Systems, methods, and compositions for targeted nucleic acid editing

Abstract

To provide systems, methods, and compositions for targeting and editing nucleic acids.SOLUTION: The invention provides non-naturally occurring or engineered RNA-targeting systems comprising a RNA-targeting Cas13 protein, at least one guide molecule, and at least one adenosine deaminase protein or catalytic domain thereof. The Cas13 protein may be a catalytically inactive Cas13 effector protein, and may be the Prevotella sp. P5-125 Cas13b protein.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 561,669, filed Sep. 21, 2017. The entire contents of the above-identified applications are hereby incorporated by reference in their entireties.

[0002] This invention was made with Federal support under Grant No. MH110049 awarded by the National Institutes of Health. The Federal Government has certain rights in this invention.

[0003] The contents of the electronic sequence listing ("BROD-2305WP_ST25.txt"; size 1,409,943 bytes, created on September 14, 2018) are hereby incorporated by reference in their entirety.

[0004] The present invention relates generally to systems, methods, and compositions for targeting and editing nucleic acids, and in particular for the programmable deamination of adenine at target loci of interest. [Background technology]

[0005] Recent advances in genome sequencing technologies and analytical methods have rapidly improved our ability to catalog and map genetic factors associated with various biological functions and diseases. Precise genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by selectively perturbing individual genetic elements and to advance synthetic biology, biotechnological, and medical applications. Although genome editing technologies such as designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases are available to generate targeted genome perturbations, there is still a need for novel genome engineering technologies that utilize novel strategies and molecular mechanisms and that are inexpensive, easy to set up, scalable, and suitable for targeting multiple locations in eukaryotic genomes. This will be a major resource for novel applications in genome engineering and biotechnology.

[0006] Programmable deamination of cytosine has been reported and can be used to correct A→G and T→C point mutations. For example, Komor et al., Nature (2016) 533:420-424, report targeted deamination of cytosine by APOBEC1 cytidine deaminase in non-targeted DNA strands displaced by binding of a Cas9-guide RNA complex to the targeted DNA strand, resulting in the conversion of cytosine to uracil. See also Kim et al., Nature Biotechnology (2017) 35:371-376; Shimatani et al., Nature Biotechnology (2017) doi:10.1038 / nbt.3833; Zong et al., Nature Biotechnology (2017) doi:10.1038 / nbt.3811; Yang Nature Communication (2016) doi:10.1038 / ncomms13330. Summary of the Invention

[0007] In one aspect, the disclosure includes an engineered, non-naturally occurring system comprising a catalytically inactive Cas13 effector protein (dCas13) or a nucleotide sequence encoding said catalytically inactive Cas13 effector protein.

[0008] In some embodiments, the dCas13 protein is truncated at the C-terminus, the N-terminus, or both. In some embodiments, the dCas13 is truncated by at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 220, at least 240, at least 260, or at least 300 amino acids on the C-terminus. In some embodiments, the dCas13 is truncated by at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 220, at least 240, at least 260, or at least 300 amino acids on the N-terminus. In some embodiments, the truncated form of the Cas13 effector protein is truncated at the C-terminus Δ984-1090, the C-terminus Δ1026-1090, the C-terminus Δ1053-1090, the C-terminus Δ934-1090, the C-terminus Δ884-1090, the C-terminus Δ834-1090, the C-terminus Δ784-1090, or the C-terminus Δ734-1090, where the amino acid positions of the truncation correspond to the amino acid positions of the Prevotella sp. P5-125 Cas13b protein. In some embodiments, the truncated form of the Cas13 effector protein is truncated at the C-terminus Δ795-1095, where the amino acid positions of the truncation correspond to the amino acid positions of the Riemerella anatipestifer Cas13b protein. In some embodiments, the truncated forms of the Cas13 effector protein are truncated at the C-terminus Δ875-1175, C-terminus Δ895-1175, C-terminus Δ915-1175, C-terminus Δ935-1175, C-terminus Δ955-1175, C-terminus Δ975-1175, C-terminus Δ995-1175, C-terminus Δ1015-1175, C-terminus Δ1035-1175, C-terminus Δ1055-1175, C-terminus Δ1075-1175, C-terminus Δ1095-1175, C-terminus Δ1115-1175, C-terminus Δ1135-1175, C-terminus Δ1155-1175, where the amino acid positions correspond to those of the Porphyromonas gulae Cas13b protein.In some embodiments, the truncated form of the Cas13 effector protein is truncated at the N-terminus Δ1-125, N-terminus Δ1-88, or N-terminus Δ1-72, where the amino acid positions of the truncation correspond to the amino acid positions of the Prevotella sp. P5-125 Cas13b protein. In some embodiments, the dCas13 comprises a truncated form of the Cas13 effector protein at the HEPN domain of the Cas13 effector protein.

[0009] In some embodiments, the Cas13 effector protein is Cas13a. In some embodiments, the Cas13 effector protein is Cas13b. In some embodiments, the Cas13 effector protein is Cas13c or Cas13d.

[0010] In some embodiments, the system further comprises a functional component, and the nucleotide sequence further encodes the functional component. In some embodiments, the functional component is a base editing component. In some embodiments, the base editing component comprises an adenosine deaminase, a cytidine deaminase, or a catalytic domain thereof. In some embodiments, the adenosine deaminase, a cytidine deaminase, or a catalytic domain thereof is fused to dCas13. In some embodiments, the adenosine deaminase, a cytidine deaminase, or a catalytic domain thereof is fused to dCas13 by a linker. In some embodiments, the adenosine deaminase, a cytidine deaminase, or a catalytic domain thereof is inserted into an internal loop of dCas13. In some embodiments, the adenosine deaminase, a cytidine deaminase, or a catalytic domain thereof is linked to an adaptor protein. In some embodiments, the adaptor protein is selected from MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, and PRR1. In some embodiments, the adenosine deaminase, cytidine deaminase, or catalytic domain thereof is a human, cephalopod, or Drosophila protein.

[0011] In some embodiments, dCas13 is a split Cas13 effector protein. In some embodiments, the split Cas13 effector protein is a first split Cas13 effector protein that can be fused to a second split Cas13 effector protein to form a catalytically active Cas13 effector protein. In some embodiments, the fusion of the first and second Cas13 effector proteins is inducible.

[0012] In some embodiments, the functional component is a transcription factor or an active domain thereof. In some embodiments, the dCas13 is fused to the transcription factor or an active domain thereof. In some embodiments, the system further comprises a guide sequence.

[0013] In another aspect, the disclosure includes a vector system comprising one or more vectors encoding dCas13 as disclosed herein.

[0014] In another aspect, the disclosure includes an in vitro or ex vivo host cell or progeny thereof or a cell line or progeny thereof comprising the system disclosed herein.

[0015] In another aspect, the disclosure includes a method for programmable and targeted base editing of a target sequence comprising delivering a system disclosed herein to a cell. In some embodiments, the method further comprises determining a target sequence of interest and selecting an adenosine deaminase, cytidine deaminase, or catalytic domain thereof, that most efficiently deaminates adenine or cytidine present in the target sequence. In some embodiments, the deamination cures a disease caused by a transcript containing a pathogenic T(U)→C, A→G, G→A, or C→T point mutation.

[0016] In another aspect, the disclosure includes a method for modifying a nucleic acid at a target locus, comprising delivering (1) the system of claim 1, where dCas13 is a first split Cas13 effector protein, and (2) a second split Cas13 effector protein, where the first and second split Cas13 effector proteins are capable of forming a catalytically active Cas13 effector protein.

[0017] In another aspect, the disclosure includes a method of regulating a target gene in a cell comprising delivering a system disclosed herein, wherein dCas13 is fused to a transcription factor or an activity domain thereof.

[0018] These and other aspects, objects, features and advantages of the example embodiments will become apparent to those skilled in the art after considering the following detailed description of the example embodiments shown.

[0019] A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief description of the drawings]

[0020] [Figure 1] 1 shows an exemplary embodiment of the present invention for targeted deamination of adenine at a target RNA sequence of interest, as exemplified herein with Cas13b protein. [Diagram 2] Figure 1 shows the development of RNA editing as a therapeutic strategy to treat human disease at the transcriptional level, for example with Cas13b. Schematic of RNA base editing with Cas13-ADAR2 fusion targeting an engineered premature termination stop codon in a luciferase transcript. [Diagram 3] Guide position and length optimization to restore luciferase expression. [Figure 4-1] Exemplary sequences of adenine deaminase proteins (SEQ ID NOs:650-656). [Figure 4-2] Exemplary sequences of adenine deaminase proteins (SEQ ID NOs:650-656). [Diagram 5] Guides used in the exemplary embodiment (SEQ ID NOs: 657-659 and 762). [Figure 6] Editing efficiency correlates with edited bases further away from the DR and with longer RNA duplexes, which is achieved by extending the guide length. [Figure 7] The higher the editing efficiency, the further away the editing site is from the DR / protein binding region. [Figure 8] Distance of the edited site from the DR. [Figure 9]Fusion of ADAR1 or ADAR2 to Cas13b12 (double R HEPN mutant) on the N- or C-terminus. The guide matches perfectly to the stop codon in luciferase. The signal appears to correlate with the distance between the edited base and the 5' end of the guide, with shorter distances resulting in better editing. [Figure 10] Cluc / Gluc tiling of Cas13a / Cas13b interference. [Figure 11] ADAR editing quantification by NGS (luciferase reporter). [Figure 12] Quantification of ADAR editing by NGS (KRAS and PPIB). [Figure 13] Cas13a / b+shRNA specificity from RNA sequencing. [Figure 14] Mismatch specificity (SEQ ID NOs: 661-668) to reduce off-target (A:A or A:G). [Figure 15] Mismatch of on-target activity. [Figure 16] ADAR motif preference. [Figure 17] Large bubbles enhance RNA editing efficiency. [Figure 18] Editing of multiple A's in the transcript (sequence numbers 669-672). [Figure 19] Guide length titration for RNA editing. [Figure 20A] Mammalian codon-optimized Cas13b orthologues mediate highly efficient RNA knockdown. Schematic diagram of representative Cas13a, Cas13b, and Cas13c loci and associated crRNAs. [Figure 20B] Mammalian codon-optimized Cas13b orthologues mediate highly efficient RNA knockdown. Schematic of the luciferase assay measuring Cas13a cleavage activity in HEK293FT cells. [Figure 20C]Mammalian codon-optimized Cas13b orthologues mediate highly efficient RNA knockdown. RNA knockdown efficiency using two different guides targeting Cluc with 19 Cas13a, 15 Cas13b, and 5 Cas13c orthologues. Luciferase expression is normalized to expression in the non-targeting guide control condition. [Figure 20D] Mammalian codon-optimized Cas13b orthologs mediate highly efficient RNA knockdown. In part C, the top seven orthologs are assessed for activity with two different guide RNAs targeting Cluc, three different NLS and NES tags. [Figure 20E] Mammalian codon-optimized Cas13b orthologues mediate highly efficient RNA knockdown. Cas13b12 and Cas13a2 (LwCas13a) are compared for knockdown activity against Gluc and Cluc. Guides are tiled along the transcript, and the guides between Cas13b12 and Cas13a2 are position-matched. [Figure 20F] Mammalian codon-optimized Cas13b orthologues mediate highly efficient RNA knockdown. Guided knockdown of Cas13a2, Cas13b6, Cas13b11, and Cas13b12 against endogenous KRAS transcripts is compared with the corresponding shRNAs. [Figure 21A] The Cas13 enzyme mediates specific RNA knockdown in mammalian cells. Schematic diagram of semi-degenerate target sequences for Cas13a / b mismatch specificity assay (SEQ ID NOs: 673-294). [Figure 21B] Cas13 enzymes mediate specific RNA knockdown in mammalian cells. Heatmap of Cas13a / b single mismatch knockdown data. Knockdown is normalized to the non-targeting (NT) guide for each enzyme. [Figure 21C]The Cas13 enzyme mediates specific RNA knockdown in mammalian cells. Cas13a double mismatch knockdown data. The position of each mismatch is shown on the X and Y axes. The knockdown data is the sum of all double mismatches at a given set of positions. Data is normalized to the NT guide per enzyme. [Figure 21D] The Cas13 enzyme mediates specific RNA knockdown in mammalian cells. Cas13b double mismatch knockdown data. For illustration, see C. [Figure 21E] The Cas13 enzyme mediates specific RNA knockdown in mammalian cells. RNA-seq data comparing the transcriptome-wide specificity of position-matched guides Cas13a / b and shRNA. The Y-axis represents read counts for the targeting condition, and the X-axis represents counts for the non-targeting condition. [Figure 21F] The Cas13 enzyme mediates specific RNA knockdown in mammalian cells. RNA expression calculated from RNA-seq data for Cas13a / b and shRNA. [Figure 21G] Cas13 enzyme mediates specific RNA knockdown in mammalian cells. Significant off-targets of Cas13a / b and shRNA from RNA-seq data. Significant off-targets were calculated using FDR<0.05. [Figure 22A] Catalytically inactive Cas13b-ADAR fusions enable targeted RNA editing in mammalian cells. Schematic of RNA editing by Cas13b-ADAR fusion proteins to remove a stop codon on a Cypridina luciferase transcript. [Figure 22B] Catalytically inactive Cas13b-ADAR fusions enable targeted RNA editing in mammalian cells. RNA editing comparison between Cas13b fused to wild-type ADAR2 and the hyperactive ADAR2 E488Q mutant for multiple guide positions. Luciferase expression is normalized to Gaussia luciferase control values. [Figure 22C] Catalytically inactive Cas13b-ADAR fusions enable targeted RNA editing in mammalian cells. Comparison of RNA editing between 30, 50, 70, and 84 nt guides designed to target various positions surrounding the editing site. [Figure 22D] Catalytically inactive Cas13b-ADAR fusions allow targeted RNA editing in mammalian cells. Schematic showing the position and length of guides used for sequencing quantification relative to the stop codon (SEQ ID NO: 695) on the Cypridina luciferase transcript. [Figure 22E] Catalytically inactive Cas13b-ADAR fusions enable targeted RNA editing in mammalian cells. On- and off-target editing efficiency for each guide design at the corresponding adenine base on the Cypridina luciferase transcript as quantified by sequencing. [Figure 22F] Catalytically inactive Cas13b-ADAR fusions allow targeted RNA editing in mammalian cells. Luciferase readout of guides that have changed the base opposite the targeted adenine. [Figure 23] A, Endogenous RNA editing by Cas13b-ADAR fusions. Next generation sequencing of endogenous Cas13b12-ADAR editing of the endogenous KRAS and PPIB loci. Two different regions per transcript were targeted and A→G editing was quantified at all adenines near the targeted adenine. [Figure 24] Strategies for determining optimal guide positions. [Figure 25A] Cas13b-huADAR2 promotes the repair of mutant luciferase transcripts. [Figure 25B] Cas13b-huADAR1 promotes the repair of mutant luciferase transcripts. [Figure 25C] Comparison of human ADAR1 and human ADAR2. [Figure 26] Comparison of E488Q vs. wild-type dADAR2 editing. E488Q is a hyperactive mutant of dADAR2. [Figure 27]The transcript targeted by Cas13b-huADAR2-E488Q contains the expected AG edit. (Figure 27A) Heat map. (Figure 27B) Template location. As in the heat map, only A sites are shown with the editing rate relative to G. [Figure 28] Endogenous tiling of guides. (Figure 28A) KRAS: Heat map. (Top) Location of template (bottom). As in heat map, only A sites are shown along with editing rate relative to G. (Figure 28B) PPIB: Heat map. (Top) Location of template (bottom). As in heat map, only A sites are shown along with editing rate relative to G. [Figure 29] Non-targeted editing. [Diagram 30] Linker optimizations. [Diagram 31] The Cas13b ADAR can be used to correct a patient-derived pathogenic A>G mutation in the expressed cDNA. [Diagram 32] Cas13b-ADAR has a slight restriction on the 5'G motif. [Figure 33-1] Screening of degenerate PFS positions for the effect of editing efficiency. All PFS (4-N) identities have higher editing than non-targeting (sequence numbers 696-699). [Figure 33-2] Screening of degenerate PFS positions for the effect of editing efficiency. All PFS (4-N) identities have higher editing than non-targeting (sequence numbers 696-699). [Diagram 34] Reduced off-target editing in targeted transcripts. [Diagram 35] Reduced off-target editing in targeted transcripts. [Diagram 36] Cas13b-ADAR transcriptome specificity. On-target editing is 71%. (Figure 36A) Targeting guide; 482 significant sites. (Figure 36B) Non-targeting guide; 949 significant sites. Note that chromosome 0 is Gluc and chromosome 1 is Cluc; the human chromosomes are in order after that. [Figure 37A]Cas13b-ADAR transcriptome specificity. Targeting guide. [Figure 37B] Cas13b-ADAR Transcriptome Specificity. Non-Targeting Guide. [Figure 37C] Cas13b-ADAR transcriptome specificity. Targeting guide. [Figure 37D] Cas13b-ADAR Transcriptome Specificity. Non-Targeting Guide. [Figure 38] Cas13b has the highest efficiency compared to competing ADAR editing strategies. [Figure 39A] Competitive RNA editing systems. Box B; On-target editing is 63%; Significant sites in Targeting Guide-2020. [Figure 39B] Competitive RNA editing systems. Box B; On-target editing is 63%; Significant sites in non-targeting guide-1805. [Figure 39C] Competitive RNA editing system. Stafforst; on-target editing is 36%; significant sites for targeting guide-176 [Figure 39D] Competitive RNA editing system. Stafforst; on-target editing is 36%; significant site in non-targeting guide-186. [Diagram 40] Dose titration of ADAR. The amount of crRNA is constant. [Figure 41A] Dose-response effect on specificity. 150ng Cas13-ADAR; on-target editing is 83%; significant site targeting guide-1231. [Figure 41B] Dose-response effect on specificity. 150ng Cas13-ADAR; on-target editing is 83%; significant site of non-targeting guide-520. [Figure 41C] Dose-response effect on specificity. 10 ng Cas13-ADAR; on-target editing is 80%; significant site targeting guide-347. [Figure 41D]Dose-response effect on specificity. 10 ng Cas13-ADAR; on-target editing is 80%; significant site in non-targeting guide-223. [Diagram 42] ADAR1 appears to be more specific than ADAR2. On-target editing is 29%. (Figure 42A) Targeting guide; 11 significant sites. (Figure 42B) Non-targeting guide; 6 significant sites. Note that chromosome 0 is Gluc and chromosome 1 is Cluc; the human chromosomes are in order after that. [Figure 43A] ADAR-specific mutants have enhanced specificity. Targeting guide. [Figure 43B] ADAR-specific mutants have enhanced specificity. Non-targeting guides. [Figure 43C] ADAR-specific mutants have enhanced specificity. Targeting vs. non-targeting ratio. [Fig. 43D] ADAR-specific mutants have enhanced specificity. Targeting and non-targeting guides. [Diagram 44] ADAR mutant luciferase results plotted along the contact points of each residue with the RNA target. [Diagram 45] The ADAR-specific mutants have enhanced specificity. The purple dots are mutants selected in the whole transcriptome off-target NGS analysis. The red dots are the starting point (i.e., E488Q mutants). Note that all additional mutants also have the E488Q mutation. [Diagram 46] ADAR mutants are more specific according to NGS. (Figure 46A) On-target. (Figure 46B) Off-target. [Figure 47]Luciferase data on ADAR-specific mutants is consistent with NGS. (Figure 47A) Targeting guides selected for NGS. (Figure 47B) Non-targeting guides selected for NGS. Luciferase data is consistent with the NGS data in Figure 46. Orthologs with less activity with non-targeting guides have less off-targets across the transcriptome, and their on-target editing efficiency can be predicted by targeting guide luciferase conditions. [Figure 48] C-terminal truncations of Cas13b 12 remain highly active in ADAR editing. [Figure 49]Characterization of highly active Cas13b orthologs for RNA knockdown. Figure 49A) Schematic of stereotyped Cas13 locus and corresponding crRNA structure. Figure 49B) Evaluation of 19 Cas13a, 15 Cas13b, and 7 Cas13c orthologs for luciferase knockdown using two different guides. Orthologs with efficient knockdown using both guides are labeled with their host organism name. Figure 49C) PspCas13b and LwaCas13a knockdown activity is compared by tiling guides against Gluc and measuring luciferase expression. Figure 49D) PspCas13b and LwaCas13a knockdown activity is compared by tiling guides against Cluc and measuring luciferase expression. Figure 49E) Expression levels of log2 (transcripts per million (TPM)) values ​​of all genes detected in the non-targeting control (x-axis) RNA-seq library compared to the Gluc targeting condition (y-axis) of LwaCas13a (red) and shRNA (black). The average of three biological replicates is shown. Gluc transcript data points are labeled. Figure 49F) Expression levels of log2 (transcripts per million (TPM)) values ​​of all genes detected in the non-targeting control (x-axis) RNA-seq library compared to the Gluc targeting condition (y-axis) of PspCas13b (blue) and shRNA (black). The average of three biological replicates is shown. Gluc transcript data points are labeled. Figure 49G) Number of significant off-targets from LwaCas13a, PspCas13b, and shRNA Gluc knockdown from transcriptome-wide analysis in Figures 49E and 49F. [Figure 50]Engineering dCas13b-ADAR fusions for RNA editing. Figure 50A) Schematic of RNA editing by dCas13b-ADAR fusion protein. Figure 50B) Schematic of Cypridina luciferase W85X target and targeting guide design (SEQ ID NO: 700 and 701). Figure 50C) Quantification of luciferase activity recovery of Cas13b-dADAR1 (left) and Cas13b-ADAR2-cd (right) with tiling guides of 30, 50, 70, or 84 nt length. Figure 50D) Schematic of target sites for targeting Cypridinia luciferase W85X. Figure 50E) Sequencing quantification of A→I editing of 50 nt guide targeting Cypridinia luciferase W85X (SEQ ID NO: 702). [Figure 51] Figure 51A) Measurement of sequence flexibility for RNA editing by REPAIRv1. Schematic of the screen to determine the protospacer adjacent site (PFS) preference of RNA editing by REPAIRv1 (SEQ ID NO: 703). Figure 51B) Distribution of RNA editing efficiency of all 4-N PFS combinations at two different editing sites. Figure 51C) Quantification of percent editing of REPAIRv1 in Cluc W85 across all possible three-base motifs (SEQ ID NO: 704). Figure 51D) Heat map of 5' and 3' base preference of RNA editing in Cluc W85 across all possible three-base motifs. [Figure 52]Correction of disease-associated mutations with REPAIRv1. Figure 52A) Schematic of target and guide design for targeting AVPR2 878G>A (sequence numbers 705-708). Figure 52B) The 878G>A mutation in AVPR2 is corrected at different percentages using REPAIRv1 with three different guide designs. Figure 52C) Schematic of target and guide design for targeting FANCC 1517G>A (sequence numbers 709-712). Figure 52D) The 1517G>A mutation in FANCC is corrected at different percentages using REPAIRv1 with three different guide designs. Figure 52E) Quantification of percent editing of 34 different disease-associated G>A mutations with REPAIRv1. Figure 52F) Analysis of all possible G>A mutations that can be corrected as annotated by the ClinVar database. The distribution of all G>A mutation editing motifs in ClinVar is shown relative to the editing efficiency by REPAIRv1 per motif as quantified in the Gluc transcript. [Figure 53] Characterization of the specificity of REPAIRv1. Figure 53A) Schematic of the KRAS target site and guide design (SEQ ID NOs: 713-720). Figure 53B) Quantification of percent editing of tiled KRAS targeting guides. Editing percentages are shown for the on-target site and adjacent adenosine sites. For each guide, regions of double-stranded RNA are indicated by red rectangles. Figure 53C) Transcriptome-wide sites of significant RNA editing by REPAIRv1 with Cluc targeting guides. The on-target site Cluc site (254 A>G) is highlighted in orange. Figure 53D) Transcriptome-wide sites of significant RNA editing by REPAIRv1 with non-targeting guides. [Figure 54]Rational mutagenesis of ADAR2 to improve the specificity of REPAIRv1. Figure 54A) Quantification of luciferase signal recovery by various dCas13-ADAR2 mutants and their specificity scores plotted along with a schematic of contacts between key ADAR2 deaminase residues and dsRNA targets. The specificity score is defined as the ratio of luciferase signal between targeting guide and non-targeting guide conditions. Figure 54B) Quantification of luciferase signal recovery by various dCas13-ADAR2 mutants versus their specificity scores. Figure 54C) Measurement of on-target editing fractions and significant off-target number per dCas13-ADAR2 mutant by transcriptome-wide sequencing of mRNA. Figure 54D) Transcriptome-wide sites of significant RNA editing by REPAIRv1 and REPAIRv2, where the guides are targeted to premature termination sites in Cluc. The on-target Cluc site (254 A>G) is highlighted in orange. Figure 54E) RNA sequencing reads surrounding the on-target Cluc editing site (254 A>G) highlighting the difference in off-target editing between REPAIRv1 and REPAIRv2. All A>G edits are highlighted in red, while sequencing errors are highlighted in blue (SEQ ID NO: 721). Figure 54F) RNA editing by REPAIRv1 and REPAIRv2, where guides target out-of-frame UAG sites in endogenous KRAS and PPIB transcripts. On-target editing fractions are shown as bar graphs on the right for each condition row. The double-stranded region formed by the guide RNA is outlined in red. [Figure 55]Bacterial screening of Cas13b orthologs for in vivo efficiency and PFS determination. Figure 55A) Schematic of bacterial analysis to determine PFS of Cas13b orthologs. Cas13b orthologs with β-lactamase targeting spacer are co-transformed with β-lactamase expression plasmid and subjected to double selection (SEQ ID NO: 722). Figure 55B) Quantification of interference activity of Cas13b orthologs targeting β-lactamase as measured by colony forming units (cfu). Figure 55C) PFS logo of Cas13b orthologs as determined by depletion sequence from bacterial analysis. [Figure 56] Optimization of Cas13b knockdown and further characterization of mismatch specificity. Figure 56A) Gluc knockdown with two different guides is measured using top 2 Cas13a and top 4 Cas13b orthologues fused to various nuclear localization and nuclear export tags. Figure 56B) KRAS knockdown is measured for LwaCas13a, RanCas13b, PguCas13b, and PspCas13b with four different guides and compared to four position-matched shRNA controls. Figure 56C) Schematic of single and double mismatched plasmid libraries used to assess the specificity of LwaCas13a and PspCas13b knockdown. All possible single and double mismatches are present in the target sequence, as well as in three positions directly adjacent to the 5' and 3' ends of the target site (SEQ ID NOs: 723-735). Figure 56D) The depletion levels of the transcripts with the indicated single mismatches are plotted as a heat map for both LwaCas13a and PspCas13b conditions. Figure 56E) The depletion levels of the transcripts with the indicated double mismatches are plotted as a heat map for both LwaCas13a and PspCas13b conditions (SEQ ID NO: 736). [Figure 57]Characterization of design parameters for dCas13-ADAR2 RNA editing. Figure 57A) Knockdown efficiency of Gluc targeting of wild-type Cas13b and catalytically inactive H133A / H1058A Cas13b (dCas13b). Figure 57B) Quantification of luciferase activity restoration by dCas13b fused to either wild-type or hyperactive E488Q mutant ADAR2 catalytic domain was tested by tiling Cluc targeting guides. Figure 57C) Guide design and sequencing quantification of A→I editing of 30 nt guide targeting Cypridinia luciferase W85X (SEQ ID NOs: 737-745). Figure 57D) Guide design and sequencing quantification of A→I editing of 50 nt guide targeting PPIB (SEQ ID NOs: 746-753). Figure 57E) Effect of linker choice on luciferase activity restoration by REPAIRv1. Figure 57F) Effect of base identity opposite the targeted adenosine on luciferase activity restoration by REPAIRv1 (sequence numbers 754-755). [Figure 58] ClinVar motif distribution of G>A mutations. Number of each possible triplet motif observed in the ClinVar database for all G>A mutations. [Figure 59] Truncations of dCas13b still have functional RNA editing. Various N- and C-terminal truncations of dCas13b allow RNA editing, as measured by restoration of luciferase signal. [Figure 60]Comparison of dCas13-ADAR2 editor with other programmable ADAR systems. Figure 60A) Schematic of two programmable ADAR schemes: box B-based targeting and full-length ADAR2 targeting. In the box B scheme (top), the ADAR2 deaminase domain (ADAR2DD(E488Q)) is fused to a small bacterial viral protein called lambda N (λN), which specifically binds to a small RNA sequence called box B-λ. A guide RNA containing two box B-λ hairpins can then guide ADAR2DD(E488Q),-λN to perform site-specific editing. In the full-length ADAR2 scheme (bottom), the dsRNA binding domain of ADAR2 binds to the hairpins in the guide RNA, allowing programmable ADAR2 editing (sequence numbers 756-760). Figure 60B) Transcriptome-wide sites of significant RNA editing by box B-ADAR2DD(E488Q) with guides targeting Cluc and non-targeting guides. The on-target Cluc site (254 A>G) is highlighted in orange. Figure 60C) Transcriptome-wide sites of significant RNA editing by ADAR2 with guides targeting Cluc and non-targeting guides. The on-target Cluc site (254 A>G) is highlighted in orange. Figure 60D) Transcriptome-wide sites of significant RNA editing by REPAIRv1 with guides targeting Cluc and non-targeting guides. The on-target Cluc site (254 A>G) is highlighted in orange. Figure 60E) Quantification of on-target editing percentages for BoxB-ADAR2DD(E488Q), ADAR2, and REPAIRv1 for guide targeting to Cluc. Figure 60F) Overlap of off-target sites between different targeting and non-targeting conditions for the programmable ADAR system. [Figure 61]Efficiency and specificity of Cas13b-ADAR2 mutants. Figure 61A) Quantification of luciferase activity recovery by dCas13b-ADAR2DD(E488Q) mutant of Cluc targeting guide and non-targeting guide. Figure 61B) Relationship between the ratio of targeting and non-targeting guide and the number of RNA editing off-targets as quantified by transcriptome-wide sequencing. Figure 61C) Quantification of the number of transcriptome-wide off-target RNA editing sites versus on-target Cluc editing efficiency of dCas13b-ADAR2DD(E488Q) mutant. [Figure 62] Transcriptome-wide specificity of RNA editing by dCas13b-ADAR2DD(E488Q) mutant. Figure 62A) Transcriptome-wide sites of significant RNA editing by dCas13b-ADAR2DD(E488Q) mutant with guide targeting Cluc. On-target Cluc site (254 A>G) is highlighted in orange. Figure 62B) Transcriptome-wide sites of significant RNA editing by dCas13b-ADAR2DD(E488Q) mutant with non-targeting guide. [Figure 63] Characterization of motif bias in off-targets of dCas13b-ADAR2DD(E488Q) editing. Figure 63A) Motifs present across all A>G off-target edits in the transcriptome for each dCas13b-ADAR2DD(E488Q) mutant. Figure 63B) Distribution of off-target A>G edits per motif identity for REPAIRv1 with targeting and non-targeting guides. Figure 63C) Distribution of off-target A>G edits per motif identity for REPAIRv2 with targeting and non-targeting guides. [Figure 64]Further characterization of REPAIRv1 and REPAIRv2 off-targets. Figure 64A) Histogram of number of off-targets per transcript for REPAIRv1. Figure 64B) Histogram of number of off-targets per transcript for REPAIRv2. Figure 64C) REPAIRv1 off-target variant effect predictions. Figure 64D) Distribution of potential oncogenic effects of REPAIRv1 off-targets. Figure 64E) REPAIRv2 off-target variant effect predictions. Figure 64F) Distribution of potential oncogenic effects of REPAIRv2 off-targets. [Figure 65] RNA editing efficiency and specificity of REPAIRv1 and REPAIRv2. Figure 65A) Quantification of percent editing of KRAS by KRAS targeting guide 1 at targeted adenosines and adjacent sites of REPAIRv1 and REPAIRv2. Figure 65B) Quantification of percent editing of KRAS by KRAS targeting guide 3 at targeted adenosines and adjacent sites of REPAIRv1 and REPAIRv2. Figure 65C) Quantification of percent editing of PPIB by PPIB targeting guide 2 at targeted adenosines and adjacent sites of REPAIRv1 and REPAIRv2. [Figure 66] Demonstration of all potential codon changes by A>G RNA editors. Figure 66A) Table of all potential codon transitions enabled by A>I editing. Figure 66B) Codon table showing all potential codon transitions enabled by A>I editing. [Figure 67] 1 shows the test results of RNA editing activity on Cas13b6. [Figure 68] 1 shows the test results of RNA editing activity on Cas13b11. [Figure 69] 1 shows the test results of RNA editing activity on Cas13b12. [Figure 70] Schematic diagram of the construct dCas13b6(Δ795-1095)-REPAIR and editing rate. [Figure 71] We demonstrate engineered editing at a site in the potassium ion channel Kcna1, which is already a natural ADAR2 substrate, by delivering dCas13b6(Δ795-1095)-REPAIR. [Figure 72] A schematic diagram of dCas13b6(Δ795-1095)-GS-HIVNES-GS-huADAR2dd is shown. [Figure 73] REPAIR assay of pgCas13b C-terminal truncations. [Figure 74] 1 shows an alignment tree of ADAR orthologs that can be used in the systems and methods herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The figures herein are for illustration purposes only and are not necessarily drawn to scale.

[0022] General definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms and techniques in molecular biology can 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) (FMA Usubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (MJ MacPherson, BD Hames, and GR Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2nd edition 2013 (EA Greenfield ed.); Animal Cell Culture (1987) (RI 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, NY 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011).

[0023] Reference is made to U.S. Provisional Patent Application Nos. 62 / 351,662 and 62 / 351,803, filed June 17, 2016, U.S. Provisional Patent Application No. 62 / 376,377, filed August 17, 2016, U.S. Provisional Patent Application No. 62 / 410,366, filed October 19, 2016, U.S. Provisional Patent Application No. 62 / 432,240, filed December 9, 2016, U.S. Provisional Patent Application No. 62 / 471,792, filed March 15, 2017, and U.S. Provisional Patent Application No. 62 / 484,786, filed April 12, 2017. Reference is made to International PCT Application No. PCT / US2017 / 038154, filed June 19, 2017. Reference is made to U.S. Provisional Patent Application No. 62 / 471,710, filed March 15, 2017 (titled "Novel Cas13B Orthologues CRISPR Enzymes and Systems"), Attorney Docket No. BI-10157 VP 47627.04.2149. Further reference is made to U.S. Provisional Patent Application No. 62 / 432,553, filed December 9, 2016, U.S. Provisional Patent Application No. 62 / 456,645, filed February 8, 2017, and U.S. Provisional Patent Application No. 62 / 471,930, filed March 15, 2017 (entitled "CRISPR Effector System Based Diagnostics"; Attorney Docket No. BI-10121 BROD 0842P), and a proposed assigned U.S. Provisional Patent Application filed April 12, 2017 (entitled "CRISPR Effector System Based Diagnostics"; Attorney Docket No. BI-10121 BROD 0843P).

[0024] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0025] The terms "optional" and "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description encompasses both instances where the event or circumstance occurs and instances where it does not occur.

[0026] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0027] The terms "about" or "approximately," as used herein, when referring to a measurable value such as a parameter, amount, time period, and the like, are meant to encompass variations of the indicated value, as well as variations of up to ±10%, up to ±5%, up to ±1%, and up to ±0.1% therefrom, insofar as such variations are appropriate to the practice of the disclosed invention. It is to be understood that the value to which the modifier "about" or "approximately" refers is itself also specifically, and preferably, disclosed.

[0028] As used herein, a "biological sample" may include whole cells and / or live cells and / or cell debris. A biological sample may include (or be derived from) a "body fluid." The invention encompasses embodiments in which the body fluid is selected from amniotic fluid, aqueous humor, vitreous humor, bile, serum, breast milk, cerebrospinal fluid, earwax (ear waste), chyle, chyme, endolymph, perilymph, exudate, feces, female ejaculatory fluid, gastric acid, gastric juice, lymph, mucus (including nasal discharge and phlegm), membranous fluid, peritoneal fluid, pleural fluid, pus, mucosal secretions, saliva, sebum (skin oil), semen, saliva, synovial fluid, sweat, tears, vaginal secretions, vomit, and mixtures of one or more thereof. Biological samples include cell cultures, body fluids, and cell cultures derived from body fluids. Body fluids may be obtained from a mammalian organism, for example, by puncture or other collection or sampling procedures.

[0029] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0030] References throughout this specification to "one embodiment," "an embodiment," or "an exemplary embodiment" mean that the detailed features, structures, or characteristics described in connection with that embodiment are included in at least one embodiment of the invention. Thus, the phrases "in one embodiment," "in an embodiment," or "an exemplary embodiment" appear in various places throughout this specification, but are not necessarily all referring to the same embodiment, although they may. Moreover, as would be apparent to one skilled in the art from this disclosure, such detailed features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Furthermore, although some embodiments described herein include certain features among other embodiments, it is intended that combinations of features from different embodiments are within the scope of the invention. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0031] Currently, C2c2 is known as Cas13a. It will be understood that the term "C2c2" herein is used synonymously with "Cas13a". As used herein, Cas13 may refer to Cas13a or Cas13b or Cas13c or Cas13d or other members of the Cas13 family. In some examples, Cas13 may be Cas13a. In some examples, Cas13 may be Cas13b. In some examples, Cas13 may be Cas13c. In some examples, Cas13 may be Cas13d.

[0032] All publications, published patent documents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication, published patent document, or patent application was specifically and individually indicated to be incorporated by reference.

[0033] Various embodiments are described below. However, it should be noted that the specific embodiments are not intended to be exhaustive or limiting of the broader aspects described herein. An aspect described in connection with a particular embodiment is not necessarily limited to that embodiment and can be implemented with any other embodiment(s).

[0034] overview The embodiments disclosed herein provide systems, constructs, and methods that use catalytically inactive Cas effector proteins (e.g., Cas13) for various applications. In general, the systems disclosed herein can include catalytically inactive Cas effector proteins (e.g., dCas13). Catalytically inactive Cas13 (dCas13) can include truncations of the Cas13 effector protein, for example, at the C-terminus, N-terminus, or both. In some embodiments, the system includes dCas13. The dCas13 can be a catalytically inactive form of any Cas13 subtype protein. For example, the dCas13 can be dCas13a, dCas13b, dCas13c, or dCas13d. In some examples, the dCas13 can be a modified Cas13 effector protein from Prevotella sp. P5-125, Riemerella anatipestifer, or Porphyromonas gulae.

[0035] The system may further include one or more functional components for a desired application. In some examples, the system may include a base editing component, such as an adenosine deaminase or a cytidine deaminase, or a catalytic domain thereof. In some examples, the system may include a transcription factor or an active domain thereof. In these cases, a catalytically inactive Cas effector protein can be used to deliver the transcription factor to the target sequence. In some examples, the catalytically inactive Cas effector protein can be a split Cas effector protein. The split Cas can be used with another split Cas for various applications. For example, two split Cas effector proteins can be fused to form a catalytically active Cas effector protein. The fusion can be controllable, for example, using an inducer.

[0036] V-type CRISPR-Cas proteins This application describes a method using V-type CRISPR-Cas protein.This is exemplified herein as Cas13, and many orthologs or homologs have been identified.It will be clear to those skilled in the art that further orthologs or homologs can be identified, and any of the functionalities described herein can be engineered into other orthologs, for example, chimeric enzymes that contain fragments from multiple orthologs.

[0037] A computational method for identifying novel CRISPR-Cas loci is described in EP3009511 or US2016208243 and may include the following steps: detecting all contigs encoding Cas1 protein; identifying all predicted protein-coding genes within 20 kB of the cas1 gene; comparing the identified genes with the Cas protein-specific profile to predict CRISPR arrays; selecting unclassified candidate CRISPR-Cas loci that contain proteins larger than 500 amino acids (>500aa); analyzing the selected candidates using methods such as PSI-BLAST and HHPred to screen for known protein domains to identify novel class 2 CRISPR-Cas loci (see also Schmakov et al. 2015, Mol Cell. 60(3):385-97). In addition to the above steps, additional analysis of the candidates may be performed by searching metagenomics databases for additional homologs. Additionally or alternatively, the same procedure can be performed on the CRISPR arrays used as seeds to extend the search to non-autonomous CRISPR-Cas systems.

[0038] In one embodiment, the step of detecting all contigs encoding Cas1 proteins is performed by GenemarkS, a gene prediction program as further described in "GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions." John Besemer, Alexandre Lomsadze and Mark Borodovsky, Nucleic Acids Research (2001) 29, pp 2607-2618, which is incorporated herein by reference.

[0039] In one embodiment, the step of identifying all predicted protein-coding genes is performed by comparing the identified genes with the Cas protein-specific profile and annotating them according to the NCBI Conserved Domain Database (CDD), a protein annotation resource consisting of a collection of well-annotated multiple sequence alignment models of ancient domains and full-length proteins. These are available as position-specific score matrices (PSSMs) for rapid identification of conserved domains in protein sequences by RPS-BLAST. The CDD content includes NCBI-curated domains that explicitly define domain boundaries using three-dimensional structural information and provide insight into sequence / structure / function relationships, as well as domain models imported from several external source databases (Pfam, SMART, COG, PRK, TIGRFAM). In a further embodiment, CRISPR arrays were predicted using the PILER-CR program, a publicly available domain software for finding CRISPR repeats, as described in "PILER-CR: fast and accurate identification of CRISPR repeats", Edgar, RC, BMC Bioinformatics, Jan 20;8:18 (2007), incorporated herein by reference.

[0040] In a further embodiment, a case-by-case analysis is performed using PSI-BLAST (Position-Specific Iterative Basic Local Alignment Search Tool). PSI-BLAST derives a Position-Specific Scoring Matrix (PSSM) or profile from a multiple sequence alignment of sequences found above a given score threshold using protein-protein BLAST. This PSSM is used to further search the database for new matches and is subsequently updated to iterate with these newly found sequences. Thus, PSI-BLAST provides a means to detect distant relationships between proteins.

[0041] In another embodiment, case-by-case analysis is performed using HHpred, a sequence database searching and structure prediction method that is as easy to use as BLAST or PSI-BLAST, but at the same time much more sensitive to finding distant homologs. In fact, the sensitivity of HHpred is comparable to the most powerful structure prediction servers currently available. HHpred is the first server based on pairwise comparison of profile hidden Markov models (HMMs). While most conventional sequence searching methods search sequence databases such as UniProt or NR, HHpred searches alignment databases such as Pfam or SMART. This greatly simplifies the list of hits to a few sequence families, rather than a cluttered pile of single sequences. All major publicly available profile and alignment databases are available in HHpred. HHpred accepts as input a single query sequence or multiple alignments. HHpred returns search results in an easy-to-read format similar to PSI-BLAST within just a few minutes. Search options include local or global alignments and scoring of secondary structure similarity. HHpred can generate pairwise query-template sequence alignments, synthetic query-template multiple alignments (eg, for transitive searches), as well as three-dimensional structural models calculated by the MODELLER software from the HHpred alignments.

[0042] Orthologs of Cas13 The terms "orthologue" (also referred to herein as "ortholog") and "homologue" (also referred to herein as "homolog") are well known in the art. As further guidance, a "homolog" of a protein as used herein is a protein of the same species that performs the same or similar function as the protein of which it is a homolog. Homologous proteins, however, may be unrelated structurally or only partially related structurally. An "orthologue" of a protein as used herein is a protein of a different species that performs the same or similar function as the protein of which it is an orthologue. Orthologous proteins may or may not be related structurally or only partially related structurally. Homologs and orthologs can be identified by homology modeling (see, for example, Greer, Science vol. 228 (1985) 1055, and Blundell et al. Eur J Biochem vol 172 (1988), 513) or "structural BLAST" (Dey F, Cliff Zhang Q, Petrey D, Honig B. Toward a "structural BLAST": using structural relationships to infer function. Protein Sci. 2013 Apr; 22 (4): 359-66. doi: 10.1002 / pro.2225). See also Shmakov et al. (2015) for applications in the field of CRISPR-Cas loci. Homologous proteins may or may not be structurally related, or may only be partially structurally related.

[0043] The Cas13 gene is found in several diverse bacterial genomes, typically at the same locus as the cas1, cas2, and cas4 genes and CRISPR cassettes (e.g., FNFX1_1431-FNFX1_1428 in Francisella cf.novicida Fx1). Thus, the layout of this putative novel CRISPR-Cas system appears to be similar to type II-B. Furthermore, like Cas9, the Cas13 protein contains a readily identifiable C-terminal region that is homologous to the transposon ORF-B, and includes an active RuvC-like nuclease, an arginine-rich region, and a Zn finger (absent in Cas9). However, unlike Cas9, Cas13 is also present in some genomes without a CRISPR-Cas context, and its relatively high similarity to ORF-B suggests that it may be a transposon component. It has been suggested that if this were a bona fide CRISPR-Cas system, Cas13 would be a functional analogue of Cas9 and a novel CRISPR-Cas type, namely type V (see Annotation and Classification of CRISPR-Cas Systems. Makarova KS, Koonin EV. Methods Mol Biol. 2015;1311:47-75). However, as described herein, Cas13 is referred to as subtype VA to distinguish it from C2c1p, which does not have the same domain structure and is therefore referred to as subtype VB.

[0044] The present invention encompasses the use of Cas13 effector proteins derived from a Cas13-encoding locus designated subtype VA. Such effector proteins are also referred to herein as "Cas13p", e.g., Cas13 proteins (and such effector proteins or Cas13 proteins or proteins derived from the Cas13 locus are also referred to as "CRISPR-Cas proteins").

[0045] In certain embodiments, the effector protein is selected from the group consisting of Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, L and Cas13 effector proteins derived from organisms of the genera including: Caenorhabditis elegans, Caenorhabditis protease, Caenorhabditis elegans, Caenorhabditis elegans, Caenorhabditis aeruginosa, Caenorhabditis nervosa ... In certain embodiments, the Cas13 effector protein is selected from an organism of a genus selected from Eubacterium, Lachnospiraceae, Leptotrichia, Francisella, Methanomethyophilus, Porphyromonas, Prevotella, Letospira, Butyvibrio, Perigrinibacterium, Pareubacterium, Moraxella, Thiomicrospira, or Acidaminococcus.

[0046] Thus, the Cas13 mutant strains of S.mutans, S.agalactiae, S.equisimilis, and S.K .sanguinis、S.pneumonia;C.jejuni、C.coli;N.salsuginis、N.tergarcus;S.auri cularis, S. carnosus, N. meningitides, N. gonorrhoeae, L. monocytogenes, L. ivanovii; 1. P. albensis, L. bacterium, B. proteoclasticus, P. bacterium, P. crevioricanis, P. disiens, and P. macacae.

[0047] The effector protein can include a chimeric effector protein comprising a first fragment from a first effector protein (e.g., Cas13) ortholog and a second fragment from a second effector (e.g., Cas13) protein ortholog, where the first and second effector protein orthologs are different. At least one of the first and second effector protein (e.g., Cas13) orthologs is selected from the group consisting of Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francis ella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium, Butyvibrio, Perigrinibacterium, Pareubacterium, Moraxella, Thiomicrospira or Acidaminococcus;For example, a chimeric effector protein comprising a first fragment and a second fragment, wherein each of the first and second fragments is selected from the group consisting of Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatr onum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium, Butyvibrio, Perigrinibacterium, Pareubacterium, Moraxella, Thiomicrospira or Acidaminococcus, wherein the first and second fragments are not from the same bacterium; e.g., a chimeric effector protein comprising the first fragment and the second fragment, wherein , each of the first and second fragments are S. mutans, S. agalactiae, S. equisimilis, S. sanguinis, S. pneumonia; C. jejuni, C. coli; N. salsuginis, N. tergarcus; S. aur icularis, S. carnosus; N. meningitides, N. gonorrhoeae; L. monocytogenes, L. ivanovii; C. botulinum, C. difficile, C. tetani, C. sordellii;Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp.SCADC, Acidaminococcus sp.BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae Cas13, wherein the first and second fragments are not from the same bacterium;

[0048] In a more preferred embodiment, Cas13p is derived from Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp.SCADC, Acidaminococcus sp.BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Moraxella bovoculi AAX08_00205, Moraxella bovoculi AAX11_00205, Butyrivibrio sp.NC3005, Thiomicrospira sp.XS5, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, and Porphyromonas macacae. In certain embodiments, Cas13p is derived from a bacterial species selected from Acidaminococcus sp.BV3L6, Lachnospiraceae bacterium MA2020. In certain embodiments, the effector protein is derived from a subspecies of Francisella tularensis 1, including but not limited to Francisella tularensis subsp. Novicida.In certain preferred embodiments, Cas13p is derived from a bacterial species selected from Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium ND2006, Lachnospiraceae bacterium MA2020, Moraxella bovoculi AAX08_00205, Moraxella bovoculi AAX11_00205, Butyrivibrio sp. NC3005, or Thiomicrospira sp. XS5.

[0049] In certain embodiments, a homolog or orthologue of Cas13 as referred to herein has at least 80%, more preferably at least 85%, even more preferably at least 90%, such as at least 95%, sequence homology or identity with an exemplary Cas13 protein disclosed herein. In further embodiments, a homolog or orthologue of Cas13 as referred to herein has at least 80%, more preferably at least 85%, even more preferably at least 90%, such as at least 95%, sequence identity with wild-type Cas13. When Cas13 has one or more mutations (mutated), said homolog or orthologue of Cas13 as referred to herein has at least 80%, more preferably at least 85%, even more preferably at least 90%, such as at least 95%, sequence identity with mutant Cas13.

[0050] In certain embodiments, the Cas13 protein can be an ortholog of an organism of a genus including, but not limited to, Acidaminococcus sp., Lachnospiraceae bacterium, or Moraxella bovoculi; in certain embodiments, the V-type Cas protein can be an ortholog of an organism of a species including, but not limited to, Acidaminococcus sp. BV3L6; Lachnospiraceae bacterium ND2006 (LbCas13), or Moraxella bovoculi 237. In certain embodiments, a homolog or ortholog of Cas13 as referred to herein has at least 80%, more preferably at least 85%, even more preferably at least 90%, such as at least 95%, sequence homology or identity with one or more of the Cas13 sequences disclosed herein. In a further embodiment, a homolog or ortholog of Cas13 as referred to herein has at least 80% sequence identity with wild-type FnCas13, AsCas13 or LbCas13, more preferably at least 85%, even more preferably at least 90%, such as at least 95%.

[0051] In certain embodiments, the Cas13 protein of the present invention has at least 60%, more particularly at least 70, such as at least 80%, more particularly at least 85%, even more particularly at least 90%, such as at least 95% sequence homology or identity with FnCas13, AsCas13 or LbCas13. In further embodiments, the Cas13 protein as referred to herein has at least 60%, such as at least 70%, more particularly at least 80%, more particularly at least 85%, even more particularly at least 90%, such as at least 95% sequence identity with wild-type AsCas13 or LbCas13. In certain embodiments, the Cas13 protein of the present invention has less than 60% sequence identity with FnCas13. Those skilled in the art will understand that this includes truncated Cas13 proteins, and thus the sequence identity is determined over the length of the truncated form. In certain embodiments, the Cas13 enzyme is not FnCas13.

[0052] Modified Cas13 enzyme In certain embodiments, it is contemplated to utilize an engineered Cas13 protein, e.g., Cas13, as defined herein, which complexes with a nucleic acid molecule comprising an RNA to form a CRISPR complex, and when in the CRISPR complex, the nucleic acid molecule targets one or more target polynucleotide loci, and the protein comprises at least one modification compared to an unmodified Cas13 protein, and wherein the CRISPR complex comprising the modified protein has an altered activity when compared to a complex comprising an unmodified Cas13 protein. When referring to a CRISPR "protein" herein, the Cas13 protein is preferably a modified CRISPR-Cas protein (having increased or decreased (or no) enzymatic activity, e.g., without limitation, Cas13. It should be understood that the term "CRISPR protein" may be used interchangeably with "CRISPR-Cas protein," regardless of whether the CRISPR protein is altered, such as having increased or decreased (or no) enzymatic activity compared to a wild-type CRISPR protein.

[0053] Computational analysis of the primary structure of Cas13 nuclease reveals three distinct regions: first, a C-terminal RuvC-like domain, which is the only functionally characterized domain, second, an N-terminal α-helical region, and third, a mixed α and β region located between the RuvC-like domain and the α-helical region.

[0054] Within the Cas13 primary structure, several small stretches of unstructured regions are predicted. The unstructured regions that are solvent exposed and not conserved among different Cas13 orthologs are preferred sides for splitting and inserting small protein sequences. In addition, these sides can be used to create chimeric proteins between Cas13 orthologs.

[0055] Based on the above information, mutations can be created that lead to inactivation of the enzyme or modify the double-stranded nuclease to nickase activity. In alternative embodiments, this information is used to develop enzymes with reduced off-target effects (as described elsewhere herein).

[0056] In certain of the above-mentioned Cas13 enzymes, the enzyme may be, but is not limited to, AsCas13 (Acidaminococcus The RuvC domain is modified by mutation of one or more residues (within the RuvC domain), including positions R909, R912, R930, R947, K949, R951, R955, K965, K968, K1000, K1002, R1003, K1009, K1017, K1022, K1029, K1035, K1054, K1072, K1086, R1094, K1095, K1109, K1118, K1142, K1150, K1158, K1159, R1220, R1226, R1242, and / or R1252 based on the amino acid position numbering of RuvC domain (BV3L6). In certain embodiments, the Cas13 enzyme comprising said one or more mutations has altered, and more preferably increased, specificity for the target.

[0057] In certain of the non-naturally occurring CRISPR-Cas proteins described above, the enzyme is modified by mutation of one or more residues (within the RAD50 domain), including but not limited to, positions K324, K335, K337, R331, K369, K370, R386, R392, R393, K400, K404, K406, K408, K414, K429, K436, K438, K459, K460, K464, R670, K675, R681, K686, K689, R699, K705, R725, K729, K739, K748, and / or K752 relative to the amino acid position numbering of AsCas13 (Acidaminococcus sp. BV3L6). In certain embodiments, the Cas13 enzyme comprising said one or more mutations has altered, and more preferably increased, specificity for the target.

[0058] In certain of the above-mentioned Cas13 enzymes, the enzyme is modified by mutation of one or more residues, including but not limited to, positions R912, T923, R947, K949, R951, R955, K965, K968, K1000, R1003, K1009, K1017, K1022, K1029, K1072, K1086, F1103, R1226, and / or R1252 based on the amino acid position numbering of AsCas13 (Acidaminococcus sp. BV3L6). In certain embodiments, the Cas13 enzyme comprising said one or more mutations has altered, more preferably increased, specificity for the target.

[0059] In certain embodiments, the Cas13 enzyme is modified by mutation of one or more residues, including but not limited to, positions R833, R836, K847, K879, K881, R883, R887, K897, K900, K932, R935, K940, K948, K953, K960, K984, K1003, K1017, R1033, R1138, R1165, and / or R1252 based on the amino acid position numbering of LbCas13 (Lachnospiraceae bacterium ND2006). In certain embodiments, the Cas13 enzyme comprising said one or more mutations has altered, more preferably increased, specificity for the target.

[0060] In certain embodiments, the Cas13 enzyme is selected from the group consisting of, but not limited to, AsCas13 (Acidaminococcus sp. BV3L6) amino acid numbering at positions K15, R18, K26, Q34, R43, K48, K51, R56, R84, K85, K87, N93, R103, N104, T118, K123, K134, R176, K177, R192, K200, K226, K273, K274, K275, K276, K277, K278, K279, K280, K281, K282, K283, K284, K285, K286, K287, K288, K289, K290, K291, K292, K293, K294, K295, K300, K301, K302, K303, K304, K305, K306, K307, K308, K310, K311, K312, K313, K314, K315, K316, K317, K320, K321, K322, K323, K324, K325, K326, K327, K328, K329, K330, K331, K332, K333, K334, K335, K336, K337, K338, K339, K340, K341, K342, K343, K344, K345, K346, K347, K348, K349, K350, K351, K352, 5, T291, R301, K307, K369, S404, V409, K414, K436, K438, K468, D482, K516, R518 , K524, K530, K532, K548, K559, K570, R574, K592, D596, K603, K607, K613, C647, R 681, K686, H720, K739, K748, K757, T766, K780, R790, P791, K796, K809, K815, T8 16, K860, R862, R863, K868, K897, R909, R912, T923, R947, K949, R951, R955, K96 5, K968, K1000, R1003, K1009, K1017, K1022, K1029, A1053, K1072, K1086, F1103, S1209, R1226, R1252, K1273, K1282, and / or K1288. In certain embodiments, the Cas13 enzyme comprising said one or more mutations has altered, and more preferably increased, specificity for the target.

[0061] In certain embodiments, the enzyme may be, but is not limited to, FnCas13 (Francisella novicida Based on the amino acid numbering of U112), the positions are K15, R18, K26, R34, R43, K48, K51, K56, K87, K88, D90, K96, K106, K107, K120, Q125, K143, R186, K187, R202, K210, K235, K296, K298, K314, K320, K326, K397, K444, K449, E454, A483, E491, K527, K541, K581, R583, K589, K595, K597, K613, K624, K635, K639, K656, K660, K6 67, K671, K677, K719, K725, K730, K763, K782, K791, R800, K809, K823, R833, K834, K839, K852, K858, K859, K869, K871, R872, K877, K905, R918, R921, K932, I960, K962, R964, R968, K978, K981, K1013, R1016, K1021, K1029, K1034, K1041, K1065, K1084, and / or K1098. In certain embodiments, the Cas13 enzyme comprising said one or more mutations has altered, and more preferably increased, specificity for the target.

[0062] In certain embodiments, the enzyme is selected from the group consisting of, but not limited to, positions K15, R18, K26, K34, R43, K48, K51, R56, K83, K84, R86, K92, R102, K103, K116, K121, R158, E159, R174, R182, K206, K251, K253, K254, K255, K256, K257, K258, K260, K261, K262, K263, K264, K265, K266, K267, K268, K269, K270, K271, K272, K273, K274, K275, K276, K277, K278, K279, K280, K281, K282, K283, K284, K285, K286, K287, K288, K289, K290, K291, K292, K293, K294, K295, K300, K301, K302, K303, K304, K305, K306, K307, K308, K310, K311, K312, K313, K314, K315, K316, K321, K322, K323, K324, K325, K326, K327, K328, K330, K331, K332, K333, K334, K335, K336, K337, K338, K340, K341, K342, K34 69, K271, K278, P342, K380, R385, K390, K415, K421, K457, K471, A506, R508, K 514, K520, K522, K538, Y548, K560, K564, K580, K584, K591, K595, K601, K634, K and / or K1208. In certain embodiments, the Cas13 enzyme comprising said one or more mutations has altered, and more preferably increased, specificity for the target.

[0063] In certain embodiments, the enzyme is selected from the group consisting of, but not limited to, positions K14, R17, R25, K33, M42, Q47, K50, D55, K85, N86, K88, K94, R104, K105, K118, K123, K131, R174, K175, R190, R198, I221, K267, Q269, K285, and the like, based on the amino acid position numbering of MbCas13 (Moraxella bovoculi 237). , K291, K297, K357, K403, K409, K414, K448, K460, K501, K515, K550, R552, K558, K564, K566, K582, K593, K604, K608, K623, K627, K633, K637, E643, K780, Y787, K7 92, K830, Q846, K858, K867, K876, K890, R900, K901, M906, K921, K927, K928, ​​K93 7, K939, R940, K945, Q975, R987, R990, K1001, R1034, I1036, R1038, R1042, K1052 , K1055, K1087, R1090, K1095, N1103, K1108, K1115, K1139, K1158, R1172, K1188, K1276, R1293, A1319, K1340, K1349, and / or K1356. In certain embodiments, a Cas13 enzyme comprising said one or more mutations has altered, and more preferably increased, specificity for the target.

[0064] In one embodiment, the Cas13 protein is modified by mutation at S1228 (e.g., S1228A) based on the amino acid position numbering of AsCas13. See Yamano et al., Cell 165:949-962 (2016), which is incorporated by reference in its entirety.

[0065] In certain embodiments, the Cas13 protein is engineered to recognize a non-native PAM, e.g., a PAM having or including the sequence YCN, YCV, AYV, TYV, RYN, RCN, TGYV, NTTN, TTN, TRTN, TYTV, TYCT, TYCN, TRTN, NTTN, TACT, TYCC, TRTC, TATV, NTTV, TTV, TSTG, TVTS, TYYS, TCYS, TBYS, TCYS, TNYS, TYYS, TNTN, TSTG, TTCC, TCCC, TATC, TGTG, TCTG, TYCV, or TCTC. In certain embodiments, the mutant Cas13 is selected from the group consisting of 11, 12, 13, 14, 15, 16, 17, 34, 36, 39, 40, 43, 46, 47, 50, 54, 57, 58, 111, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 36 69, 170, 171, 172, 173, 174, 175, 176, 177, 178, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 59 2, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 642, 643, 644, 645, 646 , 647, 648, 649, 651, 652, 653, 654, 655, 656, 676, 679, 680, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 707, 711, 714, 715, 716, 717, 718, 719, 720, 721, 722, 739, 765, 768, 769, 773, 777, 778, 779, 780, 781, 782,one or more mutated amino acid residues at positions 783, 784, 785, 786, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, or 1048, or at their corresponding positions in a Cas13 ortholog; comprising one or more mutant amino acid residues at positions 1, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 570, 571, 572, 573, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 630, 631, 632, 646, 647, 648, 649, 650, 651, 652, 653, 683, 684, 685, 686, 687, 688, 689, or 690;

[0066] In certain embodiments, the Cas13 protein is engineered to have increased activity, i.e., broader PAM specificity. In certain embodiments, the Cas13 protein comprises a mutated amino acid residue at positions 539, 542, 547, 548, 550, 551, 552, 167, 604, and / or 607 of AsCas13, or at the corresponding positions of an AsCas13 ortholog, homolog, or variant, preferably at positions 542 or 542 and 607 (said mutations are preferably 542R and 607R, e.g., S542R and K607R); or preferably at positions 542 and 548 (and optionally at position 552) (said mutations are preferably 542R and 548V (and optionally 552R), e.g., S542R and K548V (and optionally N552R)); or at positions 532, 538, 542, and / or 595 of LbCas13, Alternatively, the corresponding positions of an AsCas13 ortholog, homolog, or variant are modified by mutation of one or more residues, including but not limited to, mutant amino acid residues at positions 532 or 532 and 595 (said mutations are preferably 532R and 595R, e.g., G532R and K595R); alternatively, preferably, mutant amino acid residues at positions 532 and 538 (and optionally, 542) (said mutations are preferably 532R and 538V (and optionally 542R), e.g., G532R and K538V (and optionally Y542R), and most preferably, said mutations are S542R and K607R, S542R and K548V, or S542R, K548V, and N552R of AsCas13).

[0067] Non-activated / inactivated Cas13 protein Where the Cas13 protein has nuclease activity, the Cas13 protein can be modified to have reduced nuclease activity, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% nuclease inactivation when compared to the wild-type enzyme; or, stated differently, the Cas13 enzyme advantageously has about 0% of the nuclease activity of a non-mutated or wild-type Cas13 enzyme or CRISPR-Cas protein, or a non-mutated or wild-type Cas13 enzyme, e.g., a non-mutated or wild-type Francisella novicida U112 (FnCas13), Acidaminococcus sp. BV3L6 (AsCas13), Lachnospiraceae bacterium ND2006 (LbCas13) or Moraxella bovoculi 237 (MbCas13) has about 3%, about 5%, or about 10% or less of the nuclease activity of the Cas13 enzyme or CRISPR-Cas protein by introducing mutations into the nuclease domain of Cas13 and its orthologs.

[0068] In a preferred embodiment of the present invention, at least one Cas13 protein is used, which is a Cas13 nickase. More specifically, a Cas13 nickase is used that does not cleave the target strand, but can only cleave the strand that is complementary to the target strand, i.e., the non-target DNA strand, also referred to herein as the strand that is not complementary to the guide sequence. More specifically, the Cas13 nickase is a Cas13 protein that comprises an arginine mutation at position 1226A in the Nuc domain of Cas13 from Acidaminococcus sp., or at the corresponding position in Cas13 orthologues. Furthermore, in certain embodiments, the enzyme comprises an arginine to alanine substitution or an R1226A mutation. Those skilled in the art will understand that if the enzyme is not AsCas13, the mutation can be made at the residue at the corresponding position. In certain embodiments, the Cas13 is FnCas13, and the mutation is an arginine at position R1218. In certain embodiments, the Cas13 is LbCas13 and the mutation is an arginine at position R1138. In certain embodiments, the Cas13 is MbCas13 and the mutation is an arginine at position R1293.

[0069] In certain embodiments, CRISPR-Cas proteins can be additionally or alternatively used that can be engineered to include one or more mutations that reduce or eliminate nuclease activity. Amino acid positions in the FnCas13p RuvC domain include, but are not limited to, D917A, E1006A, E1028A, D1227A, D1255A, N1257A, D917A, E1006A, E1028A, D1227A, D1255A and N1257A. Applicants have also identified a putative second nuclease domain that is most similar to the PD-(D / E)XK nuclease superfamily and HincII endonuclease-like. Point mutations made in this putative nuclease domain to substantially reduce nuclease activity include, but are not limited to, N580A, N584A, T587A, W609A, D610A, K613A, E614A, D616A, K624A, D625A, K627A, and Y629A. In a preferred embodiment, the mutation in the FnCas13p RuvC domain is D917A or E1006A, where the D917A or E1006A mutation completely inactivates the DNA cleavage activity of the FnCas13 effector protein. In another embodiment, the mutation in the FnCas13p RuvC domain is D1255A, where the mutant FnCas13 effector protein has a significantly reduced nucleolytic activity.

[0070] More particularly, the inactivated Cas13 enzyme comprises an enzyme mutated at amino acid position As908, As993, As1263 of AsCas13 or the corresponding position in Cas13 orthologue. In addition, the inactivated Cas13 enzyme comprises an enzyme mutated at amino acid position Lb832, 925, 947 or 1180 of LbCas13 or the corresponding position in Cas13 orthologue. More particularly, the inactivated Cas13 enzyme comprises an enzyme comprising one or more of the mutations AsD908A, AsE993A, AsD1263A of AsCas13 or the corresponding mutations in Cas13 orthologue. In addition, the inactivated Cas13 enzyme comprises an enzyme comprising one or more of the mutations LbD832A, E925A, D947A ​​or D1180A of LbCas13 or the corresponding mutations in Cas13 orthologue.

[0071] Mutations can also be made to adjacent residues, e.g., amino acids in the vicinity of those indicated above that are involved in nuclease activity. In some embodiments, only the RuvC domain is inactivated, and in other embodiments, another putative nuclease domain is inactivated, where the effector protein complex functions as a nickase to cleave only one DNA strand. In a preferred embodiment, the other putative nuclease domain is a HincII-like endonuclease domain.

[0072] Inactivated Cas13 or Cas13 nickase may be associated with one or more functional domains (e.g., via a fusion protein), including, for example, adenosine deaminase or its catalytic domain. In some cases, it is advantageous to provide at least one heterologous NLS in addition. In some cases, it is advantageous to place the NLS at the N-terminus. In general, positioning one or more functional domains on inactivated Cas13 or Cas13 nickase is such that the correct spatial orientation of the functional domain allows it to affect the target with a resulting functional effect. For example, if the functional domain is its adenosine deaminase catalytic domain, the adenosine deaminase catalytic domain is placed in a spatial orientation so that it can contact and deaminate the target adenine. This may include positions other than the N / C terminus of Cas13. In some embodiments, the adenosine deaminase protein or its catalytic domain is inserted into an internal loop of Cas13.

[0073] In certain embodiments, the effector protein of the present invention (CRISPR enzyme; Cas13; effector protein) as described herein is a catalytically inactive or inactivated Cas13 effector protein (dCas13). In some embodiments, the dCas13 effector comprises a mutation in the nuclease domain. In some embodiments, the dCas13 effector protein is truncated. In some embodiments, the dCas13 comprises a truncated form of the Cas13 effector protein at one or more of the following positions: the C-terminal protein of the Cas13 effector, the N-terminal of the Cas13 effector protein, or one or more domains of the Cas13 effector protein (e.g., the HEPN domain). For example, the dCas13 can comprise a truncated form of the Cas13 effector protein at the C-terminus of the Cas13 effector protein. For example, the dCas13 can comprise a truncated form of the Cas13 effector protein at the N-terminus of the Cas13 effector protein. For example, dCas13 may comprise a truncated version of the Cas13 effector protein at the HEPN domain of the Cas13 effector protein. In some embodiments, such truncation may reduce the size of the fusion protein of the Cas13 effector and one or more functional components. In some cases, the truncated Cas13 may still retain its RNA-binding function.

[0074] In some embodiments, the C-terminus of the Cas13 effector can be truncated, for example, at least 20 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 150 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 250 amino acids, at least 260 amino acids, or at least 300 amino acids, or at least 350 amino acids, or up to 120 amino acids, or up to 140 amino acids, or up to 160 amino acids, or up to 180 amino acids, or up to 200 amino acids, or up to 250 amino acids, or up to 300 amino acids, or up to 350 amino acids, or up to 400 amino acids can be truncated at the C-terminus of the Cas13 effector. Examples of Cas13 truncations include C-terminal Δ984-1090, C-terminal Δ1026-1090, and C-terminal Δ1053-1090, C-terminal Δ934-1090, C-terminal Δ884-1090, C-terminal Δ834-1090, C-terminal Δ784-1090, and C-terminal Δ734-1090, where the amino acid positions correspond to those of the Prevotella sp. P5-125 Cas13b protein. Examples of Cas13 truncations also include C-terminal Δ795-1095, where the amino acid positions correspond to those of the Riemerella anatipestifer Cas13b protein. Further examples of Cas13 truncations include C-terminal Δ875-1175, C-terminal Δ895-1175, C-terminal Δ915-1175, C-terminal Δ935-1175, C-terminal Δ955-1175, C-terminal Δ975-1175, C-terminal Δ995-1175, C-terminal Δ1015-1175, C-terminal Δ1035-1175, C-terminal Δ1055-1175, C-terminal Δ1075-1175, C-terminal Δ1095-1175, C-terminal Δ1115-1175, C-terminal Δ1135-1175, C-terminal Δ1155-1175, where the amino acid positions correspond to those of the Porphyromonas gulae Cas13b protein.One of skill in the art will appreciate that similar truncations can be designed for other Cas13b orthologs, or other Cas13 types or subtypes, such as Cas13a, Cas13c, or Cas13d.

[0075] In some embodiments, the N-terminus of the Cas13 effector protein may be truncated, for example, at least 20 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 150 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 250 amino acids, at least 260 amino acids, or at least 300 amino acids, or at least 350 amino acids, or up to 120 amino acids, or up to 140 amino acids, or up to 160 amino acids, or up to 180 amino acids, or up to 200 amino acids, or up to 250 amino acids, or up to 300 amino acids, or up to 350 amino acids, or up to 400 amino acids may be truncated at the N-terminus of the Cas13 effector. Examples of Cas13 truncations include N-terminal Δ1-125, N-terminal Δ1-88, or N-terminal Δ1-72, where the amino acid positions of the truncations correspond to the amino acid positions of the Prevotella sp. P5-125 Cas13b protein.

[0076] In some embodiments, both the N-terminus and the C-terminus of the Cas13 effector protein may be truncated, for example, at least 20 amino acids may be truncated at the C-terminus of the Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the N-terminus of the Cas13 effector. For example, at least 40 amino acids may be truncated at the C-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the N-terminus of a Cas13 effector. For example, at least 60 amino acids may be truncated at the C-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the N-terminus of a Cas13 effector.For example, at least 80 amino acids may be truncated at the C-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the N-terminus of a Cas13 effector. For example, at least 100 amino acids may be truncated at the C-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the N-terminus of a Cas13 effector. For example, at least 120 amino acids may be truncated at the C-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the N-terminus of a Cas13 effector.For example, at least 140 amino acids may be truncated at the C-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the N-terminus of a Cas13 effector. For example, at least 160 amino acids may be truncated at the C-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the N-terminus of a Cas13 effector. For example, at least 180 amino acids may be truncated at the C-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the N-terminus of a Cas13 effector.For example, at least 200 amino acids may be truncated at the C-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the N-terminus of a Cas13 effector. For example, at least 220 amino acids may be truncated at the C-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the N-terminus of a Cas13 effector. For example, at least 240 amino acids may be truncated at the C-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the N-terminus of a Cas13 effector.For example, at least 260 amino acids may be truncated at the C-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the N-terminus of a Cas13 effector. For example, at least 280 amino acids may be truncated at the C-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the N-terminus of a Cas13 effector. For example, at least 300 amino acids may be truncated at the C-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the N-terminus of a Cas13 effector.For example, at least 350 amino acids may be truncated at the C-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the N-terminus of a Cas13 effector. For example, at least 20 amino acids may be truncated at the N-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the C-terminus of a Cas13 effector. For example, at least 40 amino acids may be truncated at the N-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the C-terminus of a Cas13 effector.For example, at least 60 amino acids may be truncated at the N-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the C-terminus of a Cas13 effector. For example, at least 80 amino acids may be truncated at the N-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the C-terminus of a Cas13 effector. For example, at least 100 amino acids may be truncated at the N-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the C-terminus of a Cas13 effector. For example, at least 120 amino acids may be truncated at the N-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the C-terminus of a Cas13 effector.For example, at least 140 amino acids may be truncated at the N-terminus of the Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the C-terminus of the Cas13 effector. For example, at least 160 amino acids may be truncated at the N-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the C-terminus of a Cas13 effector. For example, at least 180 amino acids may be truncated at the N-terminus of the Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the C-terminus of the Cas13 effector.For example, at least 200 amino acids may be truncated at the N-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the C-terminus of a Cas13 effector. For example, at least 220 amino acids may be truncated at the N-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the C-terminus of a Cas13 effector. For example, at least 240 amino acids may be truncated at the N-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the C-terminus of a Cas13 effector.For example, at least 260 amino acids may be truncated at the N-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the C-terminus of a Cas13 effector. For example, at least 280 amino acids may be truncated at the N-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the C-terminus of a Cas13 effector. For example, at least 300 amino acids may be truncated at the N-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the C-terminus of a Cas13 effector.For example, at least 350 amino acids may be truncated at the N-terminus of a Cas13 effector, and at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 120 amino acids, at least 140 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, at least 220 amino acids, at least 240 amino acids, at least 260 amino acids, at least 300 amino acids, or at least 350 amino acids may be truncated at the C-terminus of a Cas13 effector.

[0077] CRISPR-Cas proteins and guides The method and system of the present invention uses CRISPR-Cas protein and corresponding guide molecule. More specifically, the CRISPR-Cas protein is a class 2 CRISPR-Cas protein. In a particular embodiment, the CRISPR-Cas protein is Cas13. The CRISPR-Cas system does not need to create customized proteins to target specific sequences, but rather a single Cas protein can be programmed to recognize specific nucleic acid targets by guide molecules, in other words, the guide molecules can be used to recruit Cas enzyme proteins to specific nucleic acid target loci of interest.

[0078] Guide molecule The guide molecule or guide RNA of class 2 type V CRISPR-Cas proteins comprises a tracr-mate sequence (which includes a "direct repeat" in the context of an endogenous CRISPR system) and a guide sequence (also referred to as a "spacer" in the context of an endogenous CRISPR system). Indeed, in contrast to type II CRISPR-Cas proteins, Cas13 proteins do not depend on the presence of a tracr sequence. In some embodiments, a CRISPR-Cas system or complex as described herein does not comprise and / or depend on the presence of a tracr sequence (e.g., when the Cas protein is Cas13). In certain embodiments, the guide molecule may comprise, consist essentially of, or consist of a direct repeat sequence fused or linked to a guide sequence or spacer sequence.

[0079] Generally, CRISPR system is characterized by the element that promotes the formation of CRISPR complex at the site of target sequence.In the context of the formation of CRISPR complex, " target sequence " refers to the sequence that guide sequence is designed to have complementarity with, where the hybridization between target DNA sequence and guide sequence promotes the formation of CRISPR complex.

[0080] The terms "guide molecule" and "guide RNA" are used interchangeably herein and refer to an RNA-based molecule that can form a complex with a CRISPR-Cas protein and that includes a guide sequence that has sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of the complex to the target nucleic acid sequence. Guide molecule or guide RNA specifically encompasses RNA-based molecules that have one or more chemical modifications as described herein (e.g., by chemical linkage of two ribonucleotides or by substitution of one or more ribonucleotides with one or more deoxyribonucleotides).

[0081] As used herein, the term "guide sequence" in the context of CRISPR-Cas system includes any polynucleotide sequence that has sufficient complementarity with the target nucleic acid sequence to hybridize with the target nucleic acid sequence and guide the sequence-specific binding of the nucleic acid targeting complex to the target nucleic acid sequence.In the context of the present invention, the target nucleic acid sequence or target sequence is the sequence that contains the target adenosine to be deaminated, also referred to herein as "target adenosine".In some embodiments, the degree of complementarity, excluding the intended dA-C mismatch, is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more, or more than these approximate numbers, when optimally aligned using a suitable alignment algorithm. Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., the Burrows-Wheeler aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). The ability of the guide sequence (within the nucleic acid targeting guide RNA) to direct sequence-specific binding of the nucleic acid targeting complex to the target nucleic acid sequence can be assessed by any suitable assay. For example, sufficient components of a nucleic acid targeting CRISPR system to form a nucleic acid targeting complex may be provided to a host cell having a corresponding target nucleic acid sequence, including a guide sequence to be tested, such as by transfection of a vector encoding the components of the nucleic acid targeting complex, and then preferential targeting (e.g., cleavage) within the target nucleic acid sequence may be assessed, such as by a Surveyor assay as described herein.Similarly, cleavage of a target nucleic acid sequence can be determined in vitro by providing the target nucleic acid sequence, components of a nucleic acid targeting complex, including the guide sequence to be tested, and a control guide sequence that is different from the test guide sequence, and comparing the binding or cleavage rate at or near the target sequence between reactions with the test guide sequence and the control guide sequence. Other assays are possible and will occur to those skilled in the art. Guide sequences, and thus nucleic acid targeting guide RNAs, can be selected to target any target nucleic acid sequence.

[0082] In some embodiments, the guide molecule comprises a guide sequence that is designed to have at least one mismatch with the target sequence, such that the RNA duplex formed between the guide sequence and the target sequence contains an unpaired C in the guide sequence facing a target A for deamination on the target sequence. In some embodiments, apart from this AC mismatch, the degree of complementarity is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more, or greater than about these numbers, when optimally aligned using a suitable alignment algorithm.

[0083] In certain embodiments, the guide sequence or spacer length of the guide molecule is 15-50 nt. In certain embodiments, the spacer length of the guide RNA is at least 15 nucleotides. In certain embodiments, the spacer length is 15-17 nt, such as 15, 16, or 17 nt, 17-20 nt, such as 17, 18, 19, or 20 nt, 20-24 nt, such as 20, 21, 22, 23, or 24 nt, 23-25 ​​nt, such as 23, 24, or 25 nt, 24-27 nt, such as 24, 25, 26, or 27 nt, 27-30 nt, such as 27, 28, 29, or 30 nt, 30-35 nt, such as 30, 31, 32, 33, 34, or 35 nt, or 35 nt or more. In certain exemplary embodiments, the guide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 , 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nt.

[0084] In some embodiments, the guide sequence is 10-50 nt in length, but more particularly is an RNA sequence of about 20-30 nt, advantageously about 20 nt, 23-25 ​​nt or 24 nt. The guide sequence is selected to ensure that it hybridizes to the target sequence containing the adenosine to be deaminated. This is explained in more detail below. Selection may include further steps to increase the efficiency and specificity of the deamination.

[0085] In some embodiments, the guide sequence is about 20 nt to about 30 nt in length and hybridizes to the target DNA strand to form a nearly perfectly matched duplex, except for a dA-C mismatch at the target adenosine site. In particular, in some embodiments, the dA-C mismatch is located near the center of the target sequence (and thus the center of the duplex when the guide sequence hybridizes to the target sequence), thereby restricting the adenosine deaminase to a narrow editing window (e.g., about 4 bp wide). In some embodiments, the target sequence may contain two or more target adenosines to be deaminated. In further embodiments, the target sequence may further contain one or more dA-C mismatches 3' to the target adenosine site. In some embodiments, to avoid off-target editing at unintended adenine sites in the target sequence, the guide sequence may be designed to contain an unpaired guanine at the position corresponding to the unintended adenine, introducing a dA-G mismatch that is catalytically unfavorable to certain adenosine deaminases, such as ADAR1 and ADAR2. See Wong et al., RNA 7:846-858 (2001), which is incorporated by reference in its entirety.

[0086] In some embodiments, a Cas13 guide sequence with canonical length (e.g., about 24 nt for AsCas13) is used to form an RNA duplex with the target DNA. In some embodiments, a Cas13 guide molecule longer than the canonical length (e.g., more than 24 nt for AsCas13) is used to form an RNA duplex with the target DNA, for example, outside the Cas13-guide RNA-target DNA complex. This may be of interest when targeting the deamination of more than one adenine within a given stretch of nucleotides. In alternative embodiments, it is interesting to maintain the length restriction of the canonical guide sequence. In some embodiments, the guide sequence is designed to introduce a dA-C mismatch outside the canonical length of the Cas13 guide, which can reduce steric hindrance by Cas13 and increase the contact frequency between adenosine deaminase and the dA-C mismatch.

[0087] In some embodiments, the sequence of the guide molecule (direct repeat and / or spacer) is selected to reduce the degree of secondary structure within the guide molecule. In some embodiments, less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1% or less of the nucleotides of the nucleic acid targeting guide RNA are involved in self-complementary base pairing when optimally folded. Optimal folding can be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculation of minimum Gibbs free energy. An example of one such algorithm is mFold as described by Zuker and Stiegler (Nucleic Acids Res.9 (1981), 133-148). Another exemplary folding algorithm is the online web server RNAfold, developed at the Institute for Theoretical Chemistry at the University of Vienna, which uses a centroid structure prediction algorithm (see, e.g., A R Gruber et al., 2008, Cell 106(1):23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12):1151-62).

[0088] In some embodiments, it is intended to reduce the susceptibility of the guide molecule to RNA cleavage, e.g., cleavage by Cas13. Thus, in certain embodiments, the guide molecule is tailored to avoid cleavage by Cas13 or other RNA cleaving enzymes.

[0089] In certain embodiments, the guide molecule comprises non-naturally occurring nucleic acids and / or non-naturally occurring nucleotides and / or nucleotide analogs, and / or chemical modifications. Preferably, these non-naturally occurring nucleic acids and non-naturally occurring nucleotides are located outside the guide sequence. Non-naturally occurring nucleic acids can include, for example, a mixture of naturally occurring and non-naturally occurring nucleotides. Non-naturally occurring nucleotides and / or nucleotide analogs may be modified at the ribose, phosphate, and / or base moieties. In certain embodiments of the invention, the guide nucleic acid comprises ribonucleotides and non-ribonucleotides. In one such embodiment, the guide comprises one or more ribonucleotides and one or more deoxyribonucleotides. In certain embodiments of the invention, the guide comprises one or more non-naturally occurring nucleotides or nucleotide analogs, such as nucleotides with phosphorothioate bonds, locked nucleic acid (LNA) nucleotides that contain a methylene bridge between the 2' and 4' carbons of the ribose ring, or bridged nucleic acids (BNA). Other examples of modified nucleotides include 2'-O-methyl analogs, 2'-deoxy analogs, or 2'-fluoro analogs. Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine. Examples of chemical modifications of guide RNAs include, without limitation, incorporation of 2'-O-methyl (M), 2'-O-methyl 3' phosphorothioate (MS), S-constrained ethyl (cEt), or 2'-O-methyl 3' thio PACE (MSP) at one or more terminal nucleotides.Such chemically modified guides can include increased stability and activity when compared to unmodified guides, however on-target versus off-target specificity is unpredictable (Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi:10.1038 / nbt.3290, published online 29 June 2015 Ragdarm et al., 0215, PNAS, E7110-E7111; Allerson et al., J. Med. Chem. 2005, 48:901-904; Bramsen et al., Front. Genet., 2012, 3:154; Deng et al., PNAS, 2015, 112:11870-11875; Sharma et al., MedChemComm., 2014, 5:1454-1471; Hendel et al., 2015, 112:11870-11875; al., Nat. Biotechnol. (2015) 33(9):985-989; Li et al., Nature Biomedical Engineering, 2017, 1,0066 DOI:10.1038 / s41551-017-0066). In some embodiments, the 5' and / or 3' ends of the guide RNA are modified with various functional moieties, including fluorescent dyes, polyethylene glycol, cholesterol, proteins, or detection tags (see Kelly et al., 2016, J. Biotech. 233:74-83). In certain embodiments, the guide comprises ribonucleotides in the region that binds to the target DNA and one or more deoxyribonucleotides, and / or nucleotide analogs in the region that binds to Cas13. In certain embodiments of the invention, deoxyribonucleotides and / or nucleotide analogs are incorporated into the engineered guide structure, such as, but not limited to, the stem-loop region and the seed region. With respect to the Cas13 guide, in certain embodiments, the modification is not the 5'-handle of the stem-loop region. Chemical modifications in the 5'-handle of the stem-loop region of the guide can abolish its function (see Li, et al., Nature Biomedical Engineering, 2017, 1:0066).In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides of the guide are chemically modified. In some embodiments, 3-5 nucleotides at either the 3' or 5' end of the guide are chemically modified. In some embodiments, only minor modifications are introduced in the seed region, such as 2'-F modifications. In some embodiments, 2'-F modifications are introduced at the 3' end of the guide. In certain embodiments, 3-5 nucleotides at the 5' and / or 3' ends of the guide are chemically modified with 2'-O-methyl (M), 2'-O-methyl 3' phosphorothioate (MS), S-constrained ethyl (cEt), or 2'-O-methyl 3' thio PACE (MSP). Such modifications can enhance genome editing efficiency (see Hendel et al., Nat. Biotechnol. (2015) 33(9):985-989). In certain embodiments, all of the phosphodiester bonds of the guide are replaced with phosphorothioate (PS), enhancing gene disruption levels. In certain embodiments, 6 or more nucleotides at the 5' and / or 3' ends of the guide are chemically modified with 2'-O-Me, 2'-F, or S-constrained ethyl (cEt). Such chemically modified guides can mediate enhanced levels of gene disruption (see Ragdarm et al., 0215, PNAS, E7110-E7111). In certain embodiments of the invention, the guide is modified to contain a chemical moiety at its 3' and / or 5' end. Such moieties include, but are not limited to, amine, azide, alkyne, thio, dibenzocyclooctyne (DBCO), or rhodamine. In certain embodiments, the chemical moiety is conjugated to the guide by a linker, such as an alkyl chain. In certain embodiments, the chemical moiety of the modified guide can be used to attach the guide to another molecule, such as DNA, RNA, a protein, or a nanoparticle.Such chemically modified guides can be used to identify or enrich for cells that have been genetically edited by the CRISPR system (see Lee et al., eLife, 2017, 6:e25312, DOI:10.7554).

[0090] In some embodiments, the guide comprises a modified Cas13crRNA having a 5'-handle and a guide segment further comprising a seed region and a 3' end. In some embodiments, the modified guide is Acidaminococcus sp. BV3L6 Cas13(AsCas13); Francisella tularensis subsp. Novicida U112 Cas13(FnCas13); L.bacterium MC2017 Cas13(Lb3Cas13); GWC2011_GWC2_44_17 Cas13(PbCas13);Peregrinibacteria bacterium GW2011_GWA_33_10 Cas13(PeCas13);Leptospira inadai Cas13(LiCas13);Smithella sp.SC_K08D17 Cas13(SsCas13);L.bacterium MA2020 The Cas13 may be any one of the following Cas13s: Cas13 (Lb2Cas13); Porphyromonas crevioricanis Cas13 (PcCas13); Porphyromonas macacae Cas13 (PmCas13); Candidatus Methanoplasma termitum Cas13 (CMtCas13); Eubacterium eligens Cas13 (EeCas13); Moraxella bovoculi 237 Cas13 (MbCas13); Prevotella disiens Cas13 (PdCas13); or L. bacterium ND2006 Cas13 (LbCas13).

[0091] In some embodiments, the modification of the guide is a chemical modification, an insertion, a deletion, or a split. In some embodiments, the chemical modification includes, but is not limited to, the incorporation of 2'-O-methyl (M) analogs, 2'-deoxy analogs, 2-thiouridine analogs, N6-methyladenosine analogs, 2'-fluoro analogs, 2-aminopurine, 5-bromo-uridine, pseudouridine (Ψ), N1-methylpseudouridine (melΨ), 5-methoxyuridine (5moU), inosine, 7-methylguanosine, 2'-O-methyl 3' phosphorothioate (MS), S-constrained ethyl (cEt), phosphorothioate (PS), or 2'-O-methyl 3' thio PACE (MSP). In some embodiments, the guide includes one or more phosphorothioate modifications. In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 nucleotides of the guide are chemically modified. In certain embodiments, one or more nucleotides of the seed region are chemically modified. In certain embodiments, one or more nucleotides of the 3'-end are chemically modified. In certain embodiments, none of the nucleotides in the 5'-handle are chemically modified. In some embodiments, the chemical modification in the seed region is a minor modification, such as the incorporation of a 2'-fluoro analog. In certain embodiments, one nucleotide of the seed region is replaced with a 2'-fluoro analog. In some embodiments, 5-10 nucleotides of the 3'-end are chemically modified. Such chemical modification of the 3'-end of the Cas13 CrRNA can improve Cas13 activity (see Li, et al., Nature Biomedical Engineering, 2017, 1:0066). In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides at the 3' terminus are replaced with 2'-fluoro analogs.In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides at the 3' terminus are replaced with 2'-O-methyl (M) analogs.

[0092] In some embodiments, the loop of the 5'-handle of the guide is modified. In some embodiments, the loop of the 5'-handle of the guide is modified to have a deletion, insertion, split, or chemical modification. In certain embodiments, the modified loop comprises 3, 4, or 5 nucleotides. In certain embodiments, the loop comprises the sequence UCUU, UUUU, UAUU, or UGUU.

[0093] In some embodiments, the guide molecule forms a stem loop with a separate non-covalently linked sequence, which can be DNA or RNA. In certain embodiments, the sequences forming the guide are first synthesized using standard phosphoramidite synthesis protocols (Herdewijn, P., ed., Methods in Molecular Biology Col 288, Oligonucleotide Synthesis: Methods and Applications, Humana Press, New Jersey (2012)). In some embodiments, these sequences can be functionalized to include functional groups suitable for ligation using standard protocols known in the art (Hermanson, GT, Bioconjugate Techniques, Academic Press (2013)). Examples of functional groups include, but are not limited to, hydroxyl, amine, carboxylic acid, carboxylic acid halide, carboxylic acid active ester, aldehyde, carbonyl, chlorocarbonyl, imidazolylcarbonyl, hydrozide, semicarbazide, thiosemicarbazide, thiol, maleimide, haloalkyl, sulfonyl, allyl, propargyl, diene, alkyne, and azide. Once the sequence is functionalized, a covalent chemical bond or linkage can be formed between the sequence and the direct repeat sequence. Examples of chemical bonds include, but are not limited to, carbamates, ethers, esters, amides, imines, amidines, aminotolidines, hydrozones, disulfides, thioethers, thioesters, phosphorothioates, phosphorodithioates, sulfonamides, sulfonic acids, sulfones, sulfoxides, ureas, thioureas, hydrazides, oximes, triazoles, photolabile bonds, CC bond forming groups, such as those based on Diels-Alder cycloaddition pairs or ring-closing metathesis pairs, and Michael reaction pairs.

[0094] In some embodiments, these stem-loop forming sequences can be chemically synthesized using automated solid-phase oligonucleotide synthesis machines with 2'-acetoxyethyl orthoester (2'-ACE) (Scaringe et al., J. Am. Chem. Soc. (1998) 120:11820-11821; Scaringe, Methods Enzymol. (2000) 317:3-18) or 2'-thionocarbamate (2'-TC) chemistry (Dellinger et al., J. Am. Chem. Soc. (2011) 133:11540-11546; Hendel et al., Nat. Biotechnol. (2015) 33:985-989).

[0095] In certain embodiments, a guide molecule (capable of guiding Cas13 to a target locus) comprises (1) a guide sequence capable of hybridizing to a target locus, and (2) a tracr mate or direct repeat sequence, whereby the direct repeat sequence is located upstream (i.e., 5') from the guide sequence. In certain embodiments, the seed sequence of the Cas13 guide sequence (i.e., a critical sequence essential for recognition and / or hybridization to a sequence at the target locus) is approximately within about the first 10 nucleotides of the guide sequence. In certain embodiments, the Cas13 is FnCas13, and the seed sequence is approximately within the first 5 nt on the 5' end of the guide sequence.

[0096] In certain embodiments, the guide molecule comprises a guide sequence linked to a direct repeat sequence, the direct repeat sequence comprising one or more stem loops or optimized secondary structures. In certain embodiments, the direct repeat has a minimum length of 16 nt and a single stem loop. In further embodiments, the direct repeat is longer than 16 nt, preferably longer than 17 nt, in length and has two or more stem loops or optimized secondary structures. In certain embodiments, the guide molecule comprises or consists of a guide sequence linked to all or part of a natural direct repeat sequence. A typical V-type Cas13 guide molecule comprises (in the 3' to 5' direction): a guide sequence, a first complementary stretch ("repeat"), a loop (typically 4 or 5 nucleotides long), a second complementary stretch ("anti-repeat" complementary to the repeat), and a polyA (polyU-rich in RNA) tail (terminator). In certain embodiments, the direct repeat sequence retains its natural architecture and forms a single stem loop. In certain embodiments, certain aspects of the guide architecture can be altered, for example, by adding, deleting, or substituting features, while certain other aspects of the guide architecture are maintained. Preferred locations for modification (including, but not limited to, insertions, deletions, and substitutions) of engineered guide molecules include the guide ends and regions of the guide molecule, for example, stem-loops of direct repeat sequences, that are exposed when complexed with the Cas13 protein and / or target.

[0097] In certain embodiments, the stem comprises at least about 4 bp comprising complementary X and Y sequences, but more, e.g., 5, 6, 7, 8, 9, 10, 11 or 12, or less, e.g., 3, 2 base pair stems are also contemplated. Thus, for example, X2-10 and Y2-10, where X and Y represent any complementary set of nucleotides, may be contemplated. In one aspect, the stem composed of X and Y nucleotides together with the loop forms a complete hairpin in the overall secondary structure; this may be advantageous, and the amount of base pairs may be any amount that forms a complete hairpin. In one aspect, any complementary X:Y base pairing sequence (e.g., in terms of length) is allowed as long as the secondary structure of the entire guide molecule is preserved. In one aspect, the loop connecting the stem composed of X:Y base pairs may be any sequence of the same length (e.g., 4 or 5 nucleotides) or more that does not disturb the overall secondary structure of the guide molecule. In one embodiment, the stem loop may further comprise, for example, an MS2 aptamer. In one embodiment, the stem comprises about 5-7 bp including the complementary X and Y sequences, although stems of more or less base pairs are also contemplated. In one embodiment, non-Watson-Crick base pairing is contemplated, provided such pairing otherwise generally preserves the architecture of the stem loop at that position.

[0098] In certain embodiments, the natural hairpin or stem-loop structure of guide molecule is extended or replaced by extended stem-loop. It has been demonstrated that stem extension can enhance the assembly of guide molecule with CRISPR-Cas protein (Chen et al. Cell. (2013); 155 (7): 1479-1491). In certain embodiments, the stem of the stem-loop is extended by at least 1, 2, 3, 4, 5 or more complementary base pairs (i.e., corresponding to the addition of 2, 4, 6, 8, 10 or more nucleotides in the guide molecule). In certain embodiments, they are located at the end of the stem, adjacent to the loop of the stem-loop.

[0099] In certain embodiments, the sensitivity of guide molecules to RNA or reduced expression can be reduced by minor modifications of the sequence of guide molecules that do not affect their function.For example, in certain embodiments, premature termination of transcription, such as premature transcription of U6 Pol-III, can be eliminated by modifying the putative Pol-III terminator (four consecutive Us) in the guide molecule sequence.When such sequence modifications are required in the stem loop of guide molecules, they are preferably ensured by base pair flips.

[0100] In a preferred embodiment, the direct repeats can be modified to include one or more protein-binding RNA aptamers. In certain embodiments, one or more aptamers can be included, for example, as part of an optimized secondary structure. Such aptamers can be capable of binding to bacteriophage coat proteins, as further described herein.

[0101] In some embodiments, the guide molecule forms a duplex with a target DNA strand that contains at least one target adenosine residue to be edited. Upon hybridization of the guide RNA molecule to the target DNA strand, adenosine deaminase binds to the duplex and catalyzes the deamination of one or more target adenosine residues contained within the DNA-RNA duplex.

[0102] The guide sequence, and thus the nucleic acid targeting guide RNA, can be selected to target any target nucleic acid sequence. The target sequence can be DNA. The target sequence can be genomic DNA. The target sequence can be mitochondrial DNA.

[0103] In certain embodiments, the target sequence should be associated with a PAM (protospacer adjacent motif) or PFS (sequence or site adjacent to the protospacer); i.e., a short sequence recognized by the CRISPR complex. Depending on the nature of the CRISPR-Cas protein, the target sequence should be selected such that its complementary sequence in the DNA duplex (also referred to herein as the non-target sequence) is upstream or downstream of the PAM. In embodiments of the invention where the CRISPR-Cas protein is a Cas13 protein, the complementary sequence of the target sequence is downstream or 3' of the PAM. The exact sequence and length requirements of the PAM vary depending on the Cas13 protein used, but the PAM is typically a 2-5 base pair sequence adjacent to the protospacer (i.e., the target sequence). Examples of native PAM sequences for different Cas13 orthologs are described herein below, and the skilled artisan will be able to identify additional PAM sequences for use with a given Cas13 protein.

[0104] Furthermore, engineering PAM-interacting (PI) domains may allow one to program PAM specificity, improve the fidelity of target site recognition, and increase the versatility of CRISPR-Cas proteins, as described for Cas9 in, for example, Kleinstiver BP et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015 Jul 23;523(7561):481-5. doi:10.1038 / nature14592. As further detailed herein, one skilled in the art will appreciate that Cas13 proteins may be similarly modified.

[0105] In certain embodiments, the guide sequence is selected to ensure optimal efficiency of the deaminase on the adenine to be deaminated. The location of the adenine in the target strand relative to the cleavage site of Cas13 nickase may be considered. In certain embodiments, it is beneficial to ensure that the nickase acts near the adenine to be deaminated on the non-target strand. For example, in certain embodiments, Cas13 nickase cleaves the non-target strand 17 nucleotides downstream of the PAM (e.g., AsCas13, LbCas13) or 18 nucleotides downstream of the PAM (e.g., FnCas13), and it may be beneficial to design a guide in which the cytosine corresponding to the adenine to be deaminated is located in the guide sequence within 10 bp upstream or downstream of the nickase cleavage site in the sequence of the corresponding non-target strand.

[0106] In certain embodiments, the guide is an escorted guide. "Escorted" means that the Cas13 CRISPR-Cas system or complex or guide is delivered to a selected time or location within the cell, thus controlling the activity of the Cas13 CRISPR-Cas system or complex or guide spatially or temporally. For example, the activity and destination of the Cas13 CRISPR-Cas system or complex or guide can be controlled by an escort RNA aptamer sequence that has binding affinity for an aptamer ligand, such as a cell surface protein or other localized cellular component. Alternatively, the escort aptamer can be responsive to an aptamer effector on or within the cell, such as a transient effector, such as an external energy source that is added to the cell at a specific time.

[0107] The escorted Cas13 CRISPR-Cas system or complex has a guide molecule with a functional structure designed to improve the structure, architecture, stability, gene expression, or any combination thereof of the guide molecule. Such structures may include aptamers.

[0108] Aptamers are biomolecules that can be designed or selected to bind tightly to other ligands, for example, by using a technique called evolution in vitro (SELEX; ​​Tuerk C, Gold L: "Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase." Science 1990, 249: 505-510). Nucleic acid aptamers can be selected, for example, from a pool of random sequence oligonucleotides, and have high binding affinity and specificity for a wide range of biomedically relevant targets, suggesting a wide range of therapeutic utility for aptamers (Keefe, Anthony D., Supriya Pai, and Andrew Ellington. "Aptamers as therapeutics." Nature Reviews Drug Discovery 9.7 (2010): 537-550). These characteristics also suggest a wide range of applications for aptamers as drug delivery vehicles (Levy-Nissenbaum, Etgar, et al. "Nanotechnology and aptamers: applications in drug delivery." Trends in biotechnology 26.8 (2008): 442-449; and Hicke BJ, Stephens AW. "Escort aptamers: a delivery service for diagnosis and therapy." J Clin Invest 2000, 106: 923-928). Aptamers that function as molecular switches, such as RNA aptamers that bind to fluorophores to mimic the activity of green fluorescent protein (Paige, Jeremy S., Karen Y. Wu, and Samie R. Jaffrey. "RNA mimics of green fluorescent protein." Science 333.6042 (2011): 642-646), can also be constructed to respond to cues by changing their properties.It has also been suggested that aptamers can be used as components of targeted siRNA therapeutic delivery systems, for example to target cell surface proteins (Zhou, Jiehua, and John J. Rossi. "Aptamer-targeted cell-specific RNA interference." Silence 1.1 (2010):4).

[0109] Thus, in certain embodiments, the guide molecule is modified with one or more aptamer(s) designed to improve guide molecule delivery, including, for example, delivery across a cell membrane, to an intracellular compartment, or into the nucleus. Such structures may include moieties to render the guide molecule deliverable, inducible, or responsive to an effector of choice, in addition to or without one or more aptamer(s). Thus, the present invention encompasses guide molecules that respond to normal or pathological physiological conditions, including, without limitation, pH, hypoxia, O2 concentration, temperature, protein concentration, enzyme concentration, lipid structure, light exposure, mechanical disruption (e.g., ultrasound), magnetic field, electric field, or electromagnetic radiation.

[0110] The light responsiveness of the inducible system can be achieved by the activation and binding of cryptochrome-2 and CIB1. Blue light stimulation induces an activated conformational change in cryptochrome-2, which recruits its binding partner CIB1. This binding is fast and reversible, reaching saturation in less than 15 seconds after pulse stimulation and returning to baseline in less than 15 minutes after stimulation ends. These rapid binding kinetics result in a system that is temporally constrained only by the rates of transcription / translation and transcript / protein degradation, rather than inducer uptake and clearance. Cryptochrome-2 activation is also extremely sensitive, allowing the use of low light intensity stimulation, reducing the risk of phototoxicity. Furthermore, in contexts such as the intact mammalian brain, variable light intensity can be used to control the size of the stimulation region, providing greater precision than vector delivery alone can provide.

[0111] The present invention contemplates that an energy source such as electromagnetic radiation, acoustic energy, or thermal energy may induce the guide. Advantageously, the electromagnetic radiation is a component of visible light. In a preferred embodiment, the light is blue light with a wavelength of about 450 to about 495 nm. In a particularly preferred embodiment, the wavelength is about 488 nm. In another preferred embodiment, the light stimulation is pulsed. The light power may range from about 0 to 9 mW / cm2. In a preferred embodiment, a low intensity stimulation paradigm of 0.25 seconds every 15 seconds should result in maximal activation.

[0112] The chemical or energy sensitive guide may undergo a conformational change upon binding of a chemical source or induction by energy, allowing it to act as a guide and to function the Cas13 CRISPR-Cas system or complex. The invention may include applying a chemical source or energy to function the guide and to function the Cas13 CRISPR-Cas system or complex; and optionally further determining that expression of the genomic locus is altered.

[0113] There are several different designs of this chemical inducible system: 1. ABI-PYL-based systems inducible by abscisic acid (ABA) (see, for example, http: / / stke.sciencemag.org / cgi / content / abstract / sigtrans;4 / 164 / rs2), 2. FKBP-FRB-based systems inducible by rapamycin (or related chemicals based on rapamycin) (see, for example, http: / / www.nature.com / nmeth / journal / v2 / n6 / full / nmeth763.html), 3. GID1-GAI-based systems inducible by gibberellin (GA) (see, for example, http: / / www.nature.com / nchembio / journal / v8 / n5 / full / nchembio.922.html).

[0114] The chemical inducible system may be an estrogen receptor (ER)-based system that can be induced by 4-hydroxytamoxifen (4OHT) (see, for example, http: / / www.pnas.org / content / 104 / 3 / 1027.abstract). A mutant ligand-binding domain of the estrogen receptor, called ERT2, translocates into the nucleus of the cell upon binding of 4-hydroxytamoxifen. In further embodiments of the present invention, any naturally occurring or engineered derivative of any nuclear receptor, thyroid hormone receptor, retinoic acid receptor, estrogen receptor, estrogen-related receptor, glucocorticoid receptor, progesterone receptor, androgen receptor may be used in an inducible system similar to the ER-based inducible system.

[0115] Another inducible system is based on the design of using a Transient Receptor Potential (TRP) ion channel-based system that can be induced by energy, heat or radio waves (see, for example, http: / / www.sciencemag.org / content / 336 / 6081 / 604). These TRP family proteins respond to various stimuli, including light and heat. When the protein is activated by light or heat, the ion channel opens, allowing ions such as calcium to enter the cell membrane. The influx of ions binds to the intracellular ion interaction partner linked to the polypeptide, including the guide and Cas13 CRISPR-Cas complex or other components of the system, and this binding induces a change in the intracellular localization of the polypeptide, leading to the entry of the entire polypeptide into the nucleus of the cell. Once inside the nucleus, the guide protein and other components of the Cas13 CRISPR-Cas complex become active, regulating target gene expression in the cell.

[0116] Although light activation can be an advantageous embodiment, sometimes this can be a disadvantage, especially for in vivo applications where light may not penetrate the skin or other organs, in which case other energy activation methods are contemplated, especially electric field energy and / or ultrasound, which have similar effects.

[0117] The electric field energy is preferably administered using one or more electrical pulses of about 1 volt / cm to about 10 kVolt / cm under in vivo conditions substantially as described in the art. Instead of or in addition to pulses, the electric field may be delivered continuously. The electrical pulses may be applied for 1 μs to 500 ms, preferably 1 μs to 100 ms. The electric field may be applied continuously or in a pulsed manner for about 5 minutes.

[0118] As used herein, "electric field energy" is the electrical energy to which cells are exposed. Preferably, the electric field has a strength of about 1 volt / cm to about 10 kVolt / cm or more under in vivo conditions (see WO97 / 49450).

[0119] As used herein, the term "electric field" includes one or more pulses, including exponential and / or square and / or modulated and / or modulated square wave waveforms, of variable capacitance and voltage. References to electric fields and electricity should be interpreted as including references to the presence of potential differences in the environment of the cells. Such an environment may be set up by static electricity, alternating current (AC), direct current (DC), etc., as known in the art. The electric field may be uniform, non-uniform or otherwise, and may vary in strength and / or direction in a time-dependent manner.

[0120] Single or multiple applications of electric field, and single or multiple applications of ultrasound are also possible in any order and in any combination. Ultrasound and / or electric field may be delivered as single or multiple sequential applications, or as pulses (pulsed delivery).

[0121] Electroporation has been used in both in vitro and in vivo procedures to introduce exogenous materials into living cells. In in vitro applications, a living cell sample is first mixed with the agent of interest and placed between electrodes, such as parallel plates. The electrodes then apply an electric field to the cell / implant mixture. Examples of systems that perform in vitro electroporation include the Electro Cell Manipulator ECM600 product and the Electro Square Porator T820, both manufactured by the BTX Division of Genetronics, Inc. (see U.S. Patent No. 5,869,326).

[0122] Known electroporation techniques (both in vitro and in vivo) work by applying short high voltage pulses to electrodes positioned around the treatment area. The electric field generated between the electrodes makes the cell membrane temporarily porous, at which time molecules of the desired drug can enter the cell. In known electroporation applications, this electric field comprises a single square wave pulse of the order of 1000V / cm with a duration of about 100 μs. Such pulses can be generated, for example, in known applications of the Electro Square Porator T820.

[0123] Preferably, the electric field has a strength of about 1 V / cm to about 10 kV / cm under in vitro conditions. Thus, the electric field can have a strength of 1 V / cm, 2 V / cm, 3 V / cm, 4 V / cm, 5 V / cm, 6 V / cm, 7 V / cm, 8 V / cm, 9 V / cm, 10 V / cm, 20 V / cm, 50 V / cm, 100 V / cm, 200 V / cm, 300 V / cm, 400 V / cm, 500 V / cm, 600 V / cm, 700 V / cm, 800 V / cm, 900 V / cm, 1 kV / cm, 2 kV / cm, 5 kV / cm, 10 kV / cm, 20 kV / cm, 50 kV / cm or more. More preferably, the electric field has a strength of about 0.5 kV / cm to about 4.0 kV / cm under in vitro conditions. Preferably, the electric field has a strength of about 1 V / cm to about 10 kV / cm under in vivo conditions. However, the electric field strength may be lower if increasing the number of pulses delivered to the target site. Thus, pulsed electric field delivery at lower field strengths is envisioned.

[0124] Preferably, the application of the electric field is in the form of multiple pulses, such as double pulses of the same intensity and capacitance, or sequential pulses of varying intensity and / or capacitance. As used herein, the term "pulse" includes one or more electrical pulses, including exponential and / or square and / or modulated / modulated square wave waveforms, of variable capacitance and voltage.

[0125] Preferably, the electrical pulses are delivered as a waveform selected from an exponential waveform, a square waveform, a modulated waveform and a modulated square waveform.

[0126] In a preferred embodiment, low voltage direct current is used. Accordingly, applicants disclose the use of an electric field applied to the cell, tissue or tissue mass with a field strength of 1 V / cm to 20 V / cm for a period of 100 milliseconds or more, preferably 15 minutes or more.

[0127] Ultrasound is advantageously administered at a power level of about 0.05 W / cm2 to about 100 W / cm2. Diagnostic or therapeutic ultrasound, or a combination thereof, may be used.

[0128] As used herein, the term "ultrasonic wave" refers to a form of energy consisting of mechanical vibrations whose frequencies are high enough to exceed the range of human hearing. The lower frequency limit of the ultrasonic spectrum can generally be considered to be about 20 kHz. Many diagnostic applications of ultrasound utilize frequencies in the 1-15 MHz range (see Ultrasonics in Clinical Diagnosis, PNT Wells, ed., 2nd. Edition, Publ. Churchill Livingstone [Edinburgh, London & NY, 1977]).

[0129] Ultrasound has been used in both diagnostic and therapeutic applications. When used as a diagnostic tool ("diagnostic ultrasound"), ultrasound is typically used in the energy density range up to about 100 mW / cm2 (FDA recommended), although energy densities up to 750 mW / cm2 have been used. In physical therapy, ultrasound is typically used as an energy source in the range up to about 3-4 W / cm2 (WHO recommended). In other therapeutic applications, higher intensity ultrasound may be utilized, for example, 100 W / cm2 to 1 kW / cm2 (or even higher) for short periods in HIFU. The term "ultrasound" as used herein is intended to encompass diagnostic, therapeutic and focused ultrasound.

[0130] Focused ultrasound (FUS) allows the delivery of thermal energy without an invasive probe (see Morocz et al 1998 Journal of Magnetic Resonance Imaging Vol. 8, No. 1, pp. 136-142. Another form of focused ultrasound is high intensity focused ultrasound (HIFU), which is reviewed by Moussatov et al in Ultrasonics (1998) Vol. 36, No. 8, pp. 893-900 and TranHuuHue et al in Acustica (1997) Vol. 83, No. 6, pp. 1103-1106.

[0131] Preferably, a combination of diagnostic and therapeutic ultrasound is utilized. However, this combination is not intended to be limiting and one of ordinary skill in the art will appreciate that any of a variety of combinations of ultrasound may be used. In addition, the energy density, ultrasound frequency, and exposure time may also vary.

[0132] Preferably, the exposure to the ultrasonic energy source is at a power density of about 0.05 to about 100 Wcm-2. Even more preferably, the exposure to the ultrasonic energy source is at a power density of about 1 to about 15 Wcm-2.

[0133] Preferably, the exposure to the ultrasonic energy source is at a frequency of about 0.015 to about 10.0 MHz. More preferably, the exposure to the ultrasonic energy source is at a frequency of about 0.02 to about 5.0 MHz or about 6.0 MHz. Most preferably, the ultrasound is applied at a frequency of 3 MHz.

[0134] Preferably, the exposure is for a time period of about 10 milliseconds to about 60 minutes. Preferably, the exposure is for a time period of about 1 second to about 5 minutes. More preferably, the ultrasound is applied for about 2 minutes. However, depending on the specific target cells to be destroyed, the exposure may be for a longer duration, for example, 15 minutes.

[0135] Advantageously, the target tissue is exposed to an ultrasonic energy source at a frequency in the range of about 0.015 to about 10 MHz and an acoustic power density of about 0.05 Wcm-2 to about 10 Wcm-2 (see WO98 / 52609). However, alternative methods, such as exposure to an ultrasonic energy source at an acoustic power density of more than 100 Wcm-2, but for a shorter period of time, such as 1000 Wcm-2, for a period of time in the millisecond range or less, are also possible.

[0136] Preferably, the application of ultrasound is in the form of multiple pulses; thus, both continuous wave and pulsed wave (pulsed ultrasound delivery) may be utilized in any combination. For example, continuous wave ultrasound may be applied followed by pulsed wave ultrasound, or vice versa. This may be repeated any number of times, in any order, and in any combination. Pulsed wave ultrasound may be applied against a background of continuous wave ultrasound, and any pulse may be used in any sequence.

[0137] Preferably, the ultrasound may include pulsed ultrasound. In a highly preferred embodiment, the ultrasound is applied as a continuous wave with a power density of 0.7 Wcm-2 or 1.25 Wcm-2. If pulsed ultrasound is used, higher power densities may be used.

[0138] The use of ultrasound is advantageous because, like light, it can be precisely focused on a target. Moreover, unlike light, ultrasound is advantageous because it can be focused deeper into tissue. Thus, ultrasound is better suited for whole-tissue penetration therapy (such as, but not limited to, liver lobes) or whole-organ therapy (such as, but not limited to, the whole liver or whole muscle, e.g., the heart). Another important advantage is that ultrasound is a non-invasive stimulus used in a wide variety of diagnostic and therapeutic applications. For example, ultrasound is well known in medical imaging techniques, as well as in orthopedic therapy. Moreover, suitable equipment for applying ultrasound to a target vertebrate is widely available, and their use is well known in the art.

[0139] In certain embodiments, the guide molecule is modified with a secondary structure to increase the specificity of the CRISPR-Cas system, which can be added 5' to the guide sequence to protect against exonuclease activity, also referred to herein as a protective guide molecule.

[0140] In one aspect, the invention provides for hybridizing a "protector RNA" to a sequence of a guide molecule, the "protector RNA" being an RNA strand that is complementary to the 3' end of the guide molecule, thereby generating a partially double-stranded guide RNA. In certain embodiments of the invention, protecting mismatched bases (i.e., bases of the guide molecule that do not form part of the guide sequence) with a fully complementary protector sequence reduces the likelihood of target DNA binding to mismatched base pairs at the 3' end. In certain embodiments of the invention, an additional sequence comprising an extended length may be present in the guide molecule such that the guide comprises a protector sequence within the guide molecule. This "protector sequence" ensures that the guide molecule comprises a "protection sequence" in addition to the "exposed sequence" (which comprises the portion of the guide sequence that hybridizes to the target sequence). In certain embodiments, the guide molecule is modified by the presence of the protector guide to include a secondary structure such as a hairpin. Advantageously, there are 3 or 4 to 30 or more, e.g., about 10 or more, adjacent base pairs that have complementarity to the protection sequence, the guide sequence, or both. Advantageously, the protecting moiety does not interfere with the thermodynamics of the CRISPR-Cas system interacting with its target. By providing such an extension that includes a partially double-stranded guide molecule, the guide molecule is considered protected, resulting in improved specific binding of the CRISPR-Cas complex while maintaining specific activity.

[0141] In certain embodiments, a truncated guide is used, i.e., a guide molecule that includes a guide sequence that is shortened in length relative to the canonical guide sequence length. As described in Nowak et al. (Nucleic Acids Res (2016) 44 (20): 9555-9564), such a guide can allow a catalytically active CRISPR-Cas enzyme to bind to its target without cleaving the target DNA. In certain embodiments, a truncated guide is used that can bind to the target but only retains the nickase activity of the CRISPR-Cas enzyme.

[0142] CRISPR-Cas enzymes In its unmodified form, CRISPR-Cas protein is a catalytically active protein. This means that when forming a nucleic acid targeting complex (including a guide RNA hybridized to a target sequence), one or both DNA strands in or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from the target sequence) are modified (e.g., cleaved). As used herein, the term "sequence(s) associated with a target locus of interest" refers to a sequence near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from the target sequence, the target sequence being contained within the target locus of interest). Unmodified catalytically active Cas13 protein gene produces staggered cleavage, with the cleavage site typically being within the target sequence. More specifically, staggered cleavage is typically 13-23 nucleotides distal from the PAM. In certain embodiments, cleavage on the non-target strand is 17 nucleotides downstream of the PAM (i.e., between nucleotides 17 and 18 downstream of the PAM), while cleavage on the target strand (i.e., the strand that hybridizes to the guide sequence) occurs 4 nucleotides further from the sequence that is complementary to the PAM (this is 21 nucleotides upstream of the complement of the PAM on the 3' strand, or between nucleotides 21 and 22 upstream of the complement of the PAM).

[0143] In the methods of the invention, the CRISPR-Cas protein is preferably mutated relative to the corresponding wild-type enzyme such that the mutant CRISPR-Cas protein lacks the ability to cleave one or both DNA strands of the target locus containing the target sequence. In certain embodiments, one or more catalytic domains of the Cas13 protein are mutated to generate a mutant Cas protein that cleaves only one DNA strand of the target sequence.

[0144] In certain embodiments, the CRISPR-Cas protein may be mutated relative to the corresponding wild-type enzyme such that the mutant CRISPR-Cas protein lacks substantially all DNA cleavage activity. In some embodiments, a CRISPR-Cas protein is considered to be substantially lacking all DNA and / or RNA cleavage activity if the cleavage activity of the mutant enzyme is only about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less than the nucleic acid cleavage activity of the non-mutated form of the enzyme; an example may be when the nucleic acid cleavage activity of the mutant form is zero or negligible compared to the non-mutated form.

[0145] In certain embodiments of the methods described herein, the CRISPR-Cas protein is a mutant CRISPR-Cas protein that cleaves only one DNA strand, i.e., a nickase. More specifically, in the context of the present invention, the nickase ensures cleavage within a non-target sequence, i.e., within a sequence that is on the DNA strand opposite the target sequence and is 3' to the PAM sequence. By way of further guidance and without limitation, an arginine to alanine substitution (R1226A) in the Nuc domain of Cas13 from Acidaminococcus sp. converts Cas13 from a nuclease that cleaves both strands into a nickase (cleaves a single strand). Those skilled in the art will understand that if the enzyme is not AsCas13, a mutation can be made at the residue at the corresponding position. In certain embodiments, the Cas13 is FnCas13 and the mutation is an arginine at position R1218. In certain embodiments, the Cas13 is LbCas13 and the mutation is an arginine at position R1138. In certain embodiments, the Cas13 is MbCas13 and the mutation is an arginine at position R1293.

[0146] In certain embodiments of the methods described herein, the CRISPR-Cas protein has reduced or no catalytic activity.When the CRISPR-Cas protein is a Cas13 protein, the mutation can include, but is not limited to, one or more mutations in the catalytic RuvC-like domain, such as D908A or E993A, referring to the position in AsCas13.

[0147] In some embodiments, a CRISPR-Cas protein is considered to be substantially devoid of all DNA cleavage activity when the DNA cleavage activity of the mutant enzyme is only about 25%, 10%, 5%, 1%, 0.1%, 0.01% or less than the DNA cleavage activity of the non-mutated form of the enzyme; an example may be when the DNA cleavage activity of the mutant form is zero or negligible compared to the non-mutated form. In these embodiments, the CRISPR-Cas protein is used as a general DNA binding protein. The mutation may be an artificially introduced mutation, or a gain-of-function or loss-of-function mutation.

[0148] In addition to the above-mentioned mutations, CRISPR-Cas protein may be further modified.As used herein, the term "modified" with respect to CRISPR-Cas protein generally refers to CRISPR-Cas protein that has one or more modifications or mutations (including point mutations, truncations, insertions, deletions, chimeras, fusion proteins, etc.) compared to the wild-type Cas protein that is induced.Induction means that the induced enzyme is mainly based on the wild-type enzyme in the sense that it has a high degree of sequence homology, but is mutated (modified) in some way as known in the art or described herein.

[0149] Additional modifications of CRISPR-Cas proteins may or may not result in changes in functionality. By way of example, and with particular reference to CRISPR-Cas proteins, modifications that do not result in changes in functionality include, for example, codon optimization for expression in a particular host, or providing the nuclease with a particular marker (e.g., for visualization). Modifications that may result in changes in functionality may also include mutations, including point mutations, insertions, deletions, truncations (e.g., split nucleases), and the like. Fusion proteins may include, but are not limited to, fusions with, for example, heterologous or functional domains (e.g., localization signals, catalytic domains, and the like). In certain embodiments, various different modifications may be combined (e.g., a mutant nuclease may be catalytically inactive and may be further fused to a functional domain to induce, for example, DNA methylation, or another nucleic acid modification, including, but not limited to, disruption (e.g., by a different nuclease (domain)), mutation, deletion, insertion, substitution, ligation, degradation, disruption, or recombination). As used herein, "altered functionality" includes, but is not limited to, altered specificity (e.g., altered target recognition, increased (e.g., an "enhanced" Cas protein) or decreased specificity, or altered PAM recognition), altered activity (e.g., increased or decreased catalytic activity, including catalytically inactive nucleases or nickases), and / or altered stability (e.g., fusion with a destabilization domain). Suitable heterologous domains include, but are not limited to, nucleases, ligases, repair proteins, methyltransferases, (viral) integrases, recombinases, transposases, argonautes, cytidine deaminases, retrons, group II introns, phosphatases, phosphorylases, sulfurylases, kinases, polymerases, exonucleases, and the like. Examples of all these modifications are known in the art.It will be appreciated that the "modified" nuclease, and in particular the "modified" Cas or "modified" CRISPR-Cas system or complex as referred to herein, preferably still has the ability to interact with or bind to polynucleic acids (e.g., complexing with a guide molecule). Such modified Cas proteins can be combined with deaminase proteins or active domains thereof as described herein.

[0150] In certain embodiments, a CRISPR-Cas protein may include one or more modifications that result in enhanced activity and / or specificity, e.g., mutated residues that stabilize the targeting or non-targeting strand (e.g., eCas9; “Rationally engineered Cas9 nucleases with improved specificity”, Slaymaker et al. (2016), Science, 351(6268):84-88, which is incorporated herein by reference in its entirety). In certain embodiments, the altered or modified activity of an engineered CRISPR protein includes increased targeting efficiency or decreased off-target binding. In certain embodiments, the altered activity of an engineered CRISPR protein includes altered cleavage activity. In certain embodiments, the altered activity includes increased cleavage activity with respect to a target polynucleotide locus. In certain embodiments, the altered activity includes decreased cleavage activity with respect to a target polynucleotide locus. In certain embodiments, the altered activity includes decreased cleavage activity with respect to an off-target polynucleotide locus. In certain embodiments, the altered or modified activity of an engineered nuclease The altered activity includes a change in helicase dynamics. In certain embodiments, the modified nuclease includes a modification that changes the association of the protein with a nucleic acid molecule containing RNA (in the case of a Cas protein), or a strand of a target polynucleotide locus, or a strand of an off-target polynucleotide locus. In an aspect of the invention, the engineered CRISPR protein includes a modification that changes the formation of a CRISPR complex. In certain embodiments, the altered activity includes an increase in cleavage activity for an off-target polynucleotide locus. Thus, in certain embodiments, there is an increase in specificity for the target polynucleotide locus compared to the off-target polynucleotide locus. In other embodiments, there is a decrease in specificity for the target polynucleotide locus compared to the off-target polynucleotide locus.In certain embodiments, the mutations result in a decrease in off-target effects (e.g., cleavage or binding properties, activity, or kinetics), e.g., in the case of Cas proteins resulting in a decreased tolerance to mismatches between the target and the guide RNA. Other mutations may result in an increase in off-target effects (e.g., cleavage or binding properties, activity, or kinetics). Other mutations may result in an increase or decrease in on-target effects (e.g., cleavage or binding properties, activity, or kinetics). In certain embodiments, the mutations result in a change (e.g., an increase or decrease) in helicase activity, association, or formation of a functional nuclease complex (e.g., a CRISPR-Cas complex). In certain embodiments, as described above, the mutations result in an alteration in PAM recognition, i.e., they may recognize (in addition or instead) a different PAM compared to the unmodified Cas protein. Particularly preferred mutations include positively charged residues and / or (evolutionarily) conserved residues, such as conserved positively charged residues, to enhance specificity. In certain embodiments, such residues may be mutated to uncharged residues, such as alanine.

[0151] BasesEdit

[0152] In some embodiments, the system may be used for base editing. For example, the system disclosed herein includes a targeting component and a base editing component. The targeting component functions to specifically target the base editing component to a target nucleotide sequence where one or more nucleotides are to be edited. In some examples, the targeting component may be a catalytically inactive Cas13 effector protein (e.g., dCas13). The base editing component may then catalyze a chemical reaction to convert a first nucleotide in the target sequence to a second nucleotide. For example, a base editor may catalyze the conversion of an adenine so that it is read as a guanine by the transcription or translation machinery of the cell, or vice versa. Similarly, a base editing component may catalyze the conversion of a cytidine to uracil, or vice versa. In certain exemplary embodiments, a base editor may be derived by starting with a known base editor, such as an adenine deaminase or a cytidine deaminase, and modified using methods such as directed evolution to derive new functionality. Directed evolution techniques are known in the art and can include those described in WO2015 / 184016 "High-Throughput Assembly of Genetic Permuatations."

[0153] In certain exemplary embodiments, the binding moiety may be a DNA binding protein or a functional domain thereof, or an RNA binding domain or a functional domain thereof. The binding moiety may bind to a sequence, motif, or structural feature at or adjacent to the target locus. The structural feature may include a hairpin, tetraloop, or other secondary structural feature of a nucleic acid. As used herein, "adjacent" means within a certain distance and / or directional range of the target locus where adenosine deaminase can complete its base editing function. In certain exemplary embodiments, the binding moiety may bind to a sequence, motif, or structural sequence of a guide molecule. The guide molecule comprises a sequence that hybridizes to a target locus of interest to direct adenosine deaminase to the target locus.

[0154] Adenosine deaminase In certain exemplary embodiments, the base editing component is adenosine deaminase. The adenosine deaminase is recruited to the target locus by the targeting component. In certain exemplary embodiments, the adenosine deaminase is linked to an RNA binding protein or a functional fragment thereof, or a DNA binding protein or a functional fragment thereof.

[0155] The term "adenosine deaminase" or "adenosine deaminase protein," as used herein, refers to a protein, polypeptide, or one or more functional domains of a protein or polypeptide that can catalyze a hydrolytic deamination reaction that converts adenine (or the adenine portion of the molecule) to hypoxanthine (or the hypoxanthine portion of the molecule), as shown below. In some embodiments, the adenine-containing molecule is adenosine (A) and the hypoxanthine-containing molecule is inosine (I). The adenine-containing molecule can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). [ka]

[0156] According to the present disclosure, adenosine deaminases that can be used in connection with the present disclosure include, but are not limited to, members of the enzyme family known as adenosine deaminases acting on RNA (ADAR), members of the enzyme family known as adenosine deaminases acting on tRNA (ADAT), and other adenosine deaminase domain-containing (ADAD) family members. According to the present disclosure, adenosine deaminases can target adenine in RNA / DNA and RNA duplexes. Indeed, Zheng et al. (Nucleic Acids Res. 2017, 45(6):3369-3377) have shown that ADARs can carry out adenosine to inosine editing reactions on RNA / DNA and RNA / RNA duplexes. In certain embodiments, adenosine deaminases are modified to increase their ability to edit DNA in RNA / DNAn RNA duplexes, as detailed herein below.

[0157] In some embodiments, the adenosine deaminase is from one or more metazoan species, including, but not limited to, mammals, birds, frogs, squids, fish, flies, and nematodes, hi some embodiments, the adenosine deaminase is human, squid, or Drosophila adenosine deaminase.

[0158] In some embodiments, the adenosine deaminase is a human ADAR, such as hADAR1, hADAR2, hADAR3. In some embodiments, the adenosine deaminase is a Caenorhabditis elegans ADAR protein, such as ADR-1 and ADR-2. In some embodiments, the adenosine deaminase is a Drosophila ADAR protein, such as dAdar. In some embodiments, the adenosine deaminase is a squid Loligo pealeii ADAR protein, such as sqADAR2a and sqADAR2b. In some embodiments, the adenosine deaminase is a human ADAT protein. In some embodiments, the adenosine deaminase is a Drosophila ADAT protein. In some embodiments, the adenosine deaminase is a human ADAD protein, such as TENR (hADAD1) and TENRL (hADAD2).

[0159] In some embodiments, the adenosine deaminase protein recognizes one or more target adenosine residue(s) in a double-stranded nucleic acid substrate and converts it to an inosine residue(s). In some embodiments, the double-stranded nucleic acid substrate is an RNA-DNA hybrid duplex. In some embodiments, the adenosine deaminase protein recognizes a binding window on the double-stranded substrate. In some embodiments, the binding window includes at least one target adenosine residue(s). In some embodiments, the binding window ranges from about 3 bp to about 100 bp. In some embodiments, the binding window ranges from about 5 bp to about 50 bp. In some embodiments, the binding window ranges from about 10 bp to about 30 bp. In some embodiments, the binding window is about 1 bp, 2 bp, 3 bp, 5 bp, 7 bp, 10 bp, 15 bp, 20 bp, 25 bp, 30 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, 90 bp, 95 bp, or 100 bp.

[0160] In some embodiments, the adenosine deaminase protein comprises one or more deaminase domains. Without intending to be bound by theory, it is contemplated that the deaminase domain recognizes one or more target adenosine (A) residue(s) contained in a double-stranded nucleic acid substrate and converts it to inosine (I) residue(s). In some embodiments, the deaminase domain comprises an active center. In some embodiments, the active center comprises a zinc ion. In some embodiments, during the A→I editing process, base pairing of the target adenosine residue is disrupted and the target adenosine residue is "flipped" out of the double helix, becoming accessible to adenosine deaminase. In some embodiments, amino acid residues at or near the active center interact with one or more nucleotide(s) 5' to the target adenosine residue. In some embodiments, amino acid residues at or near the active center interact with one or more nucleotide(s) 3' to the target adenosine residue. In some embodiments, an amino acid residue at or near the active center further interacts with a nucleotide that is complementary to a target adenosine residue on the opposite strand, hi some embodiments, the amino acid residue forms a hydrogen bond with the 2' hydroxyl group of the nucleotide.

[0161] In some embodiments, the adenosine deaminase comprises the human ADAR2 complete protein (hADAR2) or its deaminase domain (hADAR2-D). In some embodiments, the adenosine deaminase is an ADAR family member that is homologous to hADAR2 or hADAR2-D.

[0162] In particular, in some embodiments, the homologous ADAR protein is human ADAR1 (hADAR1) or its deaminase domain (hADAR1-D). In some embodiments, glycine 1007 of hADAR1-D corresponds to glycine 487 hADAR2-D, and glutamic acid 1008 of hADAR1-D corresponds to glutamic acid 488 of hADAR2-D.

[0163] In some embodiments, the adenosine deaminase comprises a wild-type amino acid sequence of hADAR2-D, hi some embodiments, the adenosine deaminase comprises one or more mutations in the hADAR2-D sequence such that the editing efficiency and / or substrate editing preference of hADAR2-D is altered according to particular needs.

[0164] Certain mutations of the hADAR1 and hADAR2 proteins are described in Kuttan et al., Proc Natl Acad Sci USA. (2012) 109(48):E3295-304; Want et al. ACS Chem Biol. (2015) 10(11):2512-9; and Zheng et al. Nucleic Acids Res. (2017) 45(6):3369-337, which are incorporated by reference in their entireties.

[0165] In some embodiments, the adenosine deaminase comprises a mutation at glycine 336 of the hADAR2-D amino acid sequence, or the corresponding position of a homologous ADAR protein. In some embodiments, the glycine residue at position 336 is replaced with an aspartic acid residue (G336D).

[0166] In some embodiments, the adenosine deaminase comprises a mutation at glycine 487 of the hADAR2-D amino acid sequence, or at the corresponding position of a homologous ADAR protein. In some embodiments, the glycine residue at position 487 is replaced with an apolar amino acid residue having a relatively small side chain. For example, in some embodiments, the glycine residue at position 487 is replaced with an alanine residue (G487A). In some embodiments, the glycine residue at position 487 is replaced with a valine residue (G487V). In some embodiments, the glycine residue at position 487 is replaced with an amino acid residue having a relatively large side chain. In some embodiments, the glycine residue at position 487 is replaced with an arginine residue (G487R). In some embodiments, the glycine residue at position 487 is replaced with a lysine residue (G487K). In some embodiments, the glycine residue at position 487 is replaced with a tryptophan residue (G487W). In some embodiments, the glycine residue at position 487 is substituted with a tyrosine residue (G487Y).

[0167] In some embodiments, the adenosine deaminase comprises a mutation at glutamic acid 488 of the hADAR2-D amino acid sequence, or at the corresponding position of a homologous ADAR protein. In some embodiments, the glutamic acid residue at position 488 is replaced with a glutamine residue (E488Q). In some embodiments, the glutamic acid residue at position 488 is replaced with a histidine residue (E488H). In some embodiments, the glutamic acid residue at position 488 is replaced with an arginine residue (E488R). In some embodiments, the glutamic acid residue at position 488 is replaced with a lysine residue (E488K). In some embodiments, the glutamic acid residue at position 488 is replaced with an asparagine residue (E488N). In some embodiments, the glutamic acid residue at position 488 is replaced with an alanine residue (E488A). In some embodiments, the glutamic acid residue at position 488 is substituted with a methionine residue (E488M). In some embodiments, the glutamic acid residue at position 488 is substituted with a serine residue (E488S). In some embodiments, the glutamic acid residue at position 488 is substituted with a phenylalanine residue (E488F). In some embodiments, the glutamic acid residue at position 488 is substituted with a lysine residue (E488L). In some embodiments, the glutamic acid residue at position 488 is substituted with a tryptophan residue (E488W).

[0168] In some embodiments, the adenosine deaminase comprises a mutation at threonine 490 of the hADAR2-D amino acid sequence, or at the corresponding position of a homologous ADAR protein. In some embodiments, the threonine residue at position 490 is replaced with a cysteine ​​residue (T490C). In some embodiments, the threonine residue at position 490 is replaced with a serine residue (T490S). In some embodiments, the threonine residue at position 490 is replaced with an alanine residue (T490A). In some embodiments, the threonine residue at position 490 is replaced with a phenylalanine residue (T490F). In some embodiments, the threonine residue at position 490 is replaced with a tyrosine residue (T490Y). In some embodiments, the threonine residue at position 490 is replaced with a serine residue (T490R). In some embodiments, the threonine residue at position 490 is replaced with an alanine residue (T490K). In some embodiments, the threonine residue at position 490 is substituted with a phenylalanine residue (T490P).In some embodiments, the threonine residue at position 490 is substituted with a tyrosine residue (T490E).

[0169] In some embodiments, the adenosine deaminase comprises a mutation at valine 493 of the hADAR2-D amino acid sequence, or at the corresponding position of a homologous ADAR protein. In some embodiments, the valine residue at position 493 is substituted with an alanine residue (V493A). In some embodiments, the valine residue at position 493 is substituted with a serine residue (V493S). In some embodiments, the valine residue at position 493 is substituted with a threonine residue (V493T). In some embodiments, the valine residue at position 493 is substituted with an arginine residue (V493R). In some embodiments, the valine residue at position 493 is substituted with an aspartic acid residue (V493D). In some embodiments, the valine residue at position 493 is substituted with a proline residue (V493P). In some embodiments, the valine residue at position 493 is substituted with a glycine residue (V493G).

[0170] In some embodiments, the adenosine deaminase comprises a mutation at alanine 589 of the hADAR2-D amino acid sequence, or the corresponding position of a homologous ADAR protein. In some embodiments, the alanine residue at position 589 is replaced with a valine residue (A589V).

[0171] In some embodiments, the adenosine deaminase comprises a mutation at asparagine 597 of the hADAR2-D amino acid sequence, or at the corresponding position of a homologous ADAR protein. In some embodiments, the asparagine residue at position 597 is replaced with a lysine residue (N597K). In some embodiments, the adenosine deaminase comprises a mutation at position 597 of the amino acid sequence having an asparagine residue in the wild-type sequence. In some embodiments, the asparagine residue at position 597 is replaced with an arginine residue (N597R). In some embodiments, the adenosine deaminase comprises a mutation at position 597 of the amino acid sequence having an asparagine residue in the wild-type sequence. In some embodiments, the asparagine residue at position 597 is replaced with an alanine residue (N597A). In some embodiments, the adenosine deaminase comprises a mutation at position 597 of the amino acid sequence having an asparagine residue in the wild-type sequence. In some embodiments, the asparagine residue at position 597 is substituted with a glutamic acid residue (N597E). In some embodiments, the adenosine deaminase comprises a mutation at position 597 of an amino acid sequence that has an asparagine residue in the wild-type sequence. In some embodiments, the asparagine residue at position 597 is substituted with a histidine residue (N597H). In some embodiments, the adenosine deaminase comprises a mutation at position 597 of an amino acid sequence that has an asparagine residue in the wild-type sequence. In some embodiments, the asparagine residue at position 597 is substituted with a glycine residue (N597G). In some embodiments, the adenosine deaminase comprises a mutation at position 597 of an amino acid sequence that has an asparagine residue in the wild-type sequence. In some embodiments, the asparagine residue at position 597 is substituted with a tyrosine residue (N597Y). In some embodiments, the asparagine residue at position 597 is substituted with a phenylalanine residue (N597F).

[0172] In some embodiments, the adenosine deaminase comprises a mutation at serine 599 of the hADAR2-D amino acid sequence, or the corresponding position of a homologous ADAR protein. In some embodiments, the serine residue at position 599 is replaced with a threonine residue (S599T).

[0173] In some embodiments, the adenosine deaminase comprises a mutation at asparagine 613 of the hADAR2-D amino acid sequence, or at the corresponding position of a homologous ADAR protein. In some embodiments, the asparagine residue at position 613 is replaced with a lysine residue (N613K). In some embodiments, the adenosine deaminase comprises a mutation at position 613 of an amino acid sequence that has an asparagine residue in the wild-type sequence. In some embodiments, the asparagine residue at position 613 is replaced with an arginine residue (N613R). In some embodiments, the adenosine deaminase comprises a mutation at position 613 of an amino acid sequence that has an asparagine residue in the wild-type sequence. In some embodiments, the asparagine residue at position 613 is replaced with an alanine residue (N613A). In some embodiments, the adenosine deaminase comprises a mutation at position 613 of an amino acid sequence that has an asparagine residue in the wild-type sequence. In some embodiments, the asparagine residue at position 613 is substituted with a glutamic acid residue (N613E).

[0174] In some embodiments, to improve editing efficiency, the adenosine deaminase may include one or more of the following mutations based on the amino acid sequence positions of hADAR2-D: G336D, G487A, G487V, E488Q, E488H, E488R, E488N, E488A, E488S, E488M, T490C, T490S, V493T, V493S, V493A, V493R, V493D, V493P, V493G, N597K, N597R, N597A, N597E, N597H, N597G, N597Y, A589V, S599T, N613K, N613R, N613A, N613E, and the corresponding mutations of homologous ADAR proteins.

[0175] In some embodiments, to reduce editing efficiency, adenosine deaminase may include one or more of the following mutations based on the amino acid sequence positions of hADAR2-D: E488F, E488L, E488W, T490A, T490F, T490Y, T490R, T490K, T490P, T490E, N597F, and the corresponding mutations of homologous ADAR proteins. In certain embodiments, it may be beneficial to use adenosine deaminase enzymes with reduced efficacy to reduce off-target effects.

[0176] In some embodiments, to reduce off-target effects, the adenosine deaminase comprises one or more of the following mutations based on the amino acid sequence positions of hADAR2-D: R348, V351, T375, K376, E396, C451, R455, N473, R474, K475, R477, R481, S486, E488, T490, S495, R510, and the corresponding mutations of homologous ADAR proteins. In some embodiments, the adenosine deaminase comprises mutations at one or more additional positions selected from E488, and R348, V351, T375, K376, E396, C451, R455, N473, R474, K475, R477, R481, S486, T490, S495, R510. In some embodiments, the adenosine deaminase comprises a mutation at T375, and optionally at one or more additional positions. In some embodiments, the adenosine deaminase comprises a mutation at N473, and optionally at one or more additional positions. In some embodiments, the adenosine deaminase comprises a mutation at V351, and optionally at one or more additional positions. In some embodiments, the adenosine deaminase comprises a mutation at E488 and T375, and optionally at one or more additional positions. In some embodiments, the adenosine deaminase comprises a mutation at E488 and N473, and optionally at one or more additional positions. In some embodiments, the adenosine deaminase comprises a mutation at E488 and V351, and optionally at one or more additional positions. In some embodiments, the adenosine deaminase comprises a mutation at E488, and one or more of T375, N473, and V351.

[0177] In some embodiments, to reduce off-target effects, the adenosine deaminase comprises one or more mutations selected from R348E, V351L, T375G, T375S, R455G, R455S, R455E, N473D, R474E, K475Q, R477E, R481E, S486T, E488Q, T490A, T490S, S495T, and R510E based on the amino acid sequence positions of hADAR2-D, and the corresponding mutations of homologous ADAR proteins. In some embodiments, the adenosine deaminase comprises a mutation E488Q and one or more additional mutations selected from R348E, V351L, T375G, T375S, R455G, R455S, R455E, N473D, R474E, K475Q, R477E, R481E, S486T, T490A, T490S, S495T, and R510E. In some embodiments, the adenosine deaminase comprises a mutation T375G or T375S, and optionally one or more additional mutations. In some embodiments, the adenosine deaminase comprises a mutation N473D, and optionally one or more additional mutations. In some embodiments, the adenosine deaminase comprises a mutation V351L, and optionally one or more additional mutations. In some embodiments, the adenosine deaminase comprises mutations E488Q and T375G or T375G, and optionally one or more additional mutations. In some embodiments, the adenosine deaminase comprises mutations E488Q and N473D, and optionally one or more additional mutations. In some embodiments, the adenosine deaminase comprises mutations E488Q and V351L, and optionally one or more additional mutations. In some embodiments, the adenosine deaminase comprises mutations E488Q and one or more of T375G / S, N473D, and V351L.

[0178] In certain embodiments, improved editing and reduction of off-target modifications are achieved by chemical modification of gRNA. Chemically modified gRNAs, as exemplified by Vogel et al. (2014), Angew Chem Int Ed, 53:6267-6271, doi:10.1002 / anie.201402634 (incorporated herein by reference in its entirety), reduce off-target activity and improve on-target efficiency. 2'-O-methyl and phosphothioate modified guide RNAs generally improve editing efficiency in cells.

[0179] ADARs are known to exhibit preferences for adjacent nucleotides on either side of the edited A (www.nature.com / nsmb / journal / v23 / n5 / full / nsmb.3203.html, Matthews et al. (2017), Nature Structural Mol Biol, 23(5):426-433, which is incorporated herein by reference in its entirety). Thus, in certain embodiments, gRNAs, targets, and / or ADARs are selected to optimize motif preference.

[0180] Intentional mismatches have been demonstrated in vitro to enable editing of unfavorable motifs (https: / / academic.oup.com / nar / article-lookup / doi / 10.1093 / nar / gku272; Schneider et al (2014), Nucleic Acid Res, 42(10):e87; Fukuda et al. (2017), Scientific Reports, 7, doi:10.1038 / srep41478, which are incorporated herein by reference in their entirety). Thus, in certain embodiments, intentional mismatches are introduced in the flanking bases to enhance RNA editing efficiency on unfavorable 5' or 3' flanking bases.

[0181] The results suggest that AC in the targeting window of ADAR deaminase domain is preferentially edited over other bases. In addition, AU base pairs within a few bases of the targeted base show low-level editing by Cas13b-ADAR fusion, suggesting that the enzyme has the flexibility to edit multiple A. See, for example, Figure 18. These two observations suggest that multiple A in the activity window of Cas13b-ADAR fusion can be made specific for editing by mismatching all A to be edited with C. Thus, in certain embodiments, multiple A:C mismatches in the activity window are designed to generate multiple A:I edits. In certain embodiments, non-target A is paired with A or G to suppress potential off-target editing in the activity window.

[0182] The terms "editing specificity" and "editing preference" are used interchangeably herein to refer to the degree of A→I editing at a particular adenosine site in a double-stranded substrate. In some embodiments, substrate editing preference is determined by the 5'-most and / or 3'-most neighbors of the target adenosine residue. In some embodiments, the adenosine deaminase has a preference for the 5'-most neighbors of substrates ranked as U>A>C>G (">" indicates greater preference). In some embodiments, the adenosine deaminase has a preference for the 3'-most neighbors of substrates ranked as G>C to A>U (">" indicates greater preference; "~" indicates similar preference). In some embodiments, the adenosine deaminase has a preference for the 3'-most neighbors of substrates ranked as G>C>U to A (">" indicates greater preference; "~" indicates similar preference). In some embodiments, the adenosine deaminase has a preference for the 3' nearest neighbor of a substrate ranked as G>C>A>U (">" indicates greater preference). In some embodiments, the adenosine deaminase has a preference for the 3' nearest neighbor of a substrate ranked as C~G~A>U (">" indicates greater preference; "~" indicates similar preference). In some embodiments, the adenosine deaminase has a preference for triplet sequences that include target adenosine residues ranked as TAG>AAG>CAC>AAT>GAA>GAC (">" indicates greater preference), where the central A is the target adenosine residue.

[0183] In some embodiments, the substrate editing preference of adenosine deaminase is affected by the presence or absence of a nucleic acid binding domain in adenosine deaminase protein. In some embodiments, the deaminase domain is linked with a double-stranded RNA binding domain (dsRBD) or double-stranded RNA binding motif (dsRBM) to modify the substrate editing preference. In some embodiments, the dsRBD or dsRBM can be derived from an ADAR protein, such as hADAR1 or hADAR2. In some embodiments, a full-length ADAR protein is used that comprises at least one dsRBD and a deaminase domain. In some embodiments, one or more dsRBM or dsRBD is N-terminal to the deaminase domain. In other embodiments, one or more dsRBM or dsRBD is C-terminal to the deaminase domain.

[0184] In some embodiments, the substrate editing preference of adenosine deaminase is influenced by amino acid residues near or at the active center of the enzyme.In some embodiments, to modify the substrate editing preference, adenosine deaminase can include one or more of the following mutations based on the amino acid sequence positions of hADAR2-D: G336D, G487R, G487K, G487W, G487Y, E488Q, E488N, T490A, V493A, V493T, V493S, N597K, N597R, A589V, S599T, N613K, N613R, and the corresponding mutations of homologous ADAR proteins.

[0185] In particular, in some embodiments, to reduce editing specificity, adenosine deaminase may include one or more of the following mutations based on the amino acid sequence positions of hADAR2-D: E488Q, V493A, N597K, N613K, and the corresponding mutations of homologous ADAR proteins. In some embodiments, to increase editing specificity, adenosine deaminase may include the mutation T490A.

[0186] In some embodiments, to increase editing preference for a target adenosine (A) with an immediate 5' G, such as substrates containing the triplet sequence GAC (where the central A is the target adenosine residue), the adenosine deaminase can include one or more of the following mutations based on the amino acid sequence positions of hADAR2-D: G336D, E488Q, E488N, V493T, V493S, V493A, A589V, N597K, N597R, S599T, N613K, N613R, and the corresponding mutations of homologous ADAR proteins.

[0187] In particular, in some embodiments, the adenosine deaminase contains the mutation E488Q, or the corresponding mutation in a homologous ADAR protein, and edits substrates containing the following triplet sequences: GAC, GAA, GAU, GAG, CAU, AAU, UAC, where the central A is the target adenosine residue.

[0188] In some embodiments, the adenosine deaminase comprises a wild-type amino acid sequence of hADAR1-D. In some embodiments, the adenosine deaminase comprises one or more mutations in the hADAR1-D sequence such that the editing efficiency and / or substrate editing preference of hADAR1-D is altered according to specific needs.

[0189] In some embodiments, the adenosine deaminase comprises a mutation at glycine 1007 of the hADAR1-D amino acid sequence, or at the corresponding position of a homologous ADAR protein. In some embodiments, the glycine residue at position 1007 is replaced with an apolar amino acid residue having a relatively small side chain. For example, in some embodiments, the glycine residue at position 1007 is replaced with an alanine residue (G1007A). In some embodiments, the glycine residue at position 1007 is replaced with a valine residue (G1007V). In some embodiments, the glycine residue at position 1007 is replaced with an amino acid residue having a relatively large side chain. In some embodiments, the glycine residue at position 1007 is replaced with an arginine residue (G1007R). In some embodiments, the glycine residue at position 1007 is replaced with a lysine residue (G1007K). In some embodiments, the glycine residue at position 1007 is replaced with a tryptophan residue (G1007W). In some embodiments, the glycine residue at position 1007 is substituted with a tyrosine residue (G1007Y). Additionally, in other embodiments, the glycine residue at position 1007 is substituted with a leucine residue (G1007L). In other embodiments, the glycine residue at position 1007 is substituted with a threonine residue (G1007T). In other embodiments, the glycine residue at position 1007 is substituted with a serine residue (G1007S).

[0190] In some embodiments, the adenosine deaminase comprises a mutation at glutamic acid 1008 of the hADAR1-D amino acid sequence, or at the corresponding position of a homologous ADAR protein. In some embodiments, the glutamic acid residue at position 1008 is substituted with a polar amino acid residue having a relatively large side chain. In some embodiments, the glutamic acid residue at position 1008 is substituted with a glutamine residue (E1008Q). In some embodiments, the glutamic acid residue at position 1008 is substituted with a histidine residue (E1008H). In some embodiments, the glutamic acid residue at position 1008 is substituted with an arginine residue (E1008R). In some embodiments, the glutamic acid residue at position 1008 is substituted with a lysine residue (E1008K). In some embodiments, the glutamic acid residue at position 1008 is substituted with a non-polar or small polar amino acid residue. In some embodiments, the glutamic acid residue at position 1008 is substituted with a phenylalanine residue (E1008F). In some embodiments, the glutamic acid residue at position 1008 is substituted with a tryptophan residue (E1008W). In some embodiments, the glutamic acid residue at position 1008 is substituted with a glycine residue (E1008G). In some embodiments, the glutamic acid residue at position 1008 is substituted with an isoleucine residue (E1008I). In some embodiments, the glutamic acid residue at position 1008 is substituted with a valine residue (E1008V). In some embodiments, the glutamic acid residue at position 1008 is substituted with a proline residue (E1008P). In some embodiments, the glutamic acid residue at position 1008 is substituted with a serine residue (E1008S). In other embodiments, the glutamic acid residue at position 1008 is substituted with an asparagine residue (E1008N). In other embodiments, the glutamic acid residue at position 1008 is substituted with an alanine residue (E1008A). In other embodiments, the glutamic acid residue at position 1008 is substituted with a methionine residue (E1008M). In some embodiments, the glutamic acid residue at position 1008 is substituted with a leucine residue (E1008L).

[0191] In some embodiments, to improve editing efficiency, the adenosine deaminase may include one or more of the following mutations based on the amino acid sequence positions of hADAR1-D: E1007S, E1007A, E1007V, E1008Q, E1008R, E1008H, E1008M, E1008N, E1008K, and the corresponding mutations of homologous ADAR proteins.

[0192] In some embodiments, to reduce editing efficiency, the adenosine deaminase may include one or more of the following mutations based on the amino acid sequence positions of hADAR1-D: E1007R, E1007K, E1007Y, E1007L, E1007T, E1008G, E1008I, E1008P, E1008V, E1008F, E1008W, E1008S, E1008N, E1008K, and the corresponding mutations of homologous ADAR proteins.

[0193] In some embodiments, the substrate editing preference, efficiency and / or selectivity of adenosine deaminase is influenced by amino acid residues near or at the active center of the enzyme.In some embodiments, adenosine deaminase comprises a mutation at glutamic acid 1008 in the hADAR1-D sequence, or at the corresponding position of a homologous ADAR protein.In some embodiments, the mutation is E1008R, or the corresponding mutation of a homologous ADAR protein.In some embodiments, the E1008R mutant has increased editing efficiency of the target adenosine residue with a mismatched G residue on the opposite strand.

[0194] In some embodiments, the adenosine deaminase protein further comprises or is linked to one or more double-stranded RNA (dsRNA) binding motifs (dsRBM) or domains (dsRBD) for recognizing and binding to double-stranded nucleic acid substrates. In some embodiments, the interaction between adenosine deaminase and double-stranded substrates is mediated by one or more additional protein factor(s), such as CRISPR / CAS protein factors. In some embodiments, the interaction between adenosine deaminase and double-stranded substrates is further mediated by one or more nucleic acid component(s), such as guide RNA.

[0195] In certain exemplary embodiments, directed evolution methods can be used to design modified ADAR proteins that can catalyze additional reactions in addition to deamination of adenine to hypoxanthine. For example,

[0196] According to the present invention, the substrate of adenosine deaminase is an RNA / DNAn RNA duplex formed when the guide molecule binds to its DNA target, which then forms a CRISPR-Cas complex with the CRISPR-Cas enzyme. The RNA / DNA or DNA / RNAn RNA duplex is also referred to herein as an "RNA / DNA hybrid", "DNA / RNA hybrid" or "double-stranded substrate". The specific features of the guide molecule and the CRISPR-Cas enzyme are detailed below.

[0197] The term "editing selectivity" as used herein refers to the fraction of all sites on a double-stranded substrate that are edited by adenosine deaminase. Without being bound by theory, it is contemplated that the editing selectivity of adenosine deaminase is influenced by the length and secondary structure of the double-stranded substrate, such as the presence of mismatched bases, bulges, and / or internal loops.

[0198] In some embodiments, when the substrate is a fully base-paired duplex longer than 50 bp, adenosine deaminase may be able to deaminate a large number of adenosine residues within the duplex (e.g., 50% of all adenosine residues). In some embodiments, when the substrate is shorter than 50 bp, the editing selectivity of adenosine deaminase is affected by the presence of mismatches at the target adenosine site. In particular, in some embodiments, adenosine (A) residues with mismatched cytidine (C) residues on the opposite strand are deaminated with high efficiency. In some embodiments, adenosine (A) residues with mismatched guanosine (G) residues on the opposite strand are skipped without editing.

[0199] RNA editing by ADAR1 / ADAR2

[0200] Genetic diseases that have a G→A change (in the transcribed / coding region / splicing sequence) can be treated by the systems and methods disclosed herein.

[0201] ADAR mutates adenosine (A) to inosine (I), which is processed by the cellular translation machinery as guanosine (G). In some embodiments, RNA editing with dCas13-ADAR fusions can be used to reverse the effect of genetic mutations in transcripts that contain pathogenic G→A changes. ADAR RNA editing can occur in double-stranded RNA, and the edited base is opposite U or C. Cas13b-ADAR (e.g., dCas13b-ADAR) RNA editing can be achieved using guide-RNA to create a double-stranded RNA structure to genetically fuse ADAR to death-type Cas13b.

[0202] Of all premature stop pathogenic mutations in ClinVar, 985 of 9135 (10.7%) are G→A mutations and are potential candidates for correction by the Cas13-ADAR fusion editor. Of all pathogenic mutations in ClinVar, 15531 of 97941 (15.8%) are G→A mutations.

[0203] Examples of diseases with G>A mutations that can be treated by the systems and methods herein include Meyer-Gorlin syndrome, Seckel syndrome4, Joubert syndrome5, Leber congenital amaurosis10, Charcot-Marie-Tooth disease type 2, leukoencephalopathy, Usher syndrome type 2C, spinocerebellar ataxia28, glycogen storage disease type III, primary hyperoxaluria type I, extended QT syndrome 2, Sjogren-Larsson syndrome, hereditary fructosuria, neuroblastoma, amyotrophic lateral sclerosis type 9, Kallmann syndrome1, limb-girdle muscular dystrophy type 2L, familial adenomatous polyposis1, familial hyperlipoproteinemia type 3, Alzheimer's disease type 1, and metachromatic leukodystrophy.

[0204] The following diseases can also be treated by the systems and methods herein.

[0205] Premature termination disorders

[0206] Premature termination disease is characterized by mutations in premature termination codons, either through single nucleotide polymorphisms that introduce terminations, indels that change the translation frame of proteins and generate new termination codons, or alternative splicing that preferentially introduces exons with premature terminations.By removing the termination codons generated by these methods through A→I editing, RNA editing by the system and method of the present specification by ADAR can repair diseases with premature termination.When SNP is not G→A but generates nonsense mutations, some clinical benefits can be obtained by changing nonsense mutations to missense mutations.

[0207] Sterile mutation changes without germline editing

[0208] One advantage of RNA editing over DNA editing is that in the case of SNPs that affect fertility, correction by genome editing would entail germline editing, with potential ethical or safety implications. RNA editing by the systems and methods herein can avoid these issues by correcting these mutations without permanently affecting the genome.

[0209] Splicing Alterations

[0210] Pre-mRNA requires specific splice donor and acceptor sequences to undergo processing by the spliceosome. The splice acceptor site contains an invariant AG sequence that is necessary for acceptance of attack by the splice donor sequence and for intron removal. By targeting Cas13b-ADAR fusions to pre-mRNA and editing the AG splice acceptor site to IG, the splice acceptor site can be inactivated, resulting in skipping of downstream exons. This approach to splicing alteration has advantages over the current method of exon skipping with chemically modified antisense oligos. Cas13b-ADAR is genetically encoded, allowing long-term exon skipping. In addition, Cas13b-ADAR can generate mutations to promote skipping, which is likely to be more robust than masking splice donor / acceptor sites with double-stranded RNA, as is done with antisense oligos.

[0211] Neoantigens

[0212] Neoantigens in cancer are new antigens expressed in tumor cells due to mutations caused by mismatch repair deficiency. Engineering T cells against neoantigens is advantageous because the antigen is only expressed in tumor cells, so there is no off-target activity and therefore no toxicity to T cells. By RNA editing according to the system and method described herein, Cas13-ADAR fusions can target cancer cells and introduce mutations into transcripts. This can introduce amino acid changes and new antigens that can be targeted using chimeric antigen receptor T cells. This approach is better than DNA base editors because it is transient and the risk of permanently editing non-tumor cells due to off-target delivery is minimized.

[0213] Alterations in microRNA targets (tumor suppressors)

[0214] ADAR naturally edits mRNA to generate or remove microRNA targets, thereby regulating expression. Programmable RNA editing can be used to up-regulate or down-regulate microRNA targets through changes in targeting regions. In addition, microRNAs themselves are natural substrates for ADAR, and programmable RNA editing of microRNAs can reduce or enhance the function on their corresponding targets.

[0215] Create many edits along the region (many mismatches in the guides)

[0216] Cas13-ADAR fusions can be precisely targeted to edit specific adenosines by introducing mismatches in the guide region from the desired adenosine target, generating a bubble suitable for A→I editing. By introducing multiple of these mismatches across different adenosine sites in the guide / target duplex, multiple mutations can be introduced at once.

[0217] TAA (double A to G) inversion for PTC

[0218] Many diseases with premature stop codon changes involve the TAA stop codon and require an A→I change for correction, rather than a TAG or TGA stop codon, which only require one A→I edit. Two approaches can be used to invert the TAA stop codon. (1) As described herein, two mismatches can be introduced in the guide to the two adenosines in the TAA codon. (2) A two-guide array can be used to sequentially convert each of the adenosines to inosine. The first guide of the array can contain a mutation to the first adenosine, and then the second guide can have the complement of this change and a mismatch to the second adenosine in the stop codon.

[0219] Cancer (GOF, LOF mutation reversal)

[0220] Many oncogenic changes in cancer involve G→A mutations that introduce gain-of-function or loss-of-function phenotypes into mutant proteins. The RNA editing systems and methods herein can be well positioned to correct these changes and reduce oncogenicity.

[0221] Designing new base preferences

[0222] Current ADAR1 / 2 proteins are known to have peripheral base preferences for catalytic activity (pubs.acs.org / doi / 10.1021 / acschembio.5b00711), which may pose limitations for certain applications. Rational mutagenesis or directed evolution of ADAR variants with altered or relaxed base preferences could increase the versatility of programmable RNA editing.

[0223] Available ADAR orthologs

[0224] A variety of ADAR orthologs can be used in the methods and systems herein. Examples of ADAR orthologs that can be used include: TIFF2025023878000003.tif191170

[0225] Note that the above orthologs are listed with hyperactive E→Q mutations. Cephalopod orthologs can be used. Cephalopods are known to have very high rates of RNA editing, so these orthologs may have higher activity or altered base preference than the human variants.

[0226] The alignment tree of these orthologs is shown in Figure 74.

[0227] ADAR mutants with increased activity in human cells.ADAR mutants with altered activity in vitro or in yeast have been previously reported.The present disclosure further provides for screening or rational design of mutants with increased activity in the context of human cells, which can improve the efficiency or specificity of ADAR-based programmable RNA editing system or construct.

[0228] Biological application of inosine generation. ADAR RNA editing generates inosine, which, when it occurs multiple times in transcripts, can interact with endogenous biological pathways to increase inflammation in cells and tissues. The generation of multiple inosine bases can increase inflammation, especially in cells where inflammation can lead to clearance. Additional inosine generation can also be used to destabilize transcripts.

[0229] The upstream start codon is removed to promote protein expression (ATG mutation) of the downstream ORF. Antisense oligos can be used to block the upstream start codon site to promote protein expression at the downstream start codon. This can allow boosting of endogenous protein levels for therapeutic purposes. Cas13-ADAR fusions can achieve a similar effect by converting the ATG site to an ITG (GTG) site, thereby removing the upstream codon in the endogenous transcript and boosting protein translation. This can be an application that allows boosting of gene expression. An example is boosting fetal hemoglobin levels in sickle cell disease and thalassemia.

[0230] Mutagenesis of ADARs for C→U or any transition. ADARs (e.g., those described in the orthologs section) can be made into C→U editors or editors of any base transition through rational mutagenesis or directed evolution methods.

[0231] APOBEC RNA editing

[0232] C→U conversion. The system can include Cas13b-APOBEC fusions (e.g., dCas13b-APOBEC fusions). These fusions allow C→U editing of RNA. APOBEC substrates are ssRNA, and these systems can target regions of RNA around the guide / target duplex.

[0233] C→U knockdown via stop codon introduction. In addition to correcting pathogenic U→C mutations that arise during the cell cycle, Cas13-APOBEC fusions can introduce stop codons by converting CAA, CGA, or CAG to TAA, TGA, or TAG, respectively.

[0234] APOBEC orthologs. APOBEC orthologs can be screened using the system. Screening for additional APOBEC orthologs fused to Cas13 (e.g., dCas13) can increase the efficiency of C→U editing or allow additional types of base conversion.

[0235] Mutation of APOBEC for dsRNA / base flip / increased activity. RNA insertion / deletion by editosome. The system can further include an RNA editosome (e.g., an RNA editosome fused to Cas13, such as dCas13). Such a system can guide targeted deletion or targeted insertion of sequences into transcripts.

[0236] References

[0237] Human SNPs resulting in premature stop codons and protein truncation.Hum Genomics.2006;2(5):274-286.

[0238] The genetics of human infertility by functional interrogation of SNPs in mice.PNAS August 18,2015 112(33)10431-10436

[0239] microRNA editing in seed region aligns with cellular changes in hypoxic conditions.Nucleic Acids Research,Volume 44,Issue 13,27 July 2016,Pages 6298-6308

[0240] RNA Editing Modulates Human Hepatic Aryl Hydrocarbon Receptor Expression by Creating MicroRNA Recognition Sequence.January 8,2016,The Journal of Biological Chemistry,291,894-903.

[0241] Mechanistic insights into editing-site specificity of ADARs.PNAS November 27,2012 109(48)E3295-E3304.

[0242] A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase Acting on RNA 1.ACS Chem.Biol.,2015、10(11),pp 2512-2519.

[0243] Inosine-Containing RNA Is a Novel Innate Immune Recognition Element and Reduces RSV Infection.PLoS One.2011;6(10):e26463.

[0244] Inosine-Mediated Modulation of RNA Sensing by Toll-Like Receptor 7 (TLR7) and TLR8.J Virol.2014 Jan;88(2):799-810.

[0245] Specific cleavage of hyper-edited dsRNAs.The EMBO Journal(2001)20,4243-4252.

[0246] Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames.Nature Biotechnology volume 34,pages 875-880(2016).

[0247] The mechanism of U insertion / deletion RNA editingin kinetoplastid mitochondria.Nucleic Acids Res.1997 Oct 1;25(19):3751-3759.

[0248] Functions and Regulation of RNA Editing by ADAR Deaminases.Annual Review of Biochemistry,Vol.79:321-349(Volume publication date 7 / 7 / 2010).

[0249] Trade-off between Transcriptome Plasticity and Genome Evolution in Cephalopods.Cell.Volume 169,Issue 2,6 April 2017,Pages 191-202.e11.

[0250] An adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U deamination of DNA.PNAS May 8,2007 104(19)7821-7826.

[0251] シチジンデアミナーゼ The term "cytidine deaminase" or "cytidine deaminase protein" as used herein refers to a protein, polypeptide, or one or more functional domains of a protein or polypeptide capable of catalyzing a hydrolytic deamination reaction that converts cytosine (or the cytosine portion of a molecule) to uracil (or the uracil portion of a molecule), as shown below. In some embodiments, the cytosine-containing molecule is cytidine (C) and the uracil-containing molecule is uridine (U). The cytosine-containing molecule can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). [ka]

[0252] In accordance with the present disclosure, cytidine deaminases that can be used in connection with the present disclosure include, but are not limited to, members of the enzyme family known as apolipoprotein B mRNA-editing complex (APOBEC) family deaminases, activation-induced deaminases (AIDs), or cytidine deaminase 1 (CDA1). In certain embodiments, the deaminase is APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, and APOBEC3D deaminase, APOBEC3E deaminase, APOBEC3F deaminase, APOBEC3G deaminase, APOBEC3H deaminase, or APOBEC4 deaminase.

[0253] In the methods and systems of the present invention, the cytidine deaminase can target cytosines in a single strand of DNA. In certain exemplary embodiments, the cytidine deaminase can edit a single strand that exists outside of a binding component, e.g., a bound Cas13. In other exemplary embodiments, the cytidine deaminase can edit at a localization bubble, e.g., a localization bubble formed by a mismatch of the target editing site rather than the guide sequence. In certain exemplary embodiments, the cytidine deaminase can include mutations that serve to focus activity, such as those disclosed in Kim et al., Nature Biotechnology (2017) 35(4):371-377 (doi:10.1038 / nbt.3803.

[0254] In some embodiments, the cytidine deaminase is from one or more metazoan species, including but not limited to mammals, birds, frogs, squids, fish, flies, and nematodes, hi some embodiments, the cytidine deaminase is human, primate, bovine, canine, rat, or mouse cytidine deaminase.

[0255] In some embodiments, the cytidine deaminase is a human APOBEC, such as hAPOBEC1 or hAPOBEC3. In some embodiments, the cytidine deaminase is human AID.

[0256] In some embodiments, the cytidine deaminase protein recognizes one or more target cytosine residue(s) in the single-stranded bubble of the RNA duplex and converts it to uracil residue(s). In some embodiments, the cytidine deaminase protein recognizes a binding window on the single-stranded bubble of the RNA duplex. In some embodiments, the binding window comprises at least one target cytosine residue(s). In some embodiments, the binding window ranges from about 3 bp to about 100 bp. In some embodiments, the binding window ranges from about 5 bp to about 50 bp. In some embodiments, the binding window ranges from about 10 bp to about 30 bp. In some embodiments, the binding window is about 1 bp, 2 bp, 3 bp, 5 bp, 7 bp, 10 bp, 15 bp, 20 bp, 25 bp, 30 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, 90 bp, 95 bp, or 100 bp.

[0257] In some embodiments, the cytidine deaminase protein comprises one or more deaminase domains. Without intending to be bound by theory, it is contemplated that the deaminase domain functions to recognize one or more target cytosine (C) residue(s) contained in a single-stranded bubble of an RNA duplex and convert it to a uracil (U) residue(s). In some embodiments, the deaminase domain comprises an active center. In some embodiments, the active center comprises a zinc ion. In some embodiments, amino acid residues at or near the active center interact with the target cytosine residue from one or more nucleotide(s) 5'. In some embodiments, amino acid residues at or near the active center interact with the target cytosine residue from one or more nucleotide(s) 3'.

[0258] In some embodiments, the cytidine deaminase comprises human APOBEC1 full protein (hAPOBEC1), or its deaminase domain (hAPOBEC1-D), or its C-terminal truncated form (hAPOBEC-T). In some embodiments, the cytidine deaminase is an APOBEC family member that is homologous to hAPOBEC1, hAPOBEC-D, or hAPOBEC-T. In some embodiments, the cytidine deaminase comprises human AID1 full protein (hAID), or its deaminase domain (hAID-D), or its C-terminal truncated form (hAID-T). In some embodiments, the cytidine deaminase is an AID family member that is homologous to hAID, hAID-D, or hAID-T. In some embodiments, the hAID-T is hAID that is C-terminally truncated by about 20 amino acids.

[0259] In some embodiments, the cytidine deaminase comprises a wild-type amino acid sequence of a cytosine deaminase, In some embodiments, the cytidine deaminase comprises one or more mutations in the cytosine deaminase sequence such that the editing efficiency and / or substrate editing preference of the cytosine deaminase is altered according to specific needs.

[0260] Certain mutations of APOBEC1 and APOBEC3 proteins are described in Kim et al., Nature Biotechnology (2017) 35(4):371-377 (doi:10.1038 / nbt.3803); and Harris et al. Mol. Cell (2002) 10:1247-1253, each of which is incorporated by reference in its entirety.

[0261] In some embodiments, the cytidine deaminase is an APOBEC1 deaminase that includes one or more mutations at amino acid positions corresponding to W90, R118, H121, H122, R126, or R132 in rat APOBEC1, or an APOBEC3G deaminase that includes one or more mutations at amino acid positions corresponding to W285, R313, D316, D317X, R320, or R326 in human APOBEC3G.

[0262] In some embodiments, the cytidine deaminase comprises a mutation at tryptophan 90 of the rat APOBEC1 amino acid sequence, or the corresponding position in a homologous APOBEC protein, e.g., tryptophan 285 of APOBEC3G. In some embodiments, the tryptophan residue at position 90 is substituted with a tyrosine or phenylalanine residue (W90Y or W90F).

[0263] In some embodiments, the cytidine deaminase comprises a mutation at arginine 118 of the rat APOBEC1 amino acid sequence, or the corresponding position in a homologous APOBEC protein. In some embodiments, the arginine residue at position 118 is substituted with an alanine residue (R118A).

[0264] In some embodiments, the cytidine deaminase comprises a mutation at histidine 121 of the rat APOBEC1 amino acid sequence, or the corresponding position in a homologous APOBEC protein. In some embodiments, the histidine residue at position 121 is substituted with an arginine residue (H121R).

[0265] In some embodiments, the cytidine deaminase comprises a mutation at histidine 122 of the rat APOBEC1 amino acid sequence, or the corresponding position in a homologous APOBEC protein. In some embodiments, the histidine residue at position 122 is replaced with an arginine residue (H122R).

[0266] In some embodiments, the cytidine deaminase comprises a mutation at arginine 126 of the rat APOBEC1 amino acid sequence, or the corresponding position in a homologous APOBEC protein, e.g., arginine 320 of APOBEC3G. In some embodiments, the arginine residue at position 126 is substituted with an alanine residue (R126A) or a glutamic acid (R126E).

[0267] In some embodiments, the cytidine deaminase comprises a mutation at arginine 132 of the APOBEC1 amino acid sequence, or the corresponding position in a homologous APOBEC protein. In some embodiments, the arginine residue at position 132 is substituted with a glutamic acid residue (R132E).

[0268] In some embodiments, to narrow the editing window, the cytidine deaminase can include one or more of the following mutations based on the amino acid sequence positions of rat APOBEC1: W90Y, W90F, R126E, and R132E, as well as the corresponding mutations in homologous APOBEC proteins.

[0269] In some embodiments, to reduce editing efficiency, the cytidine deaminase may include one or more of the following mutations based on the amino acid sequence positions of rat APOBEC1: W90A, R118A, R132E, and the corresponding mutations in homologous APOBEC proteins. In certain embodiments, it may be beneficial to use a cytidine deaminase enzyme with reduced efficacy to reduce off-target effects.

[0270] In some embodiments, the cytidine deaminase is wild-type rat APOBEC1 (rAPOBEC1, or a catalytic domain thereof. In some embodiments, the cytidine deaminase comprises one or more mutations in the rAPOBEC1 sequence such that the editing efficiency and / or substrate editing preference of rAPOBEC1 is altered according to particular needs.

[0271] rAPOBEC1:MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHV TLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK

[0272] In some embodiments, the cytidine deaminase is wild-type human APOBEC1 (hAPOBEC1) or a catalytic domain thereof. In some embodiments, the cytidine deaminase comprises one or more mutations in the hAPOBEC1 sequence such that the editing efficiency and / or substrate editing preference of hAPOBEC1 is altered according to specific needs.

[0273] APOBEC1:MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVI YVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR

[0274] In some embodiments, the cytidine deaminase is wild-type human APOBEC3G (hAPOBEC3G) or a catalytic domain thereof. In some embodiments, the cytidine deaminase comprises one or more mutations in the hAPOBEC3G sequence such that the editing efficiency and / or substrate editing preference of hAPOBEC3G is altered according to particular needs.

[0275] hAPOBEC3G:MELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNEPWVRGRH ETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN

[0276] In some embodiments, the cytidine deaminase is wild-type Petromyzon marinus CDA1 (pmCDAl) or a catalytic domain thereof. In some embodiments, the cytidine deaminase comprises one or more mutations in the pmCDA1 sequence such that the editing efficiency and / or substrate editing preference of pmCDA1 is altered according to specific needs.

[0277] pmCDAl:MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADC AEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV

[0278] In some embodiments, the cytidine deaminase is wild-type human AID (hAID) or a catalytic domain thereof. In some embodiments, the cytidine deaminase comprises one or more mutations in the pmCDA1 sequence such that the editing efficiency and / or substrate editing preference of pmCDA1 is altered according to specific needs.

[0279] hAID:MDSLLMNRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPYLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGLLD

[0280] In some embodiments, the cytidine deaminase is a truncated form of hAID (hAID-DC) or its catalytic domain. In some embodiments, the cytidine deaminase contains one or more mutations in the hAID-DC sequence such that the editing efficiency and / or substrate editing preference of hAID-DC is altered according to specific needs.

[0281] hAID-DC:MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILL

[0282] Additional embodiments of cytidine deaminases are disclosed in WO2017 / 070632, entitled "Nucleobase Editors and Uses Thereof," which is incorporated herein by reference in its entirety.

[0283] In some embodiments, the cytidine deaminase has an efficient deamination window that confines nucleotides susceptible to deamination editing. Thus, in some embodiments, "editing window width" refers to the number of nucleotide positions in a given target site where the editing efficiency of the cytidine deaminase exceeds half-maximal value for that target site. In some embodiments, the cytidine deaminase has an editing window width in the range of about 1 to about 6 nucleotides. In some embodiments, the editing window width of the cytidine deaminase is 1, 2, 3, 4, 5, or 6 nucleotides.

[0284] Without intending to be bound by theory, in some embodiments, it is contemplated that the length of the linker sequence affects the editing window width. In some embodiments, as the linker length increases (e.g., from about 3 to about 21 amino acids), the editing window width increases (e.g., from about 3 to about 6 nucleotides). In a non-limiting example, a 16-residue linker provides an efficient deamination window of about 5 nucleotides. In some embodiments, the length of the guide RNA affects the editing window width. In some embodiments, shortening the guide RNA leads to a narrowing of the efficient deamination window of cytidine deaminase.

[0285] In some embodiments, the mutation of the cytidine deaminase affects the editing window width. In some embodiments, the cytidine deaminase component of the CD-functionalized CRISPR system comprises one or more mutations that reduce the catalytic efficiency of the cytidine deaminase, so that the deaminase does not undergo the deamination of a large number of cytidines per DNA binding event. In some embodiments, the tryptophan at residue 90 (W90) of APOBEC1 or the corresponding tryptophan residue in a homologous sequence is mutated. In some embodiments, the catalytically inactive Cas13 is fused or linked to an APOBEC1 mutant comprising a W90Y or W90F mutation. In some embodiments, the tryptophan at residue 285 (W285) of APOBEC3G or the corresponding tryptophan residue in a homologous sequence is mutated. In some embodiments, the catalytically inactive Cas13 is fused or linked to an APOBEC3G mutant comprising a W285Y or W285F mutation.

[0286] In some embodiments, the cytidine deaminase component of the CD-functionalized CRISPR system comprises one or more mutations that reduce the tolerance of non-optimal presentation of cytidine to the deaminase active site. In some embodiments, the cytidine deaminase comprises one or more mutations that change the substrate binding activity of the deaminase active site. In some embodiments, the cytidine deaminase comprises one or more mutations that change the conformation of the DNA recognized and bound by the deaminase active site. In some embodiments, the cytidine deaminase comprises one or more mutations that change the substrate accessibility to the deaminase active site. In some embodiments, the arginine at residue 126 (R126) of APOBEC1, or the corresponding arginine residue in a homologous sequence, is mutated. In some embodiments, a catalytically inactive Cas13 is fused or linked to APOBEC1 that comprises a R126A or R126E mutation. In some embodiments, the tryptophan at residue 320 (R320) of APOBEC3G, or the corresponding arginine residue in a homologous sequence, is mutated. In some embodiments, a catalytically inactive Cas13 is fused or linked to an APOBEC3G mutant comprising an R320A or R320E mutation. In some embodiments, the arginine at residue 132 (R132) of APOBEC1, or the corresponding arginine residue in a homologous sequence, is mutated. In some embodiments, a catalytically inactive Cas13 is fused or linked to an APOBEC1 mutant comprising an R132E mutation.

[0287] In some embodiments, the APOBEC1 domain of the CD-functionalized CRISPR system comprises one, two, or three mutations selected from W90Y, W90F, R126A, R126E, and R132E. In some embodiments, the APOBEC1 domain comprises a double mutation of W90Y and R126E. In some embodiments, the APOBEC1 domain comprises a double mutation of W90Y and R132E. In some embodiments, the APOBEC1 domain comprises a double mutation of R126E and R132E. In some embodiments, the APOBEC1 domain comprises a triple mutation of W90Y, R126E, and R132E.

[0288] In some embodiments, one or more mutations in cytidine deaminase as disclosed herein reduce the editing window width to about 2 nucleotides. In some embodiments, one or more mutations in cytidine deaminase as disclosed herein reduce the editing window width to about 1 nucleotide. In some embodiments, one or more mutations in cytidine deaminase as disclosed herein reduce the editing window width, while only minimally or modestly affecting the editing efficiency of the enzyme. In some embodiments, one or more mutations in cytidine deaminase as disclosed herein reduce the editing window width without reducing the editing efficiency of the enzyme. In some embodiments, one or more mutations in cytidine deaminase as disclosed herein allow discrimination of adjacent cytidine nucleotides that would otherwise be edited with similar efficiency by cytidine deaminase.

[0289] In some embodiments, the cytidine deaminase protein further comprises or is linked to one or more double-stranded RNA (dsRNA) binding motifs (dsRBM) or domains (dsRBD) to recognize and bind to double-stranded nucleic acid substrates. In some embodiments, the interaction between the cytidine deaminase and the substrate is mediated by one or more additional protein factor(s), such as CRISPR / CAS protein factor(s). In some embodiments, the interaction between the cytidine deaminase and the substrate is further mediated by one or more nucleic acid component(s), such as guide RNA.

[0290] According to the present invention, the substrate of cytidine deaminase is a DNA single-stranded bubble of RNA duplex containing the desired cytosine, which is made accessible to cytidine deaminase when guide molecule binds to its DNA target, and forms a CRISPR-Cas complex with CRISPR-Cas enzyme.Thereby, cytosine deaminase can be fused to or bound to one or more components of the CRISPR-Cas complex, i.e., CRISPR-Cas enzyme and / or guide molecule.The specific features of guide molecule and CRISPR-Cas enzyme are detailed below.

[0291] RNA-binding proteins In certain exemplary embodiments, the RNA binding protein or its functional domain comprises an RNA recognition motif.In some examples, the RNA binding protein can be a Cas effector protein, such as Cas13 effector protein.In some cases, the RNA binding protein can be a catalytically inactive Cas effector protein, such as dCas13.

[0292] In certain exemplary embodiments, the RNA binding protein comprises a zinc finger motif. The RNA binding protein or functional domain thereof may comprise a Cys2-His2, Gag knuckle, Treble-clet, zinc ribbon, Zn2 / Cys6 class motif.

[0293] In certain exemplary embodiments, the RNA binding protein may comprise a Pumilio homology domain.

[0294] In one aspect, the present invention provides a method for the targeted deamination of adenine in RNA, more particularly in RNA sequence of interest.According to the method of the present invention, adenosine deaminase (AD) protein is specifically recruited to the relevant adenine in RNA sequence of interest by CRISPR-Cas complex, which can specifically bind to target sequence.To achieve this, adenosine deaminase protein can be covalently linked to CRISPR-Cas enzyme or provided as a separate protein, but ensure its recruitment to CRISPR-Cas complex.

[0295] In certain embodiments of the method of the present invention, the recruitment of adenosine deaminase to target locus is ensured by fusing adenosine deaminase or its catalytic domain to CRISPR-Cas protein, which is Cas13 protein.The method of generating fusion protein from two separated proteins is known in the art, and typically involves the use of spacer or linker.Cas13 protein can be fused to adenosine deaminase protein or its catalytic domain at either its N-terminus or C-terminus.In certain embodiments, CRISPR-Cas protein is inactive or inactivated Cas13 protein, and is linked to the N-terminus of deaminase protein or its catalytic domain.

[0296] The term "linker", when used in reference to a fusion protein, refers to a molecule that binds to the proteins to form the fusion protein. Generally, such molecules have no specific biological activity other than linking the proteins or preserving a minimum distance or other spatial relationship between the proteins. However, in certain embodiments, the linker may be selected to affect some property of the linker and / or the fusion protein, such as the folding, net charge, or hydrophobicity of the linker. The linker (e.g., the non-nucleotide loop) can be of any length. In some embodiments, the linker has a length equal to about 0-16 nucleotides. In some embodiments, the linker has a length equal to about 0-8 nucleotides. In some embodiments, the linker has a length equal to about 0-4 nucleotides. In some embodiments, the linker has a length equal to about 2 nucleotides. Examples of linker designs are also described in WO2011 / 008730.

[0297] Linkers suitable for use in the methods of the present invention are well known to those skilled in the art and include, but are not limited to, straight or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. However, as used herein, a linker can also be a covalent bond (carbon-carbon bond or carbon-heteroatom bond). In certain embodiments, a linker is used to separate the CRISPR-Cas protein and the adenosine deaminase by a distance sufficient to ensure that each protein retains its required functional properties. A preferred peptide linker sequence accommodates a flexible, extended conformation and does not tend to develop ordered secondary structures. In certain embodiments, the linker can be a chemical moiety, which can be a monomer, dimer, multimer, or polymer. The linker preferably comprises an amino acid. Exemplary amino acids in flexible linkers include Gly, Asn, and Ser. Thus, in certain embodiments, the linker comprises one or more combinations of Gly, Asn, and Ser amino acids. Other near-neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. Exemplary linkers are disclosed in Maratea et al. (1985), Gene 40:39-46; Murphy et al. (1986) Proc. Nat'l. Acad. Sci. USA 83:8258-62; U.S. Pat. No. 4,935,233; and U.S. Pat. No. 4,751,180. For example, the GlySer linkers GGS, GGGS, or GSG can be used. The GGS, GSG, GGGS, or GGGGS linkers can be used in 3 (e.g., (GGS)3 (SEQ ID NO:12, (GGGGS)3) (SEQ ID NO:1) or 5, 6, 7, 9, or even 12 or more repeats to provide suitable length. In certain embodiments, linkers such as (GGGGS)3) (SEQ ID NO:1) are preferred for use herein. (GGGGS)6) (SEQ ID NO: 4), (GGGGS)9) (SEQ ID NO: 7) or (GGGGS)12) (SEQ ID NO: 13) may be preferred for use as alternatives.Other preferred alternatives are (GGGGS)1) (SEQ ID NO: 14), (GGGGS)2) (SEQ ID NO: 15), (GGGGS)4) (SEQ ID NO: 2), (GGGGS)5) (SEQ ID NO: 3), (GGGGS)7) (SEQ ID NO: 5), (GGGGS)8) (SEQ ID NO: 6), (GGGGS)10) (SEQ ID NO: 8), or (GGGGS)11) (SEQ ID NO: 9). In yet another embodiment, LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO: 11) is used as a linker. In yet further embodiments, the linker is an XTEN linker. In certain embodiments, the CRISPR-cas protein is a Cas13 protein and is linked to the deaminase protein or its catalytic domain by a LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO: 11) linker. In a further specific embodiment, the Cas13 protein is C-terminally linked to the N-terminus of the deaminase protein or its catalytic domain by a LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO: 11) linker. In addition, the N- and C-terminal NLS can also function as a linker (e.g., PKKKRKVEASSPKKRKVEAS (SEQ ID NO: 16)).

[0298] In certain embodiments of the method of the present invention, adenosine deaminase protein or its catalytic domain is delivered to cells or expressed in cells as a separate protein, but modified so that it can be linked to either Cas13 protein or guide molecule. In certain embodiments, this is ensured by the use of orthogonal RNA binding proteins or adaptor protein / aptamer combinations present in various bacteriophage coat proteins. Examples of such coat proteins include, but are not limited to, MS2, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s and PRR1. Aptamers can be naturally occurring or synthetic oligonucleotides that have been engineered through iterative rounds of in vitro selection or SELEX (systematic evolution of ligands by exponential enrichment) to bind to a specific target.

[0299] In certain embodiments of the methods and systems of the present invention, the guide molecule is provided with one or more unique RNA loop(s) or unique sequence(s) that can recruit an adaptor protein. The guide molecule can be extended without colliding with the Cas13 protein by the insertion of unique RNA loop(s) or unique sequence(s) that can recruit an adaptor protein that can bind to the unique RNA loop(s) or unique sequence(s). Examples of modified guides and their use to recruit effector domains to CRISPR-Cas complexes are provided in Konermann (Nature 2015,517(7536):583-588). In certain embodiments, the aptamer is a minimal hairpin aptamer that selectively binds to the dimerized MS2 bacteriophage coat protein in mammalian cells and is introduced into the guide molecule, such as a stem loop and / or tetraloop. In these embodiments, an adenosine deaminase protein is fused to MS2. The adenosine deaminase protein is then co-delivered together with the CRISPR-Cas protein and the corresponding guide RNA.

[0300] The term "AD-functionalized CRISPR system" as used herein refers to a nucleic acid targeting and editing system comprising: (a) a CRISPR-Cas protein, more specifically a catalytically inactive Cas13 protein; (b) a guide molecule comprising a guide sequence; and (c) an adenosine deaminase protein or its catalytic domain, which is covalently or non-covalently bound to the CRISPR-Cas protein or guide molecule, or is adapted to be linked thereto after delivery; the guide sequence is substantially complementary to the target sequence, but comprises an unpaired C corresponding to the A targeted for deamination, resulting in an AC mismatch in the RNA duplex formed by the guide sequence and the target sequence. When applied to eukaryotic cells, the CRISPR-Cas protein and / or adenosine deaminase are preferably NLS-tagged.

[0301] In some embodiments, components (a), (b), and (c) are delivered to a cell as a ribonucleoprotein complex. The ribonucleoprotein complex may be delivered via one or more lipid nanoparticles.

[0302] In some embodiments, components (a), (b), and (c) are delivered to cells as one or more RNA molecules, such as one or more guide RNAs and one or more mRNA molecules encoding a CRISPR-Cas protein, an adenosine deaminase protein, and optionally an adaptor protein. The RNA molecules can be delivered via one or more lipid nanoparticles.

[0303] In some embodiments, components (a), (b), and (c) are delivered to cells as one or more DNA molecules. In some embodiments, the one or more DNA molecules are contained within one or more vectors, such as viral vectors (e.g., AAV). In some embodiments, the one or more DNA molecules comprise one or more regulatory elements operably configured to express a CRISPR-Cas protein, a guide molecule, and an adenosine deaminase protein or a catalytic domain thereof, and optionally, the one or more regulatory elements comprise an inducible promoter.

[0304] In some embodiments, the CRISPR-Cas protein is an inactivated Cas13. In some embodiments, the inactivated Cas13 is an inactivated Cas13a protein comprising one or more mutations in the HEPN domain. In some embodiments, the inactivated Cas13a comprises a mutation corresponding to R474A and R1046A in Leptotrichia wadei (LwaCas13a). In some embodiments, the inactivated Cas13 is an inactivated Cas13b protein comprising one or more of the following amino acid positions: R116A, H121A, R1177A, H1182A of the Cas13b protein arising from Bergeyella zoohelcum ATCC 43767, or the corresponding amino acid positions of the Cas13b ortholog.

[0305] In some embodiments, the guide molecule can hybridize with the target sequence that comprises the adenine to be deaminated in the RNA sequence to form an RNA duplex that comprises the unpaired cytosine opposite the adenine.Once the RNA duplex is formed, the guide molecule forms a complex with Cas13 protein, and directs the complex to bind to the RNA polynucleotide at the target RNA sequence of interest.Details of the guide aspect of the AD-functionalized CRISPR-Cas system are provided herein below.

[0306] In some embodiments, for example, a Cas13 guide RNA having the canonical length of LawCas13 is used to form an RNA duplex with the target DNA. In some embodiments, for example, a Cas13 guide molecule longer than the canonical length of LawCas13a is used to form an RNA duplex with the target DNA, for example, outside of the Cas13-guide RNA-target DNA complex.

[0307] In at least a first design, the AD-functionalized CRISPR system comprises (a) an adenosine deaminase fused or linked to a CRISPR-Cas protein (the CRISPR-Cas protein is catalytically inactive), and (b) a guide molecule comprising a guide sequence designed to introduce an AC mismatch into the RNA duplex formed between the guide sequence and the target sequence. In some embodiments, the CRISPR-Cas protein and / or the adenosine deaminase are NLS-tagged at either or both the N- or C-terminus.

[0308] In at least a second design, the AD-functionalized CRISPR system includes (a) a catalytically inactive CRISPR-Cas protein, (b) a guide molecule including a guide sequence designed to introduce an AC mismatch into an RNA duplex formed between the guide sequence and the target sequence, and an aptamer sequence (e.g., MS2 RNA motif or PP7 RNA motif) capable of binding to an adaptor protein (e.g., MS2 coat protein or PP7 coat protein), and (c) an adenosine deaminase fused or linked to the adaptor protein (the binding of the aptamer to the adaptor protein recruits the adenosine deaminase to the RNA duplex formed between the guide sequence and the target sequence for targeted deamination at the A of the AC mismatch). In some embodiments, the adaptor protein and / or the adenosine deaminase are NLS-tagged at either or both the N- or C-terminus. The CRISPR-Cas protein can also be NLS-tagged.

[0309] In some embodiments, a Cas13 guide RNA having a canonical length (e.g., about 15-30 nt) is used to form an RNA duplex with the target RNA. In some embodiments, a Cas13 guide molecule longer than the canonical length (e.g., >30 nt) is used to form an RNA duplex with the target RNA, e.g., outside of the Cas13-guide RNA-target RNA complex.

[0310] In at least a first design, the CD-functionalized CRISPR system comprises (a) a cytidine deaminase fused or linked to a CRISPR-Cas protein (the CRISPR-Cas protein is a catalytically inactive Cas13), and (b) an optional guide molecule comprising a guide sequence designed to either (A) be upstream or downstream of a cytosine of interest or (B) introduce a CA / U mismatch in the RNA duplex formed between the guide sequence and the target sequence. In some embodiments, the CRISPR-Cas protein and / or the cytidine deaminase are NES-tagged at either or both the N- or C-terminus.

[0311] In at least a second design, the CD-functionalized CRISPR system includes (a) a CRISPR-Cas protein that is a catalytically inactive Cas13, (b) an optional guide molecule that includes a guide sequence that is designed to either (A) be upstream or downstream of a cytosine of interest or (B) introduce a CA / U mismatch into the RNA duplex formed between the guide sequence and the target sequence, and an aptamer sequence (e.g., MS2 RNA motif or PP7 RNA motif) that can bind to an adaptor protein (e.g., MS2 coat protein or PP7 coat protein), and (c) a cytidine deaminase fused or linked to the adaptor protein (binding of the aptamer to the adaptor protein recruits the cytidine deaminase to the RNA duplex formed between the guide sequence and the target sequence for targeted deamination at either the C outside the target sequence or the C of the optional CA / U mismatch). In some embodiments, the adaptor protein and / or the cytidine deaminase are NES-tagged at either or both the N- or C-terminus. The CRISPR-Cas proteins are also NES-tagged.

[0312] The use of different aptamers and corresponding adaptor proteins also allows for orthogonal gene editing to be implemented. In one example of using adenosine deaminase in combination with cytidine deaminase to edit / deaminate orthogonal genes, different loci targeted by sgRNAs are engineered with unique RNA loops to recruit MS2-adenosine deaminase and PP7-cytidine deaminase (or PP7-adenosine deaminase and MS2-cytidine deaminase), respectively, resulting in orthogonal deamination of A or C, respectively, at the desired target locus. PP7 is an RNA-binding envelope protein of the bacteriophage Pseudomonas. Like MS2, PP7 binds to specific RNA sequences and secondary structures. The PP7 RNA recognition motif is distinct from that of MS2. As a result, PP7 and MS2 can be multiplexed to simultaneously mediate different effects at different genomic loci. For example, locus A targeted by sgRNA can be modified with MS2 loop and recruit MS2 adenosine deaminase, while locus B targeted by another sgRNA can be modified with PP7 loop and recruit PP7 cytidine deaminase. In the same cell, thus, orthogonal locus-specific modification is realized. This principle can be extended to incorporate other orthogonal RNA-binding proteins.

[0313] In at least a third design, an AD-functionalized CRISPR system includes (a) an adenosine deaminase that is inserted into an internal loop or unstructured region of the CRISPR-Cas protein (the CRISPR-Cas protein is either catalytically inactive or is a nickase), and (b) a guide molecule that includes a guide sequence designed to introduce an AC mismatch into the RNA duplex formed between the guide sequence and the target sequence.

[0314] CRISPR-Cas protein split sites suitable for insertion of adenosine deaminase can be identified with the aid of crystal structures. In cases where a relatively high degree of homology exists between the ortholog and the intended CRISPR-Cas protein, the crystal structure of the ortholog can be used.

[0315] The split position can be located in a region or loop. Preferably, the split position is located where the interruption of the amino acid sequence does not result in partial or complete destruction of structural features (e.g., α-helix or β-sheet). Unstructured regions (regions that do not appear in the crystal structure because they are not structured enough to be "frozen" in the crystal) are often the preferred choice. The positions in the unstructured region or outside the loop do not have to be exactly the number mentioned above, but can differ, for example, by 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 amino acids on either side of the positions given above, depending on the size of the loop, as long as the split position still fits within the unstructured region or outside the loop.

[0316] The system described herein can be used to target and deaminate specific adenines in an RNA polynucleotide sequence. For example, the guide molecule can form a complex with CRISPR-Cas protein, and the complex is directed to bind to the target RNA sequence in the RNA polynucleotide of interest. The guide sequence is designed to have an unpaired C, so that the RNA duplex formed between the guide sequence and the target sequence contains an AC mismatch, which directs adenosine deaminase to contact and deaminate the A opposite the unpaired C, converting it to inosine (I). Inosine (I) base pairs with C function like G in cellular processes, so the targeted deamination of A described herein is useful for correcting undesired GA and CT mutations, as well as obtaining desired AG and TC mutations.

[0317] In some embodiments, the system is used to carry out target deamination in RNA polynucleotide molecules in vitro.In some embodiments, the AD functionalized CRISPR system is used to carry out target deamination in DNA molecules in cells.The cells can be eukaryotic cells, such as animal cells, mammalian cells, human cells, or plant cells.

[0318] The present invention also relates to a method for treating or preventing disease by targeted deamination using an AD-functionalized CRISPR system, where deamination of A cures disease caused by transcripts containing pathogenic G→A or C→T point mutations. Examples of diseases that can be treated or prevented by the present invention include cancer, Meyer-Gorlin syndrome, Seckel syndrome4, Joubert syndrome5, Leber congenital amaurosis10, Charcot-Marie-Tooth disease type 2, Charcot-Marie-Tooth disease type 2, Usher syndrome type 2C, spinocerebellar ataxia28, spinocerebellar ataxia28, spinocerebellar ataxia28, extended QT syndrome2, Sjogren-Larsson syndrome, hereditary fructosuria, hereditary fructosuria, neuroblastoma, neuroblastoma, Kallmann syndrome1, Kallmann syndrome1, Kallmann syndrome1, metachromatic leukodystrophy.

[0319] The present invention also relates to a method for knocking out or knocking down the undesired activity of a gene, wherein the deamination of A in the transcript of the gene results in loss of function.For example, in one embodiment, the targeted deamination by the AD-functionalized CRISPR system can cause nonsense mutations, resulting in premature stop codons in endogenous genes.This can change the expression of endogenous genes, resulting in desired traits in edited cells.In another embodiment, the targeted deamination by the AD-functionalized CRISPR system can cause nonconservative missense mutations, resulting in the coding of different amino acid residues in endogenous genes.This can change the expression of expressed endogenous genes, resulting in desired traits in edited cells.

[0320] The present invention also relates to modified cells, or progeny thereof, obtained by targeted deamination using an AD-functionalized CRISPR system, which contain an I or G in place of an A in a target RNA sequence of interest compared to the corresponding cell before targeted deamination. The modified cell may be a eukaryotic cell, such as an animal cell, a plant cell, a mammalian cell, or a human cell.

[0321] In some embodiments, the modified cells are therapeutic T cells, such as T cells suitable for CAR-T therapy. The modifications can result in one or more desired traits of the therapeutic T cells, including, but not limited to, down-regulation of immune checkpoint receptors (e.g., PDA, CTLA4), down-regulation of HLA proteins (e.g., B2M, HLA-A), and down-regulation of endogenous TCRs.

[0322] In some embodiments, the modified cells are antibody-producing B cells. The modification can result in one or more desired traits in the B cells, including but not limited to enhanced antibody production.

[0323] The present invention also relates to modified non-human animals or modified plants. The modified non-human animals may be livestock. The modified plants may be crops.

[0324] The present invention further relates to a method of cell therapy comprising administering the modified cells described herein to a patient in need thereof, where the presence of the modified cells treats a disease in the patient. In one embodiment, the modified cells of the cell therapy are CAR-T cells capable of recognizing and / or attacking tumor cells. In another embodiment, the modified cells of the cell therapy are stem cells, such as neural stem cells, mesenchymal stem cells, hematopoietic stem cells, or iPSC cells.

[0325] The invention additionally relates to an engineered, non-naturally occurring system suitable for modifying an adenine at a target locus of interest comprising a guide molecule comprising a guide sequence, or a nucleotide sequence encoding the guide molecule; a CRISPR-Cas protein, or one or more nucleotide sequences encoding a CRISPR-Cas protein; an adenosine deaminase protein or catalytic domain thereof, covalently or non-covalently linked to the CRISPR-Cas protein or guide molecule, or one or more nucleotide sequences encoding same, wherein the adenosine deaminase protein or catalytic domain thereof is adapted to be linked thereto after delivery; and the guide sequence is capable of hybridizing with a target sequence comprising an adenine within an RNA polynucleotide of interest, but comprises a cytosine at the position corresponding to the adenine.

[0326] The invention additionally relates to an engineered non-naturally occurring system suitable for modifying an adenine at a target locus of interest, comprising: a first regulatory element operably linked to one or more nucleotide sequences encoding a guide molecule comprising a guide sequence; a second regulatory element operably linked to a nucleotide sequence encoding a CRISPR-Cas protein; and optionally one or more vectors comprising a nucleotide sequence encoding an adenosine deaminase protein or catalytic domain thereof under the control of the first or second regulatory element or operably linked to a third regulatory element, wherein when the nucleotide sequence encoding the adenosine deaminase protein or catalytic domain thereof is operably linked to the third regulatory element, the adenosine deaminase protein or catalytic domain thereof is adapted to be linked to the guide molecule or the Crispr-Cas protein after expression; the guide sequence is capable of hybridizing with a target sequence comprising an adenine within the target locus, but comprises a cytosine at the position corresponding to the adenine; and components (a), (b), and (c) are located on the same or different vectors of the system.

[0327] The present invention additionally relates to in vitro, ex vivo or in vivo host cells or cell lines or their progeny, comprising the engineered non-naturally occurring system or vector system described herein. The host cell may be a eukaryotic cell, such as an animal cell, a plant cell, a mammalian cell, or a human cell.

[0328] Base excision repair inhibitors In some embodiments, the system further comprises a base excision repair (BER) inhibitor. Without wishing to be bound by any particular theory, the cell DNA repair response to the presence of I:T pairing may cause the decrease in nucleic acid base editing efficiency in cells. Alkyl adenine DNA glycosylase (also known as DNA-3-methyladenine glycosylase, 3-alkyladenine DNA glycosylase, or N-methylpurine DNA glycosylase) catalyzes the removal of hypoxanthine from DNA in cells, which can initiate base excision repair, resulting in the inversion of I:T pairing to A:T pairing.

[0329] In some embodiments, the BER inhibitor is an inhibitor of alkyladenine DNA glycosylase. In some embodiments, the BER inhibitor is an inhibitor of human alkyladenine DNA glycosylase. In some embodiments, the BER inhibitor is a polypeptide inhibitor. In some embodiments, the BER inhibitor is a protein that binds to hypoxanthine. In some embodiments, the BER inhibitor is a protein that binds to hypoxanthine in DNA. In some embodiments, the BER inhibitor is a catalytically inactive alkyladenine DNA glycosylase protein or a binding domain thereof. In some embodiments, the BER inhibitor is a catalytically inactive alkyladenine DNA glycosylase protein or a binding domain thereof that does not remove hypoxanthine from DNA. Other proteins that can inhibit (e.g., sterically block) alkyladenine DNA glycosylase base excision repair enzyme are within the scope of this disclosure. In addition, any protein that blocks or inhibits base excision repair is also within the scope of this disclosure.

[0330] Without wishing to be bound by any particular theory, base excision repair can be inhibited by molecules that bind to the edited strand, block the edited base, inhibit alkyladenine DNA glycosylase, inhibit base excision repair, protect the edited base, and / or promote fixation of the non-edited strand. It is believed that the use of BER inhibitors described herein can increase the editing efficiency of adenosine deaminase that can catalyze the A→I change.

[0331] Thus, in the first design of the AD-functionalized CRISPR system described above, the CRISPR-Cas protein or adenosine deaminase can be fused or linked to a BER inhibitor (e.g., an inhibitor of alkyladenine DNA glycosylase). In some embodiments, the BER inhibitor can be composed of one of the following structures (nCas13 = Cas13 nickase; dCas13 = inactivated Cas13): [AD]-[any linker]-[nCas13 / dCas13]-[any linker]-[BER inhibitor]; [AD]-[any linker]-[BER inhibitor]-[any linker]-[nCas13 / dCas13]; [BER inhibitor]-[any linker]-[ [anchor]-[AD]-[any linker]-[nCas13 / dCas13];[BER inhibitor]-[any linker]-[nCas13 / dCas13]-[any linker]-[AD];[nCas13 / dCas13]-[any linker]-[AD]-[any linker]-[BER inhibitor];[nCas13 / dCas13]-[any linker]-[BER inhibitor]-[any linker]-[AD].

[0332] Similarly, in the second design of the AD-functionalized CRISPR system above, the CRISPR-Cas protein, adenosine deaminase, or adapter protein can be fused or linked to a BER inhibitor (e.g., an inhibitor of alkyladenine DNA glycosylase). In some embodiments, the BER inhibitor may be comprised of one of the following structures (nCas13 = Cas13 nickase; dCas13 = inactivated Cas13): [nCas13 / dCas13]-[optional linker]-[BER inhibitor]; [BER inhibitor]-[optional linker]-[nCas13 / dCas13]; [AD]-[optional linker]-[adapter]-[optional linker]-[BER inhibitor]; [AD]-[optional linker]-[BER inhibitor]-[optional linker]-[adapter]; [BER inhibitor]-[optional linker]-[AD]-[optional linker]-[adapter]; [BER inhibitor]-[optional linker]-[adapter]-[optional linker]-[AD]; [adapter]-[optional linker]-[AD]-[optional linker]-[BER inhibitor]; [adapter]-[optional linker]-[BER inhibitor]-[optional linker]-[AD].

[0333] In the third design of the AD-functionalized CRISPR system above, BER inhibitors can be inserted into internal loops or unstructured regions of the CRISPR-Cas protein.

[0334] Nuclear targeting In some embodiments, the methods of the invention relate to modifying an adenine in a target locus of interest (the target locus is within a cell). To improve nuclear targeting of the CRISPR-Cas protein and / or adenosine deaminase protein or catalytic domain thereof used in the methods of the invention, it may be advantageous to provide one or both of these components with one or more nuclear localization sequences (NLS).

[0335] In a preferred embodiment, the NLS used in the context of the present invention is heterologous to the protein. Non-limiting examples of NLSs include the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 17) or PKKKRKVEAS (SEQ ID NO: 18); an NLS from nucleoplasmin (e.g., nucleoplasmic bisection having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 19)); a c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 20) or RQRRNELKRSP (SEQ ID NO: 21); a hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 22); the IBB domain from importin alpha having the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 23); the sequences VSRKRPRP (SEQ ID NO: 24) and PPKKARED (SEQ ID NO: 25) of the sarcoma T protein; the sequence PQPKKKPL (SEQ ID NO: 26) of human p53; the mouse c-abl Examples of NLS sequences include those derived from the sequence SALIKKKKKMAP (SEQ ID NO: 27) of IV; the sequences DRLRR (SEQ ID NO: 28) and PKQKKRK (SEQ ID NO: 29) of influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 30) of hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 31) of mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 32) of human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 33) of steroid hormone receptor (human) glucocorticoid. Generally, one or more NLSs are strong enough to drive the accumulation of a detectable amount of DNA-targeting Cas protein in the nucleus of a eukaryotic cell. In general, the strength of the nuclear localization activity may be derived from the number of NLSs in the CRISPR-Cas protein, the particular NLS(s) used, or a combination of these factors. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the nucleic acid targeting protein, such that its location within the cell can be visualized, for example, in combination with a means to detect the location of the nucleus (e.g., a nuclear-specific stain such as DAPI).Cell nuclei may also be isolated from cells, and their content may then be analyzed by any suitable process for detecting proteins, such as immunohistochemistry, Western blot, or enzyme activity assays. Accumulation in nuclei may also be determined indirectly, for example, by assaying for the effect of nucleic acid targeting complex formation at the target sequence (e.g., assaying for deaminase activity), or assaying for changes in gene expression activity affected by DNA targeting complex formation and / or DNA targeting), compared to a control that is not exposed to CRISPR-Cas protein and deaminase protein, or a control that is exposed to CRISPR-Cas and / or deaminase protein with one or more NLS deleted.

[0336] CRISPR-Cas and / or adenosine deaminase proteins may be provided with one or more heterologous NLSs, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more heterologous NLSs. In some embodiments, the protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino terminus, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy terminus, or a combination thereof (e.g., 0 or at least one or more NLSs at the amino terminus and 0 or at least one or more NLSs at the carboxy terminus). When more than one NLS is present, each can be selected independently of the other, and thus a single NLS may be present in more than one copy and / or in combination with one or more other NLSs present in one or more copies. In some embodiments, an NLS is considered to be near the N- or C-terminus if the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more amino acids from the N- or C-terminus along the polypeptide chain. In preferred embodiments of the CRISPR-Cas protein, the NLS is attached to the C-terminus of the protein.

[0337] In certain embodiments of the methods described herein, the CRISPR-Cas protein and the deaminase protein are delivered to the cell or expressed in the cell as separate proteins. In these embodiments, each of the CRISPR-Cas and the deaminase protein can be provided with one or more NLSs as described herein. In certain embodiments, the CRISPR-Cas and the deaminase protein are delivered to the cell or expressed in the cell as a fusion protein. In these embodiments, one or both of the CRISPR-Cas and the deaminase protein are provided with one or more NLSs. When the adenosine deaminase is fused to an adaptor protein (e.g., MS2), as described above, the one or more NLSs can be provided on the adaptor protein, so long as they do not interact with aptamer binding. In certain embodiments, the one or more NLS sequences can also function as linker sequences between the adenosine deaminase and the CRISPR-Cas protein.

[0338] In certain embodiments, the guide of the present invention comprises a specific binding site (e.g., an aptamer) of an adaptor protein, which can be linked or fused to an adenosine deaminase or its catalytic domain. When such a guide forms a CRISPR complex (i.e., a CRISPR-Cas protein that binds to the guide and the target), the adaptor protein binds and the adenosine deaminase or its catalytic domain associated with the adaptor protein is advantageously positioned in a spatial orientation to allow for effective function.

[0339] Those skilled in the art will understand that modifications to the guide that allow for the binding of the adaptor + adenosine deaminase but do not properly position the adaptor + adenosine deaminase (e.g., due to steric hindrance within the three-dimensional structure of the CRISPR complex) are unintended modifications. One or more modified guides may be modified at the tetraloop, stem loop 1, stem loop 2, or stem loop 3 as described herein, preferably at either the tetraloop or stem loop 2, and most preferably at both the tetraloop and stem loop 2.

[0340] Use of orthogonal catalytically inactive CRISPR-Cas proteins In certain embodiments, Cas13 nickase is used in combination with an orthogonal catalytically inactive CRISPR-Cas protein to increase the efficiency of said Cas13 nickase (as described in Chen et al. 2017, Nature Communications 8:14958; doi:10.1038 / ncomms14958). More specifically, the orthogonal catalytically inactive CRISPR-Cas protein is characterized by a PAM recognition site different from the Cas13 nickase used in the system, and the corresponding guide sequence is selected to bind to the target sequence proximal to the Cas13 nickase of the system. When used in the context of the present invention, the orthogonal catalytically inactive CRISPR-Cas protein does not form part of the system, but only functions to increase the efficiency of said Cas13 nickase, and is used in combination with a standard guide molecule as described in the art for said CRISPR-Cas protein. In certain embodiments, the orthogonal catalytically inactive CRISPR-Cas protein is an inactivated CRISPR-Cas protein, i.e., it contains one or more mutations that abolish the nuclease activity of the CRISPR-Cas protein. In certain embodiments, the catalytically inactive orthogonal CRISPR-Cas protein is provided with two or more guide molecules that can hybridize to a target sequence that is proximal to the target sequence of Cas13 nickase. In certain embodiments, at least two guide molecules are used to target the catalytically inactive CRISPR-Cas protein, at least one of which can hybridize to the target sequence 5' of the target sequence of the Cas13 nickase of the system, and at least one of which can hybridize to the target sequence 3' of the target sequence of the Cas13 nickase of the system. Thereby, the one or more target sequences may be on the same or opposite DNA strand as the target sequence of the Cas13 nickase.In certain embodiments, the guide sequence of one or more guide molecules of the orthogonal catalytically inactive CRISPR-Cas protein is selected so that the target sequence is proximal to that of the guide molecule for targeting AD-functionalized CRISPR, i.e., targeting Cas13 nickase. In certain embodiments, the target sequence of one or more of the orthogonal catalytically inactive CRISPR-Cas enzyme is separated from the target sequence of Cas13 nickase by more than 5 but less than 450 base pairs, respectively. The optimal distance between the target sequence of the guide used in the orthogonal catalytically inactive CRISPR-Cas protein and the target sequence of the system can be determined by those skilled in the art. In certain embodiments, the orthogonal CRISPR-Cas protein is a class II, type II CRISPR protein. In certain embodiments, the orthogonal CRISPR-Cas protein is a class II, type V CRISPR protein. In certain embodiments, the orthogonal CRISPR-Cas protein is a catalytically inactive orthogonal CRISPR-Cas protein. In certain embodiments, the catalytically inactive orthogonal CRISPR-Cas protein is modified to change its PAM specificity as described elsewhere herein. In certain embodiments, the Cas13 protein nickase is a nickase that has limited activity in human cells by itself, but ensures the required nickase activity by combining the inactive orthogonal CRISPR-Cas protein with one or more corresponding proximal guides.

[0341] Cas13 effector protein complexes can deliver functional effectors Unlike CRISPR-Cas13-mediated knockout, which eliminates expression by mutating at the DNA level, CRISPR-Cas13 knockdown can temporarily reduce gene expression using artificial transcription factors, for example, through mutation of residues in the cleavage domain(s) of Cas13 protein, resulting in the generation of catalytically inactive Cas13 protein. Catalytically inactive Cas13 forms a complex with guide RNA or crRNA and localizes to the DNA sequence specified by the targeting domain of guide RNA or crRNA, but does not cleave the target. Fusing the inactive Cas13 protein to an effector domain, for example, a transcription repression domain, allows the effector to be recruited to any site specified by the guide RNA.

[0342] In some embodiments, dCas13 may be used to deliver transcription factors or their active domains. dCas13 may be fused to transcription factors or their active domains. The resulting complex may be delivered under the guidance of nucleic acid, for example, a guide sequence.

[0343] Split Protein In this connection, and more generally for various applications as described herein, it is noted that the use of split versions of RNA targeting effector proteins may be envisaged. Indeed, this may not only allow for increased specificity, but may also be advantageous for delivery. Cas13 (e.g., Cas13b) may be split in the sense that the two parts of the Cas13 enzyme essentially contain functional Cas13. Ideally, the split should always be such that the catalytic domain(s) are not affected. This Cas13c may function as a nuclease, or it may be a dead Cas13c, which is an RNA binding protein that has essentially little or no catalytic activity, typically due to mutation(s) in its catalytic domain.

[0344] Each half of split Cas13 may be fused to a dimerization partner. By way of example and not limitation, a rapamycin-sensitive dimerization domain can be used to create a chemically inducible split Cas13 for temporal control of Cas13 activity. Thus, Cas13 can be made chemically inducible by splitting it into two fragments, and the rapamycin-sensitive dimerization domain can be used to reassemble Cas13 in a controlled manner. The two parts of split Cas13c can be considered as the N'-terminal part and the C'-terminal part of split Cas13c. This fusion is typically the split point of Cas13. In other words, the C'-terminus of the N'-terminal part of split Cas13c is fused to one of the dimer halves, while the N'-terminus of the C'-terminal part is fused to the other dimer half.

[0345] Cas13 does not have to be split in the sense that the cleavage point is created de novo. The split point is typically designed in silico and cloned into the construct. Together, the two parts of the split Cas13c, the N'-terminal part and the C'-terminal part, form a complete Cas13 that preferably contains at least 70% or more of wild-type amino acids (or nucleotides encoding them), preferably at least 80% or more of wild-type amino acids (or nucleotides encoding them), preferably at least 90% or more, preferably at least 95% or more, and most preferably at least 99% or more. There may be some trimming and mutants are envisioned. Non-functional domains may be removed completely. The important point is that the two parts can be brought together and the desired Cas13c function is restored or restored. The dimer may be a homodimer or a heterodimer.

[0346] In some cases, the first and second split Cas13 effector proteins can be fused to each other to form a catalytically active Cas13 effector protein. The fusion can be inducible.

[0347] Optimized functional RNA targeting system

[0348] In one embodiment, the present invention provides a system for specifically delivering functional components to RNA environment.This can be achieved by using the CRISPR system that comprises the RNA targeting effector protein of the present invention, which can specifically target different components to RNA.More specifically, such components include activators or repressors, such as activators or repressors of RNA translation, degradation, etc.

[0349] According to one aspect, the present invention provides a non-naturally occurring or engineered composition comprising a guide RNA or crRNA comprising a guide sequence capable of hybridizing to a target sequence of interest in a cell, wherein the guide RNA or crRNA is modified by the insertion of one or more unique RNA sequence(s) that bind to an adaptor protein. In certain embodiments, the RNA sequence can bind to two or more adaptor proteins (e.g., aptamers), each adaptor protein being associated with one or more functional domains. When two or more functional domains are present, the functional domains can be the same or different, e.g., two the same or two different activators or repressors. In an aspect, the present invention provides a composition as described herein, wherein one or more functional domains are attached to an RNA targeting enzyme such that, upon binding to the target RNA, the functional domain is in a spatial orientation that allows the functional domain to function in its ascribed function. In certain aspects, the invention provides a composition as described herein, wherein the composition comprises a CRISPR-Cas13 complex having at least three functional domains, at least one of which is associated with an RNA targeting enzyme and at least two of which are associated with a gRNA or a crRNA.

[0350] CRISPR Development and Use The present invention can be further exemplified and extended based on aspects of the development and use of CRISPR-Cas as set out in the following papers, and in particular as they relate to the delivery of CRISPR protein complexes and the use of RNA-guided endonucleases in cells and organisms: ·Multiplex genome engineering using CRISPR / Cas systems.Cong,L.,Ran,FA,Cox,D.,Lin,S.,Barretto,R.,Habib,N.,Hsu,PD,Wu,X.,Jiang,W.,Marraffini,LA,& Zhang,F.Science Feb 15;339(6121):819-23(2013); ·RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Jiang W.,Bikard D.,Cox D.,Zhang F,Marraffini LA.Nat Biotechnol Mar;31(3):233-9(2013); ·One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR / Cas-Mediated Genome Engineering.Wang H., Yang H., Shivaila CS., Dawlaty MM., Cheng AW., Zhang F., Jaenisch R. Cell May 9;153(4):910-8(2013); ·Optical control of mammalian endogenous transcription and epigenetic states.Konermann S,Brigham MD,Trevino AE,Hsu PD,Heidenreich M,Cong L,Platt RJ,Scott DA,Church GM,Zhang F.Nature.Aug 22;500(7463):472-6.doi:10.1038 / Nature12466.Epub 2013 Aug 23(2013); ·Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity.Ran,FA.,Hsu,PD.,Lin,CY.,Gootenberg,JS.,Konermann,S.,Trevino,AE.,Scott,DA.,Inoue,A.,Matoba,S.,Zhang,Y.,& Zhang,F.Cell Aug 28.pii:S0092-8674(13)01015-5(2013-A); ·DNA targeting specificity of RNA-guided Cas9 nucleases.Hsu,P.,Scott,D.,Weinstein,J.,Ran,FA.,Konermann,S.,Agarwala,V.,Li,Y.,Fine,E.,Wu,X.,Shalem,O.,Cradick,TJ.,Marraffini,LA.,Bao,G.,& Zhang,F.Nat Biotechnol doi:10.1038 / nbt.2647(2013); ·Genome engineering using the CRISPR-Cas9 system.Ran,FA.,Hsu,PD.,Wright,J.,Agarwala,V.,Scott,DA.,Zhang,F.Nature Protocols Nov;8(11):2281-308(2013-B); ·Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells.Shalem,O.,Sanjana,NE.,Hartenian,E.,Shi,X.,Scott,DA.,Mikkelson,T.,Heckl,D.,Ebert,BL.,Root,DE.,Doench,JG.,Zhang,F.Science Dec 12.(2013).; ·Crystal structure of cas9 in complex with guide RNA and target DNA.Nishimasu,H.,Ran,FA.,Hsu,PD.,Konermann,S.,Shehata,SI.,Dohmae,N.,Ishitani,R.,Zhang,F.,Nureki,O.Cell Feb 27,156(5):935-49(2014); ·Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.Wu X.,Scott DA.,Kriz AJ.,Chiu AC.,Hsu PD.,Dadon DB.,Cheng AW.,Trevino AE.,Konermann S.,Chen S.,Jaenisch R.,Zhang F.,Sharp PA.Nat Biotechnol.Apr 20.doi:10.1038 / nbt.2889(2014); ·CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling.Platt RJ,Chen S,Zhou Y,Yim MJ,Swiech L,Kempton HR,Dahlman JE,Parnas O,Eisenhaure TM,Jovanovic M,Graham DB,Jhunjhunwala S,Heidenreich M,Xavier RJ,Langer R,Anderson DG,Hacohen N,Regev A,Feng G,Sharp PA,Zhang F.Cell 159(2):440-455 DOI:10.1016 / j.cell.2014.09.014(2014); ·Development and Applications of CRISPR-Cas9 for Genome Engineering,Hsu PD,Lander ES,Zhang F.,Cell.Jun 5;157(6):1262-78(2014) ·Doi:10.1126 / science.1246981(2014), Wang T, Wei JJ, Sabatini DM, Lander ES, Science.January 3;343(6166):80-84; ·Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation, Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, Sullender M, Ebert BL, Xavier RJ, Root DE 2014)Night Biotechnol.Dec;32(12):1262-7(2014); ·In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9,Swiech L,Heidenreich M,Banerjee A,Habib N,Li Y,Trombetta J,Sur M,Zhang F.,(Published 19 October 2014)Nat Biotechnol.Jan;33(1):102-6(2015); ·Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex,Konermann S,Brigham MD,Trevino AE,Joung J,Abudayyeh OO,Barcena C,Hsu PD,Habib N,Gootenberg JS,Nishimasu H,Nureki O,Zhang F.,Nature.Jan 29;517(7536):583–8(2015) ·A split-Cas9 architecture for inducible genome editing and transcription modulation,Zetsche B,Volz SE,Zhang F.,(August 02 February 2015)Nat Biotechnol.Feb;33(2):139-42(2015); ·Chen S,Sanjana NE,Zheng K,Shalem O,Lee K,Shi X,Scott DA,Song J,Pan JQ,Weissleder R,Lee H,Zhang F,Sharp PA.Cell 160,1246-1260,March 12,2015(August 2015). ·In vivo genome editing using Staphylococcus aureus Cas9, Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F 2015), Nature.Apr 9;520(7546):186-91(2015) ·Shalem et al.,“High-throughput functional genomics using CRISPR-Cas9,”Nature Reviews Genetics 16,299-311(May 2015); ·Xu et al.,“Sequence determinants of improved CRISPR sgRNA design,”Genome Research 25,1147-1157(August 2015); ·Parnas et al.,“A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks,”Cell 162,675-686(July 30,2015) ·Ramanan et al.,CRISPR / Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus,Scientific Reports 5:10833.doi:10.1038 / srep10833(June 2,2015) ·Nishimasu et al.,Crystal Structure of Staphylococcus aureus Cas9,Cell 162,1113-1126(Aug.27,2015) ·BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis,Canver et al.,Nature 527(7577):192-7(Nov.12,2015)doi:10.1038 / nature15521.Epub 2015 Sep 16 ·Cas13 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System,Zetsche et al.,Cell 163,759-71(Sep 25,2015) ·Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems,Shmakov et al.,Molecular Cell,60(3),385-397 doi:10.1016 / j.molcel.2015.10.008 Epub October 22,2015 ·Rationally engineered Cas9 nucleases with improved specificity,Slaymaker et al.,Science 2016 Jan 1 351(6268):84-88 doi:10.1126 / science.aad5227.Epub 2015 Dec 1. ·Gao et al., “Engineered Cas13 Enzymes with Altered PAM Specificities,” bioRxiv 091611;doi:http: / / dx.doi.org / 10.1101 / 091611(Dec.4,2016). Each of these is incorporated herein by reference, may be considered in the practice of the present invention, and is briefly discussed below: ·Cong et al. engineered a type II CRISPR / Cas system for use in eukaryotic cells based on both Streptococcus thermophilus Cas9 and also Streptococcus pyogenes Cas9, and demonstrated that Cas9 nuclease can be directed by small RNAs to induce precise DNA cleavage in human and mouse cells. The authors' work further showed that Cas9 converted into a nicking enzyme can be used to facilitate homologous recombination repair in eukaryotic cells with minimal mutagenic activity. In addition, the authors' work demonstrated that it may be possible to simultaneously edit several endogenous genomic locus sites within mammalian genomes by encoding multiple guide sequences into a single CRISPR sequence, demonstrating the easy programmability and broad applicability of RNA-guided nuclease technology. Thus, it is now possible to program sequence-specific DNA cleavage in cells using RNA, defining a new class of genome engineering tool. These studies further showed that other CRISPR loci may be transplantable into mammalian cells and may also mediate mammalian genome cleavage. Importantly, it can be envisioned that several aspects of the CRISPR / Cas system could be further improved to increase its efficiency and versatility. · Jiang et al. used clustered regularly interspaced short random repeats (CRISPR)-associated Cas9 endonuclease complexed with dual RNA to introduce precise mutations into the genomes of Streptococcus pneumoniae and Escherichia coli. The approach relies on dual RNA:Cas9-induced cleavage at targeted genomic sites to kill non-mutated cells, circumventing the need for selectable markers or counterselection systems. The study reported that reprogramming dual RNA:Cas9 specificity by altering the sequence of a short CRISPR RNA (crRNA) to generate single and multiple nucleotide changes results in template editing. The study showed that multiplexing mutagenesis is possible when two crRNAs are used simultaneously. Furthermore, when this approach was combined with recombination, in S. pneumoniae, nearly 100% of cells recovered using the described approach contained the desired mutations, and in E. coli, 65% of cells recovered contained mutations. · Wang et al. (2013) used the CRISPR / Cas system to generate mice carrying mutations in multiple genes in a single step, which was previously generated in multiple steps by sequential recombination in embryonic stem cells and / or by time-consuming crossbreeding of mice carrying single mutations. The CRISPR-Cas system can greatly accelerate in vivo studies of functionally redundant genes and epistatic interactions. Konermann et al. (2013) addressed the need in the art for a versatile and robust technique that allows optical and chemical modulation of CRISPR Cas9 enzymes and also DNA binding domains based on transcription activator-like effectors. Ran et al. (2013-A) described a method in which Cas9 nickase mutants were combined with paired guide RNAs to introduce targeted double-strand breaks. This addresses the problem that the Cas9 nuclease of microbial CRISPR-Cas systems is targeted to specific genomic loci by guide sequences, which can tolerate some mismatches with the DNA target and therefore promote undesired off-target mutagenesis. Because individual nicks in the genome are repaired with high fidelity, double-strand breaks require simultaneous nicking by appropriately offset guide RNAs, increasing the number of bases that are specifically recognized for targeted cleavage. The authors demonstrated that paired nicking can be used to reduce off-target activity in cellular systems by 50- to 1,500-fold and promote gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity. ·Hsu et al. (2013) characterized the specificity of SpCas9 targeting in human cells to inform target site selection and avoid off-target effects. The study assessed SpCas9-induced indel mutation levels in over 700 guide RNA variants and over 100 predicted genomic off-target loci in 293T and 293FT cells. The authors showed that SpCas9 tolerates mismatches between guide RNA and target DNA at various positions in a sequence-dependent manner, with sensitivity to the number, position, and distribution of mismatches. The authors further showed that SpCas9-mediated cleavage is not affected by DNA methylation, and that the dosage of SpCas9 and guide RNA can be titrated to minimize off-target modifications. In addition, to facilitate mammalian genome engineering applications, the authors reported the provision of a web-based software tool to guide target sequence selection and validation as well as off-target analysis. Ran et al. (2013-B) described a set of tools for Cas9-mediated genome editing using non-homologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells and for generating engineered cell lines for downstream functional studies. To minimize off-target cleavage, the authors further described a double-nicking strategy using a Cas9 nickase mutant with paired guide RNAs. From the protocol provided by the authors, guidelines for target site selection, evaluation of cleavage efficiency, and analysis of off-target activity were experimentally derived. The study showed that starting from target design, genetic modification could be achieved within only 1-2 weeks, and engineered clonal cell lines could be derived within 2-3 weeks. ·Shalem et al. described a new method to interrogate gene function on a genome-wide scale. Their study showed that delivery of a genome-wide CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enabled both negative and positive selection screens in human cells. First, the authors demonstrated the identification of genes essential for cell survival in cancer and pluripotent stem cells using the GeCKO library. Second, they screened for genes whose loss is involved in resistance to the therapeutic drug vemurafenib, which inhibits mutant protein kinase BRAF, in a melanoma model. Their study showed that the top ranked candidates included previously validated genes NF1 and MED12 as well as novel hits NF2, CUL3, TADA2B, and TADA1. They observed a high degree of concordance and a high rate of hit confirmation between independent guide RNAs targeting the same gene, thus demonstrating the promise of genome-wide screening with Cas9. ·Nishimasu et al. reported the crystal structure of Streptococcus pyogenes Cas9 complexed with an sgRNA and its target DNA at 2.5 Å resolution. The structure reveals a two-lobe organization composed of a target recognition lobe and a nuclease lobe that accepts the sgRNA:DNA RNA duplex in a positively charged groove at their interface. The recognition lobe is critical for sgRNA and DNA binding, whereas the nuclease lobe contains the HNH and RuvC nuclease domains that are appropriately positioned to cleave the complementary and non-complementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain involved in the interaction with the protospacer adjacent motif (PAM). This high-resolution structural analysis and accompanying functional analysis reveal the molecular mechanism of RNA-guided DNA targeting by Cas9, which is in turn paving the way for the rational design of novel and versatile genome editing technologies. ·Wu et al. mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNA (sgRNA) in mouse embryonic stem cells (mESCs). The authors showed that each of the four sgRNAs tested targeted dCas9 to tens to thousands of genomic sites, often characterized by a 5-nucleotide seed region and a NGG protospacer adjacent motif (PAM) in the sgRNA. Inaccessibility of chromatin reduces dCas9 binding to other sites containing matching seed sequences; thus, 70% of off-target sites are associated with genes. The authors showed that targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site that was mutated above background levels. The authors propose a two-state model of Cas9 binding and cleavage, in which seed matching triggers binding, but cleavage requires extensive pairing with the target DNA. Platt et al. established Cre-dependent Cas9 knock-in mice. The authors demonstrated in vivo and ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Hsu et al. (2014) is a review article that broadly explores the history of CRISPR-Cas9 from yogurt to genome editing, including genetic screening of cells. ·Wang et al. (2014) describes a pooled loss-of-function genetic screening method suitable for both positive and negative selection using genome-scale lentiviral single guide RNA (sgRNA) libraries. ·Doench et al. created a pool of sgRNAs tiling across all possible target sites in a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target genes by antibody counterstaining and flow cytometry. The authors showed that optimization of the PAM improved activity and also provided an online tool for designing sgRNAs. · Swiech et al. demonstrate that AAV-mediated SpCas9 genome editing may enable reverse genetic studies of gene function in the brain. Konermann et al. (2015) discuss the ability to add multiple effector domains, e.g., transcriptional activators, functional and epigenetic regulators, with and without linkers at appropriate positions on the guide, such as the stem or tetraloop. · Zetsche et al. demonstrate that the Cas9 enzyme can be split into two halves, thus controlling the assembly of Cas9 for activation. · Chen et al. on multiplex screening by demonstrating that genome-wide in vivo CRISPR-Cas9 screening in mice reveals regulatory genes of lung metastasis. Ran et al. (2015) demonstrated that SaCas9 and its genome editing capabilities cannot be inferred from biochemical assays. Shalem et al. (2015) described methods to synthetically silence (CRISPRi) or activate (CRISPRa) expression using fusions of catalytically inactive Cas9 (dCas9), demonstrating advances in using Cas9 for genome-wide screens, including arrayed and pooled screens, knockout approaches to inactivate genomic loci, and strategies to modulate transcriptional activity. Xu et al. (2015) evaluated DNA sequence features that contribute to the efficiency of single guide RNAs (sgRNAs) in CRISPR-based screening. The authors investigated the efficiency of CRISPR / Cas9 knockout and nucleotide preferences at the cleavage site. They also found that the sequence preferences of CRISPRi / a differed substantially from those of CRISPR / Cas9 knockout. (2015) identified genes that control tumor necrosis factor (Tnf) induction by bacterial lipopolysaccharide (LPS) by introducing a genome-wide pooled CRISPR-Cas9 library into dendritic cells (DCs). Known regulators of Tlr4 signaling and previously unknown candidates were identified and grouped into three functional modules with distinct effects on the canonical response to LPS. Ramanan et al (2015) demonstrated cleavage of viral episomal DNA (cccDNA) in infected cells. The HBV genome is present in the nuclei of infected hepatocytes as a 3.2 kb double-stranded episomal DNA species called covalently closed circular DNA (cccDNA), and cccDNA is a major component of the HBV life cycle whose replication is not inhibited by current therapies. The authors showed that sgRNAs specifically targeting highly conserved regions of HBV robustly inhibited viral replication and depleted cccDNA. (2015) reported the crystal structure of SaCas9 in complex with a single guide RNA (sgRNA) and its double-stranded DNA target, containing 5'-TTGAAT-3'PAM and 5'-TTGGGT-3'PAM. Structural comparison of SaCas9 and SpCas9 revealed both structural conservation and diversity, explaining their distinct PAM specificity and orthologous sgRNA recognition. · Canver et al. (2015) demonstrated CRISPR-Cas9-based functional studies of non-coding genomic elements. The authors developed a pooled CRISPR-Cas9 guide RNA library and performed in situ saturation mutagenesis of the human and mouse BCL11A enhancer, which revealed key features of the enhancer. (2015) reported the characterization of Cas13, a class 2 CRISPR nuclease from Francisella novicida U112 that has distinct characteristics from Cas9. Cas13 is a single RNA-guided endonuclease that lacks tracrRNA and cleaves DNA by utilizing T-rich protospacer adjacent motifs and staggered DNA double-strand breaks. Shmakov et al. (2015) reported three distinct class 2 CRISPR-Cas systems. Two of the system CRISPR enzymes (C2c1 and C2c3) contain RuvC-like endonuclease domains that are distantly related to Cas13. Unlike Cas13, C2c1 depends on both crRNA and tracrRNA for DNA cleavage. The third enzyme (C2c2) contains two predicted HEPN RNase domains and is tracrRNA-independent. · Slaymaker et al. (2016) reported improving the specificity of Streptococcus pyogenes Cas9 (SpCas9) using structure-guided protein engineering. The authors developed an “enhanced specificity” SpCas9 (eSpCas9) mutant that maintains robust on-target cleavage while exhibiting reduced off-target effects.

[0351] The methods and tools provided herein are exemplified for Cas13, a type II nuclease that does not use tracrRNA. Orthologs of Cas13 have been identified in different bacterial species as described herein. Furthermore, type II nucleases with similar properties can be identified using methods described in the art (Shmakov et al. 2015, 60:385-397; Abudayeh et al. 2016, Science, 5; 353 (6299)). In certain embodiments, such a method for identifying novel CRISPR effector proteins may include the steps of selecting a sequence from a database that encodes a seed that identifies the presence of a CRISPR Cas locus, identifying a locus located within 10 kb of the seed that contains an open reading frame (ORF) in the selected sequence, and selecting a locus from the identified locus that contains an ORF, of which only one ORF encodes a novel CRISPR effector that has more than 700 amino acids and has 90% or less homology to a known CRISPR effector. In certain embodiments, the seed is a protein common to CRISPR-Cas systems, such as Cas1. In further embodiments, a CRISPR array is used as a seed to identify novel effector proteins.

[0352] Since then, the efficacy of the present invention has been demonstrated. Pre-assembled recombinant CRISPR-Cas13 complexes containing Cas13 and crRNA may be transfected, for example, by electroporation, resulting in high mutation rates and no detectable off-target mutations. Hur, JK et al, Targeted mutagenesis in mice by electroporation of Cas13 ribonucleoproteins, Nat Biotechnol. 2016 Jun 6. doi:10.1038 / nbt.3596. Genome-wide analysis shows that Cas13 is highly specific. In one measure, significantly fewer in vitro cleavage sites were determined for Cas13 in human HEK293T cells compared to SpCas9. Kim, D. et al., Genome-wide analysis reveals specificities of Cas13 endonucleases in human cells, Nat Biotechnol. 2016 Jun 6. doi:10.1038 / nbt.3609. An efficient multiplexing system using Cas13 has been demonstrated in Drosophila using gRNAs processed from arrays containing the inventive tRNAs. Port, F. et al, Expansion of the CRISPR toolbox in an animal with tRNA-flanked Cas9 and Cas13 gRNAs. doi:http: / / dx.doi.org / 10.1101 / 046417.

[0353] See also “Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing”, Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter, Jennifer A. Foden, Vishal Thapar, Deepak Reyon, Mathew J. Goodwin, Martin J. Aryee, J. Keith Joung Nature Biotechnology 32(6):569-77 (2014), which relates to dimeric RNA-guided FokI nucleases that recognize extension sequences and can edit endogenous genes in human cells with high efficiency.

[0354] For general information regarding CRISPR-Cas systems, their components, and delivery of such components, including methods, materials, delivery vehicles, vectors, particles, and amounts and formulations thereof, as well as their production and use, and CRISPR-Cas expressing eukaryotic cells, eukaryotic organisms such as mice, see U.S. Pat. Nos. 8,999,641, 8,993,233, 8,697,359, 8,77 Nos. 1,945, 8,795,965, 8,865,406, 8,871,445, 8,889,356, 8,889,418, 8,895,308, 8,906,616, 8,932,814, and 8,945,839; U.S. Patent Application Publication No. US2014-0310830 (U.S. Patent Application No. 14 / 105,031), US2014-0287938 A1 (U.S. Patent Application No. 14 / 213,991), US2014-0273234 A1 (U.S. Patent Application No. 14 / 293,674), US2014-0273232 A1 (U.S. Patent Application No. 14 / 290,575), US2014-0273231 (U.S. Patent Application No. 14 / 259,420), US2014-0256046 A1 (U.S. Patent Application No. 14 / 226,274), US2014-0248702 A1 (U.S. Patent Application No. 14 / 258,458), US2014-0242700 A1 (U.S. Patent Application No. 14 / 222,930), US2014-0242699 A1 (U.S. Patent Application No. 14 / 183,512), US2014-0242664 A1 (U.S. Patent Application No. 14 / 104,990), US2014-0234972 A1 (U.S. Patent Application No. 14 / 183,471), US2014-0227787 A1 (U.S. Patent Application No. 14 / 256,912), US2014-0189896 A1 (U.S. Patent Application No. 14 / 105,035), US2014-0186958 (U.S. Patent Application No. 14 / 105,017), US2014-0186919 A1 (U.S. Patent Application No. 14 / 104,977), US2014-0186843 A1 (U.S. Patent Application No. 14 / 104,900), US2014-0179770 A1 (U.S. Patent Application No. 14 / 104,837) and US2014-0179006 A1 (U.S. Patent Application No. 14 / 183,486),US2014-0170753 (U.S. Patent Application Serial No. 14 / 183,429); US2015-0184139 (U.S. Patent Application Serial No. 14 / 324,960); 14 / 054,414, European Patent Applications EP2 771 468 (EP13818570.7), EP2 764 103 (EP13824232.6), and EP2 784 162 (EP14170383.5); and PCT patent publications WO2014 / 093661 (PCT / US2013 / 074743), WO2014 / 093694 (PCT / US2013 / 074790), WO2014 / 093595 (PCT / US2013 / 074611), WO2014 / 093718 (PCT / US2013 / 074825), WO2014 / 093709 (PCT / US2013 / 074812), WO2014 / 0936 22(PCT / US2013 / 074667), WO2014 / 093635(PCT / US2013 / 074691), WO2014 / 093655(PCT / US2013 / 074736), WO2014 / 093712( PCT / US2013 / 074819), WO2014 / 093701(PCT / US2013 / 074800), WO2014 / 018423(PCT / US2013 / 051418), WO2014 / 204723(PCT / US2014 / 041790), WO2014 / 204724(PCT / US2014 / 041800), WO2014 / 204725(PCT / US2014 / 041803), WO2014 / 204726(PCT / US2 014 / 041804), WO2014 / 204727(PCT / US2014 / 041806), WO2014 / 204728(PCT / US2014 / 041808), WO2014 / 204729(PCT / US2014 / 041809), WO2015 / 089351(PCT / US2014 / 069897), WO2015 / 089354(PCT / US2014 / 069902), WO2015 / 089364(PCT / US2014 / 0699 25), WO2015 / 089427(PCT / US2014 / 070068), WO2015 / 089462(PCT / US2014 / 070127), WO2015 / 089419(PCT / US2014 / 070057),WO2015 / 089465(PCT / US2014 / 070135), WO2015 / 089486(PCT / US2014 / 070175), WO2015 / 058052(PCT / US2014 / 061077), WO2015 / 070083(PCT / US2014 / 0 64663), WO2015 / 089354(PCT / US2014 / 069902), WO2015 / 089351(PCT / US2014 / 069897), WO2015 / 089364(PCT / US2014 / 069925), WO2015 / 089427(PCT / US See WO2014 / 070068), WO2015 / 089473 (PCT / US2014 / 070152), WO2015 / 089486 (PCT / US2014 / 070175), WO2016 / 049258 (PCT / US2015 / 051830), WO2016 / 094867 (PCT / US2015 / 065385), WO2016 / 094872 (PCT / US2015 / 065393), WO2016 / 094874 (PCT / US2015 / 065396), and WO2016 / 106244 (PCT / US2015 / 067177).

[0355] Also, U.S. Patent Application No. 62 / 180,709, June 17, 2015, PROTECTED GUIDE RNAS (PGRNAS); U.S. Patent Application No. 62 / 091,455, filed December 12, 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. Patent Application No. 62 / 096,708, December 24, 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. Patent Application No. 62 / 091,462, December 12, 2014, U.S. Patent Application No. 62 / 096,324, December 23, 2014, U.S. Patent Application No. 62 / 180,681, June 17, 2015, and U.S. Patent Application No. 62 / 237,496, October 5, 2015, DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS; U.S. Patent Application No. 62 / 091,456, Dec. 12, 2014 and No. 62 / 180,692, Jun. 17, 2015, ESCORTED AND FUNCTIONALIZED GUIDES FOR CRISPR-CAS SYSTEMS; U.S. Patent Application No. 62 / 091,461, Dec. 12, 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR GENOME EDITING AS TO HEMATOPOETIC STEM CELLS (HSCs); U.S. Patent Application No. 62 / 094,903, Dec. 19, 2014, UNBIASED IDENTIFICATION OF DOUBLE-STRAND BREAKS AND GENOMIC REARRANGEMENT BY GENOME-WISE INSERT CAPTURE SEQUENCING; U.S. Patent Application No. 62 / 096,761, Dec. 24, 2014, ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED ENZYME AND GUIDE SCAFFOLDS FOR SEQUENCE MANIPULATION; U.S. Patent Application No. 62 / 098,059, Dec. 30, 2014, U.S. Patent Application No. 62 / 181,641, Jun. 18, 2015, and U.S. Patent Application No. 62 / 181,667, Jun. 18, 2015, RNA-TARGETING SYSTEM;U.S. Patent Application No. 62 / 096,656, Dec. 24, 2014 and No. 62 / 181,151, Jun. 17, 2015, CRISPR HAVING OR ASSOCIATED WITH DESTABILIZATION DOMAINS; U.S. Patent Application No. 62 / 096,697, Dec. 24, 2014, CRISPR HAVING OR ASSOCIATED WITH AAV; U.S. Patent Application No. 62 / 098,158, Dec. 30, 2014, ENGINEERED CRISPR COMPLEX INSERTIONAL TARGETING SYSTEMS; U.S. Patent Application No. 62 / 151,052, Apr. 22, 2015, CELLULAR TARGETING FOR EXTRACELLULAR EXOSOMAL REPORTING; U.S. Patent Application No. 62 / 054,490, Sep. 24, 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING PARTICLE DELIVERY COMPONENTS; U.S. Patent Application No. 61 / 939,154, 02 / 12 / 14, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. Patent Application No. 62 / 055,484, 09 / 25 / 14, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. Patent Application No. 62 / 087,537, 12 / 04 / 14, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS;U.S. Patent Application No. 62 / 054,651, filed Sep. 24, 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OF MULTIPLE CANCER MUTATIONS IN VIVO; U.S. Patent Application No. 62 / 067,886, filed Oct. 23, 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OF MULTIPLE CANCER MUTATIONS IN VIVO; U.S. Patent Application No. 62 / 054,675, filed Sep. 24, 2014 and U.S. Patent Application No. 62 / 181,002, filed Jun. 17, 2015, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS IN NEURONAL CELLS / TISSUES; U.S. Patent Application No. 62 / 054,528, 9 / 24 / 14, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS IN IMMUNE DISEASES OR DISORDERS; U.S. Patent Application No. 62 / 055,454, 9 / 25 / 14, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING CELL PENETRATION PEPTIDES (CPP); U.S. Patent Application No. 62 / 055,460, 9 / 25 / 14, MULTIFUNCTIONAL-CRISPR COMPLEXES AND / OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES;U.S. Patent Application No. 62 / 087,475, Dec. 4, 2014 and No. 62 / 181,690, Jun. 18, 2015, FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. Patent Application No. 62 / 055,487, Sep. 25, 2014, FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. Patent Application No. 62 / 087,546, Dec. 4, 2014 and No. 62 / 181,687, Jun. 18, 2015, MULTIFUNCTIONAL CRISPR COMPLEXES AND / OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; and U.S. Patent Application No. 62 / 098,285, Dec. 30, 2014, CRISPR-MEDIATED IN VIVO MODELING AND GENETIC SCREENING OF TUMOR GROWTH AND METASTASIS.

[0356] No. 62 / 181,659, filed June 18, 2015, and U.S. Patent Application No. 62 / 207,318, filed August 19, 2015, ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS, ENZYME AND GUIDE SCAFFOLDS OF CAS9 ORTHOLOGS AND VARIANTS FOR SEQUENCE MANIPULATION. US Patent Application No. 62 / 181,663, June 18, 2015 and US Patent Application No. 62 / 245,264, October 22, 2015, NOVEL CRISPR ENZYMES AND SYSTEMS, U.S. Patent Application No. 62 / 181,675, June 18, 2015, 62 / 285...

Claims

1. An engineered, non-naturally occurring system comprising a catalytically inactive Cas13 effector protein (dCas13) or a nucleotide sequence encoding said catalytically inactive Cas13 effector protein.

2. The system of claim 1, wherein the dCas13 protein is truncated at the C-terminus, N-terminus, or both.

3. The system of claim 2, wherein the dCas13 is truncated by at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 220, at least 240, at least 260, or at least 300 amino acids on the C-terminus.

4. The system of claim 2, wherein the dCas13 is truncated by at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 220, at least 240, at least 260, or at least 300 amino acids on the N-terminus.

5. The system of claim 2, wherein the truncated form of the Cas13 effector protein is truncated at the C-terminus Δ984-1090, the C-terminus Δ1026-1090, the C-terminus Δ1053-1090, the C-terminus Δ934-1090, the C-terminus Δ884-1090, the C-terminus Δ834-1090, the C-terminus Δ784-1090, or the C-terminus Δ734-1090, and the amino acid positions of the truncations correspond to the amino acid positions of the Prevotella sp. P5-125 Cas13b protein.

6. The system of claim 2, wherein the truncated form of the Cas13 effector protein is truncated at the C-terminus Δ795-1095, the amino acid positions of the truncation corresponding to the amino acid positions of the Riemerella anatipestifer Cas13b protein.

7. 3. The system of claim 2, wherein the truncated forms of the Cas13 effector protein are truncated at the C-terminus Δ875-1175, C-terminus Δ895-1175, C-terminus Δ915-1175, C-terminus Δ935-1175, C-terminus Δ955-1175, C-terminus Δ975-1175, C-terminus Δ995-1175, C-terminus Δ1015-1175, C-terminus Δ1035-1175, C-terminus Δ1055-1175, C-terminus Δ1075-1175, C-terminus Δ1095-1175, C-terminus Δ1115-1175, C-terminus Δ1135-1175, C-terminus Δ1155-1175, and the amino acid positions correspond to the amino acid positions of the Porphyromonas gulae Cas13b protein.

8. The system of claim 2, wherein the truncated form of the Cas13 effector protein is truncated at the N-terminus Δ1-125, the N-terminus Δ1-88, or the N-terminus Δ1-72, and the amino acid positions of the truncations correspond to the amino acid positions of Prevotella sp. P5-125 Cas13b protein.

9. The system of claim 1 , wherein the dCas13 comprises a truncated form of the Cas13 effector protein with a HEPN domain of the Cas13 effector protein.

10. The system of claim 1 , wherein the Cas13 effector protein is Cas13a.

11. The system of claim 1 , wherein the Cas13 effector protein is Cas13b.

12. The system of claim 1 , wherein the Cas13 effector protein is Cas13c or Cas13d.

13. The system of claim 1 further comprising a functional component or wherein the nucleotide sequence further encodes said functional component.

14. The system of claim 13 , wherein the functional component is a base editing component.

15. The system of claim 14, wherein the base editing component comprises an adenosine deaminase, a cytidine deaminase, or a catalytic domain thereof.

16. The system of claim 15, wherein the adenosine deaminase, the cytidine deaminase, or the catalytic domain thereof is fused to the dCas13.

17. The system of claim 16, wherein the adenosine deaminase, the cytidine deaminase, or the catalytic domain thereof is fused to the dCasl3 by a linker.

18. The system of claim 15, wherein the adenosine deaminase, the cytidine deaminase, or the catalytic domain thereof is inserted into an internal loop of the dCasl3.

19. The system of claim 15, wherein the adenosine deaminase, the cytidine deaminase, or the catalytic domain thereof is linked to an adaptor protein.

20. 20. The system of claim 19, wherein the adaptor protein is selected from MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s and PRR1.

21. 16. The system of claim 15, wherein the adenosine deaminase, the cytidine deaminase, or the catalytic domain thereof is a human, cephalopod, or Drosophila protein.

22. The system of claim 1 , wherein the dCas13 is a split Cas13 effector protein.

23. 23. The system of claim 22, wherein the split Cas13 effector protein is a first split Cas13 effector protein that can be fused to a second split Cas13 effector protein to form a catalytically active Cas13 effector protein.

24. 24. The system of claim 23, wherein the fusion of the first and second Cas13 effector proteins is inducible.

25. The system according to claim 13 , wherein the functional component is a transcription factor or an active domain thereof.

26. The system of claim 25, wherein the dCas13 is fused to the transcription factor or its active domain.

27. The system of claim 1 further comprising a guide sequence.

28. A vector system comprising one or more vectors encoding dCas13 according to any one of the preceding claims.

29. An in vitro or ex vivo host cell or its progeny or a cell line or its progeny comprising a system according to any one of claims 1 to 27.

30. 22. A method for programmable and targeted base editing of a target sequence comprising delivering to a cell the system of any one of claims 1 to 21.

31. 31. The method of claim 30, further comprising determining the target sequence of interest and selecting an adenosine deaminase, cytidine deaminase, or catalytic domain thereof, that most efficiently deaminates adenine or cytidine present in the target sequence.

32. 32. The method of claim 31 , wherein the deamination cures a disease caused by a transcript containing a pathogenic T(U)→C, A→G, G→A, or C→T point mutation.

33. 1. A method for modifying a nucleic acid at a target locus, comprising: (1) The system of claim 1, wherein the dCas13 is a first split Cas13 effector protein; and (2) the method comprising delivering a second split Cas13 effector protein, wherein the first and second split Cas13 effector proteins are capable of forming a catalytically active Cas13 effector protein.

34. 1. A method for regulating a target gene in a cell, comprising: The method comprises delivering the system described in claim 1, wherein the dCas13 is fused to a transcription factor or an activity domain thereof.

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