGRR3 (spacer sequence of SEQ ID NO: 25), alGRR4 (spacer sequence of SEQ ID NO: 26), alGRR5 (spacer sequence of SEQ ID NO: 27), and alGRR6 (spacer sequence of SEQ ID NO: 28). The target sites for alGRRl-alGRR6 are shown in FIG. 2E and described in Table 1 below. As shown in FIGS. 2A and 2C, in cells transfected with dCas9 and sgRR2 the expression of mRFP was almost completely abolished (FIG. 2A), while expression of GFP increased slightly (FIG. 2C). BBl(Cm): expression backbone of dCas14al-l; BB (Carb): expression backbone of sgRR2. As shown in FIGS. 2B and 2D, in cells transfected with dCasl4al-l and any of sgRNAs alGRRl-alGRR6 the expression levels of both mRFP (FIG. 2B) and GFP (FIG. 2D) were significantly reduced compared to those when using control non-targeting guides alNT2 and alNT3, which lack binding sites in the mRFP and GFP expression cassettes. These results demonstrate the feasibility of using dCasl4 systems for multiplexed control of multiple genes.

Table 1

[0169] In another study, different lengths of guide RNAs targeting different sites in GFP were tested for GFP repression. EC001 was transformed with plasmids encoding dCasl4al-l and one of the following sgRNAs targeting GFP but not RFP: alGR4 (spacer sequence of SEQ ID NO: 4), alGR5 (spacer sequence of SEQ ID NO: 5), alGR6 (spacer sequence of SEQ ID NO: 6), alGR7 (spacer sequence of SEQ ID NO: 7), alGR8 (spacer sequence of SEQ ID NO: 8), alGR9 (spacer sequence of SEQ ID NO: 9), alGRIO (spacer sequence of SEQ ID NO: 10), alGR12 (spacer sequence of SEQ ID NO: 11), alGR13 (spacer sequence of SEQ ID NO: 12), alGR14 (spacer sequence of SEQ ID NO: 13), alGRI5 (spacer sequence of SEQ ID NO: 14), alGR16 (spacer sequence of SEQ ID NO: 15), alGR17 (spacer sequence of SEQ ID NO: 16), alGRl 8 (spacer sequence of SEQ ID NO: 17), alGR19 (spacer sequence of SEQ ID NO: 18), alGR21 (spacer sequence of SEQ ID NO: 19), alGR22 (spacer sequence of SEQ ID NO: 20), alGR23 (spacer sequence of SEQ ID NO: 21), and alGR24 (spacer sequence of SEQ ID NO: 22). The target sites for alGR4-alGR24 are shown in FIG. 3G and described in Table 2 below. As shown in FIGS. 3A and 3B, in cells transfected with dCas9 and sgRR2 the expression of mRFP was almost completely abolished (FIG. 3A), while expression of GFP increased (FIG. 3B). BBl(Cm): expression backbone of dCasl4al-l; BB (Carb): expression backbone of sgRR2. As shown in FIGS. 3C and 3E, in cells transfected with dCasl4al-l and certain sgRNAs (alGRIO, alGR12, alGR13-alGR18, and alGR22-alGR24), the expression level of GFP was reduced compared to that when using control non-targeting guides alNT2 and alNT3, which lack binding sites in the GFP expression cassettes. No such reduction was observed for RFP levels (FIGS. 3D and 3F). These results demonstrate a dependence on target site and guide length for repression of gene expression.

Table 2

[0170] In another study, different lengths of guide RNAs targeting different sites in RFP were tested for RFP repression. EC001 was transformed with plasmids encoding dCasl4al-l and one of the following sgRNAs targeting RFP but not GFP: alRRl (spacer sequence of SEQ ID NO: 29), alRR2 (spacer sequence of SEQ ID NO: 29), alRR3 (spacer sequence of SEQ ID

NO: 30), alRR4 (spacer sequence of SEQ ID NO: 31), alRR6 (spacer sequence of SEQ ID

NO: 32), alRR4 (spacer sequence of SEQ ID NO: 31), alRR6 (spacer sequence of SEQ ID

NO: 32), alRR7 (spacer sequence of SEQ ID NO: 33), alRR8 (spacer sequence of SEQ ID

NO: 34), alRR9 (spacer sequence of SEQ ID NO: 35), alRRl 1 (spacer sequence of SEQ ID NO: 36), alRR12 (spacer sequence of SEQ ID NO: 37), alRR13 (spacer sequence of SEQ ID NO: 38), alRR14 (spacer sequence of SEQ ID NO: 39), alRR15 (spacer sequence of SEQ ID NO: 40), alRR16 (spacer sequence of SEQ ID NO: 41), alRRl? (spacer sequence of SEQ ID NO: 42), alRRl 8 (spacer sequence of SEQ ID NO: 43), alRR19 (spacer sequence of SEQ ID NO: 44), alRR20 (spacer sequence of SEQ ID NO: 45), alRR21 (spacer sequence of SEQ ID NO: 46), alRR22 (spacer sequence of SEQ ID NO: 47), alRR23 (spacer sequence of SEQ ID NO: 48), alRR25 (spacer sequence of SEQ ID NO: 49), alRR26 (spacer sequence of SEQ ID NO: 50), alRR27 (spacer sequence of SEQ ID NO: 51), alRR28 (spacer sequence of SEQ ID NO: 52), and alRR29 (spacer sequence of SEQ ID NO: 53). The target sites for alRRl - alRR29 are shown in FIG. 4F and described in Table 3 below. As shown in FIGS. 4B, 4D, and 4E, in cells transfected with dCasl4al-l and certain sgRNAs (alRRl -alRRl 8), the expression level of RFP was reduced compared to that when using control non-targeting guides (alNTl, alNT2, or alNT3), which lack binding sites in the RFP expression cassettes. No such reduction was observed for GFP levels (FIGS. 4A, 4C, and 4E). These results further demonstrate the dependence on target site and guide length for repression of gene expression, and show that guides with lengths ranging from 20-34 can be effective in this system.

Table 3

[0171] In another study, the hypothesis that Casl4 is an RNA-guided DNA- and RNA- targeting system was tested. EC001 was transformed with plasmids encoding dCasl4al-l and one of the following sgRNAs: alGRl (spacer sequence of SEQ ID NO: 1), alGRZ (spacer sequence of SEQ ID NO: 2), and alGR3 (spacer sequence of SEQ ID NO: 3). The target sites for these sgRNAs are shown in FIG. 5C and described in Table 4 below. alGRl-alGR3 can target the 5 ’ UTR region on the sense strand of the GFP expression cassette. They also partially match a region of the 5’ UTR of RFP (18-bp to 24-bp match with 2-bp mismatch), though importantly there is no PAM in the RFP expression cassette to allow for DNA binding. Surprisingly, gene activation of RFP was observed (multiple replicates), as shown in FIG. 5B. There was almost no change in expression for GFP (FIG. 5 A). This suggests that dCasl4 can bind to both DNA and RNA. Binding to DNA relies on both guide RNA matching and the PAM, whereas binding to RNA only reties on guide RNA matching without the need for a PAM. In the case for RFP, it binds only to RNA and activates gene expression, possibly byaltering the RNA conformation to allow for better translation. In the case for GFP, it can bind to both DNA and RNA with opposing effects, with DNA binding repressing transcription and RNA binding activating translation, leading to an overall lack of any change in gene expression. These results support the hypothesis that Casl4 is an RNA-guided DNA- and RNA-targeting system.

Table 4

[0172] In another study, the ability of dCasl4 to bind single stranded DNA was tested. An electrophoretic mobility' shift assay (EMSA) was used in the study, with 2 nM of a cy'5-labeled 40-bp ssDNA test substrate added to each lane of the gel. Different gel lanes further included one or both of dCas 14 and sgRNA, at concentrations ranging from 60 nM to 600 nM. As shown in the image of FIG. 7, dCasl4 was demonstrated as exhibiting strong binding activity' to ssDNA.

[0173] Accordingly, the dCasl4al -mediated CRISPRi systems described herein can be used in different configurations to specifically target DNA and RNA, to target DNA only, or to target RNA only. In a system for targeting both DNA and RNA, the guide RNA can be designed to have a spacer complementary to a target sequence in the sense strand (non-template strand) having a compatible PAM (e.g., 5'-TTTG-3' or 5'-TTTA-3' located immediately 5' of the target protospacer). See, e.g., FIG. 6A. In a system for targeting DNA only, the guide RNA can be designed to have a spacer complementary to a target sequence in the anti-sense strand (template strand) having a compatible PAM (e.g., 5'-TTTG-3' or 5'-TTTA-3' located immediately 5' of the target protospacer). See, e.g., FIG. 6B. In a system for targeting RNA only, the guide RNA can be designed to have a spacer complementary to a target sequence in the sense strand (nontemplate strand) without a compatible PAM. See, e.g., FIG. 6C.

[0174] Although the foregoing disclosure has been described in some detail by way of illustration and example for purpose of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications within the spirit and scope of the disclosure may be practiced, e.g., within the scope of the appended claims. It should also be understood that aspects of the disclosure and portions of various recited embodiments and features can be combined or interchanged either in whole or in part. In the foregoing descriptions of the various embodiments, those embodiments which refer to another embodiment may be appropriately combined with other embodiments as will be appreciated by one of skill in the art. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the disclosure. In addition, each reference provided herein is incorporated by reference in its entirety for all purposes to the same extent as if each reference was individually incorporated by reference.

SEQUENCE LISTING

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A system comprising:. a Cas 14 protein or variant thereof, or nucleic acid encoding the Cas 14 protein or variant, wherein the Cas 14 protein or variant is a dCas 14 protein having reduced or eliminated nuclease activity as compared to a parental Casl4 protein from which it is derived; and a guide RNA (gRNA) or nucleic acid encoding the gRNA, wherein the gRNA comprises a spacer having sufficient complementary to a target nucleotide sequence in a target nucleic acid such that it can hybridize to the target nucleic acid.

2. The system of claim 1, wherein the gRNA is a single-guide RNA (sgRNA).

3. The system of claim 1, wherein the gRNA comprises a spacer having a length ranging from 20 nucleotides to 34 nucleotides.

4. The system of claim 1, wherein the Cas 14 protein or variant thereof recognizes a protospacer adjacent motif (PAM) having the sequence 5'-TTTG-3' or 5 -T1TA-3'.

5. The system of claim 1, wherein the parental Casl4 protein comprises the amino acid sequence of SEQ ID NO: 58.

6. The system of claim 1, wherein the dCasl4 protein comprises one or more amino acids that have been altered or otherwise removed to reduce or eliminate its nuclease activity.

7. The system of claim 6, wherein the one or more amino acids include D326 and D510 with respect to SEQ ID NO: 58.

8. The system of claim 7, wherein one or both of D326 and D510 are substituted with an amino acid that reduces, substantially eliminates, or eliminates nuclease activity.

9. The system of claim 8, wherein one or both of D326 and D510 are substituted with alanine.

10. The system of claim 1, wherein the dCasl4 protein comprises the amino acid sequence of SEQ ID NO: 60.

11. The system of claim 1, wherein the target nucleic acid is a target dsDNA.

12. The system of claim 11, wherein a protospacer in the target dsDNA targeted by the gRNA spacer has a PAM selected from 5'-TTTG-3' and 5'-TTTA-3'.

13. The system of claim 11, wherein the target dsDNA is a genomic DNA and the target nucleotide sequence is present in the sense strand of the genomic DNA corresponding to an mRNA transcribed from the genomic DNA.

14. The system of claim 11, wherein the target dsDNA is a genomic DNA and the target nucleotide sequence is present in the anti-sense strand of the genomic DNA complementary to an mRNA transcribed from the genomic DNA.

15. The system of claim 1, wherein the target nucleic acid is a target RNA.

16. The system of claim 15, wherein the target RNA is an mRNA transcribed from a genomic DNA, and the target nucleotide sequence is present in the sense strand of the genomic DNA corresponding to the mRNA.

17. The system of claim 16, wherein any protospacers in the genomic DNA targeted by the gRNA spacer do not have a PAM selected from 5'-lllG-3' and 5'-TTTA-3'.

18. The system of claim 1, wherein the target nucleic acid is a target ssDNA.

19. The system of claim 13, wherein targeting of the genomic DNA by the Casl4 systems is capable of modulating transcription of the mRNA.

20. The system of claim 19, wherein targeting of the genomic DNA by the Casl4 system is capable of repressing transcription of the mRNA.

21. The system of claim 19, wherein targeting of the genomic DNA by the Cas 14 system is capable of enhancing transcription of the mRNA.

22. The system of claim 13, wherein targeting of the mRNA by the Cas 14 system is capable of modulating translation of the mRNA.

23. The system of claim 22, wherein targeting of the mRNA by the Cas 14 system is capable of repressing translation of the mRNA.

24. The system of claim 22, wherein targeting of the mRNA by the Cas 14 system is capable of enhancing translation of the mRNA.

25. The system of claim 1, wherein the target nucleotide sequence is present in a gene sequence.

26. The system of claim 25, wherein the target nucleotide sequence is present in a promoter region of the gene.

27. The system of claim 25, wherein the target nucleotide sequence is present in a 5’ UTR region of the gene.

28. The system of claim 25, wherein the target nucleotide sequence is present in a 5’ UTR/RBS region of the gene.

29. The system of claim 25, wherein the target nucleotide sequence is present in a coding region of the gene.

30. A method for modulating the expression of a target gene in a cell to produce an engineered cell, the method comprising providing to the cell the system of claim 1 , wherein the target nucleotide sequence is present in the target gene, thereby producing an engineered cell having modulated expression of the target gene.

31. The method of claim 30, wherein the engineered cell is a prokaryotic cell.

32. The method of claim 31, wherein the prokaryotic cell is a bacterial cell.

33. An engineered cell produced by the method of claim 30.

34. The engineered cell of claim 33, wherein the engineered cell is a prokaryotic cell.

35. The engineered cell of claim 34, wherein the prokaryotic cell is a bacterial cell.

36. An engineered cell comprising the system of claim 1.

37. The engineered cell of claim 36, wherein the engineered cell is a prokaryotic cell

38. The engineered cell of claim 37, wherein the prokaryotic cell is a bacterial cell

39. A kit comprising one or more components of the system of claim 1.