Methods for producing genetically modified cells

Abstract

The present invention relates to an RNA-mediated base editing system that allows the recruitment of multiple effector proteins to a single target site, enabling highly efficient base editing. The present invention further relates to methods and kits for genetically modifying cells.

Description

[Technical field]

[0001] The present invention relates to methods for producing genetically modified cells, including plant cells, animal cells, mammalian cells, immune cells and stem cells, using RNA-mediated base editing. [Background technology]

[0002] Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas technology has emerged as an innovative nucleic acid-guided gene editing tool to precisely edit genes of interest. Directed by guide RNA (gRNA), the CRISPR-Cas system induces double-strand breaks (DSBs) within genes of interest, thereby forming small insertions or deletions (collectively referred to as "indels") that are created by highly variable repair via the non-homologous end joining (NHEJ) pathway. Alternatively, precise genome recombination can be achieved by introducing DSBs together with the simultaneous delivery of DNA templates for repair by homology-directed repair (HDR). Although this approach is efficient and reliable for simply disrupting a single gene by NHEJ, precise recombination of single nucleotides by HDR is extremely inefficient. Furthermore, induction of multiple DSBs during multiplexed gene editing procedures can result in the formation of undesirable genotoxic and potentially carcinogenic macroscopic chromosomal translocations. Thus, there remains a need in the art for more controlled and safer methods of multiple genetic manipulation of cells, particularly human cells, that limit the induction of toxic DSBs.

[0003] Recently, an RNA-mediated base editing system has also been developed and is described in US Patent Publication 1 ("Nuclease-independent targeted gene editing platform and its application" (Rutgers)). This system recruits a base editing enzyme to a target DNA sequence via the guide RNA component of the CRISPR-Cas complex. This system contains a modified guide RNA with a reprogrammable RNA motif (also known in the art as an RNA aptamer) at its 3' end that recruits a cognate RNA binding domain (also known in the art as an RNA aptamer ligand) fused to an effector (such as a deaminase effector). Using this system, targeted nucleotide modifications have been achieved with high precision in eukaryotic cells, including prokaryotic and mammalian cells; see US Patent Publications 1 and 2. As disclosed in US Patent Publication 3, a new second-generation base editing system with enhanced specificity and efficacy in prokaryotic cells has been tested and further improved in mammalian cells. The second-generation system / platform exhibits high specificity, high efficacy, and low off-target liability. With a modular design that completely separates the nucleic acid modification module from the nucleic acid recognition module, as well as other advantages disclosed herein, the RNA-mediated base editing platform provides an alternative to the recruitment of effectors via fusion to or direct interaction with sequence-targeting proteins, which could not effectively separate the sequence targeting function from the nucleic acid modification function.Without the requirement of DNA DSBs and HDR, the new system provides a powerful tool for genetic engineering and therapeutic development.

[0004] Compared with recruitment by direct protein fusion (prior art BE systems) or other recruitment approaches by protein components, the RNA scaffold-mediated effector recruitment mechanism has several distinct features that are potentially advantageous both for system engineering and for achieving better functionality. For example, the modular design, where the nucleic acid sequence targeting function and the effector function are present on different molecules, allows the functional modules to be reprogrammed independently. This does not require re-engineering of individual functional Cas9 fusion proteins. Thus, the combination of different modules can be achieved by exchanging the nucleotide sequences of the recruitment RNA motif and the cognate RNA binding domain. On the other hand, recruitment of effectors by direct fusion or direct interaction with the protein components of the sequence targeting unit always requires the re-engineering of new fusion proteins, which often shows unpredictable results and is technically more challenging. Moreover, the fused effectors are small in size, potentially allowing functional effectors to oligomerize more efficiently, whereas the formation of oligomers is hindered due to steric hindrance by direct fusion. Furthermore, because RNA scaffold-mediated base editing does not require the generation of Cas fusion proteins that would further increase the gene / transcript size of Cas, RNA-mediated base editing systems could potentially be constructed in a more efficient manner for packaging and delivery by viral vectors.

[0005] Despite the advantages of the above-mentioned RNA-mediated base editing system, significant optimization is still required.The efficiency of base editing enzymes such as deaminase is highly dependent on the sequence of target nucleic acid.Therefore, when using RNA-mediated base editing system, effector must be carefully selected based on target sequence.The process of selecting optimal effector and testing the system can be time-consuming, so it is desirable to develop a more efficient base editing system. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 017 / 011721 Brochure [Patent Document 2] International Publication No. 2018 / 129129 Brochure [Patent Document 3] US Patent Application Publication No. 62 / 901,584 Summary of the Invention [Problem to be solved by the invention]

[0007] The inventors of the present invention have developed a novel RNA-mediated base editing system that allows the recruitment of multiple effectors to a single target site, enabling highly efficient base editing.

[0008] The RNA-aptamer-mediated base editing system described in the prior patent application WO 2005 / 023363 was assumed to involve irreversible recruitment of a single aptamer ligand that precisely recombines into the target genome locus. However, the present inventors surprisingly found that the recruitment mechanism is actually reversible, which is responsible for improving the efficiency of base editing. Based on this finding, the present inventors carefully devised a system that can recruit multiple effectors to a single genome locus to perform single or multiple highly efficient editing within this region. Furthermore, the system can be applied to edit multiple different genome loci simultaneously (multiplexed base editing). [Means for solving the problem]

[0009] In a first aspect, the present invention provides a method for genetic modification of a cell by base editing, comprising: i) sequence-targeted proteins; ii) a) a guide RNA, and b) a ligand-binding moiety; and iii) c) a ligand capable of reversibly binding to said ligand binding moiety, and d) two or more effector proteins, each of which comprises an effector domain; The present invention provides a method for the preparation of a method for the preparation of a nucleic acid sequence comprising:

[0010] In one embodiment, there is provided a method for genetic modification of a cell by base editing, the method comprising: i) sequence-targeted proteins; ii) e) a guide RNA, and f) a ligand-binding moiety; and iii) g) a ligand capable of reversibly binding to said ligand binding moiety, and h) two or more effector proteins, each of which comprises an effector domain; The present invention provides a method comprising: bringing into a cell, e.g., introducing into a cell

[0011] In one embodiment, the guide RNA (or gRNA) comprises a crRNA that comprises a targeting sequence complementary to the target nucleic acid. In one embodiment, the guide RNA further comprises a tracrRNA or a scoutRNA that can bind to a sequence-targeting protein. Thus, in one embodiment, the guide RNA comprises only a crRNA. In other embodiments, the guide RNA comprises a crRNA and a tracrRNA. In an alternative embodiment, the guide RNA comprises a crRNA and a scoutRNA.

[0012] In one embodiment, the sequence-targeting protein is a Cas protein. When the effector is bound to (or contains) a ligand, the system has a modular design. The presence of a ligand-binding moiety in an RNA-ligand binding complex allows the complex to associate with a corresponding ligand associated with (or contained in) the effector. Thus, the ligand-binding moiety associates with the guide RNA in a manner and orientation that allows it to reversibly associate with the ligand. Similarly, the ligand is attached or bound to the effector in a manner that allows it to reversibly bind with the ligand-binding moiety. Where an effector (or "effector domain") is bound to (or contains) a ligand, the system has a modular design. The presence of a ligand-binding moiety in an RNA-ligand binding complex allows the complex to associate with a corresponding ligand associated with (or contained within) the effector domain. Thus, the ligand-binding moiety is associated with the guide RNA in a manner and orientation such that it can reversibly associate with the ligand. Similarly, the ligand is attached or bound to the effector domain in a manner such that it can reversibly bind with the ligand-binding moiety. When the ligand and the ligand-binding moiety are associated with each other, the effector attached to or associated with the ligand becomes part of any base editing complex that contains the RNA-ligand binding complex. When the base editing complex also contains a Cas protein, the Cas protein and the effector can be held in the same location, e.g., at or near a target site of interest.

[0013] Therefore, in order to use a particular effector with a Cas protein, the effector must be associated with a ligand that can reversibly associate with the ligand-binding moiety that is part of the base editing complex containing the Cas protein. To change the effector from one system to the next, only the effector-ligand needs to be changed. As a result, the same RNA-ligand binding complex and its associated Cas protein can be used with multiple different effectors. Multiple different effectors can be used simultaneously or sequentially in the same system, or simultaneously or sequentially in different systems, by associating and dissociating their ligands with the ligand-binding moiety.

[0014] In a preferred embodiment, the ligand binding portion of the RNA-ligand binding complex is an RNA motif and the ligand of the effector protein is an RNA binding domain. In a preferred embodiment, the ligand binding portion of the RNA-ligand binding complex is an RNA motif and the ligand of the effector protein is an RNA binding domain that can recognize and bind to the RNA motif. In an embodiment, the RNA-ligand binding complex comprises a guide RNA and an RNA motif, it is referred to as an RNA scaffold.

[0015] When the effector is bound to (or contains) an RNA binding domain, the system has a modular design. The presence of an RNA motif in the RNA-ligand binding complex (RNA scaffold) allows this complex to associate with the corresponding RNA binding domain associated with (or contained in) the effector. Thus, the RNA motif associates with the guide RNA in such a manner and orientation that it can reversibly associate with the RNA binding domain. Similarly, the RNA binding domain attaches to or binds to the effector in such a manner that it can reversibly bind with the RNA motif.

[0016] When an effector (or "effector domain") is bound to (or contains) a ligand, the system has a modular design. The presence of an RNA motif in an RNA-ligand binding complex (RNA scaffold) allows the complex to associate with a corresponding ligand associated with (or contained within) the effector domain. Thus, the RNA motif associates with the guide RNA in a manner and orientation that allows it to reversibly associate with the RNA binding domain. Similarly, the RNA binding domain is attached or bound to the effector domain in a manner that allows it to reversibly bind with the RNA motif.

[0017] When the RNA-binding domain and the RNA motif are associated with each other, the effector associated with the RNA-binding domain becomes part of any base-editing complex that contains the RNA-ligand binding complex (RNA scaffold). When the base-editing complex also contains a Cas protein, the Cas protein and the effector can be held in the same location, e.g., at or near the target site of interest.

[0018] Thus, the present invention provides a method for using a particular effector with a Cas protein, where the effector comprises an RNA binding domain that can reversibly associate with an RNA motif that is part of a base editing complex that contains said Cas protein. To change the effector from one system to the next, only the effector-RNA binding domain needs to be changed. As a result, the same RNA-ligand binding complex (RNA scaffold) and its associated Cas protein can be used with multiple different effectors. Multiple different effectors can be used simultaneously or sequentially in the same system, or simultaneously or sequentially in different systems, by associating and dissociating their RNA binding domains with the RNA motif.

[0019] In any embodiment of the present invention, each of the two or more effector proteins comprises a ligand that can reversibly bind to the same ligand binding portion in the RNA-ligand binding complex.In other words, each of the two or more effector proteins can interact with a common ligand binding portion, and therefore each can be recruited by the same RNA-ligand binding complex.Usually, each of the two or more effector proteins comprises a different effector domain, and each comprises a ligand that can reversibly bind to the same ligand binding portion.

[0020] In one embodiment of the present invention, each of the two or more effector proteins comprises an RNA binding domain that can reversibly bind to the same RNA motif in the RNA scaffold.In other words, each of the two or more effector proteins can interact with a common RNA motif, and therefore each can be recruited by the same RNA scaffold.Usually, each of the two or more effector proteins comprises a different effector domain, and each comprises an RNA binding domain that can reversibly bind to the same RNA motif.

[0021] In one embodiment, the RNA binding domain may be monomeric, dimeric or multimeric. In a preferred embodiment, the RNA binding domain may form a dimer such that the dimeric RNA binding protein and its associated effector domain(s) reversibly associate with a single RNA motif.

[0022] The binding affinity of the RNA motif and / or RNA binding domain has a dissociation constant (Kd) of less than 150 nM. Typically, Kd is between 1-150 nM, 5-150 nM, 10-150 nM; 15-150 nM; 20-150 nM, 25-150 nM, 30-150 nM, 40-150 nM, 50-150 nM, and 60-150 nM, and more typically, Kd is between 1-10 nM: 1-20 nM, 1-30 nM, 1-40 nM, 1-50 nM; 1-60 nM, 1-70 nM; 1-80 nM; and 1-90 nM. If the dissociation constant exceeds 150 nM, the efficiency of base editing decreases.

[0023] In one embodiment of the invention, the RNA motif and the RNA binding domain are as follows: telomerase Ku binding motif and Ku protein or its RNA binding section; telomerase Sm7 binding motif and Sm7 protein or its RNA binding section; MS2 phage operator stem loop and MS2 coat protein (MCP) or its RNA-binding section; PP7 phage operator stem loop and PP7 coat protein (PCP) or its RNA-binding section; BoxB binding motif and lambda bacteriophage protein N (LambdaN-(1-22)) or an RNA-binding section thereof; a Csy4-binding motif and Csy4[H29A] or an RNA-binding section thereof; or Qbeta phage operator stem loop and Qbeta coat protein [Q65H] or a section of its RNA-binding portion; wherein the pair is selected from the group consisting of:

[0024] In some embodiments, the functions of the effector domains of two or more effector proteins are the same or different. In one embodiment, the effector domain is an enzyme or a protein fragment. In one embodiment, the effector domain is an enzyme or a protein fragment that has enzymatic activity. For example, in one embodiment, the effector domain has a deamination activity, such as, in one embodiment, a cytidine or adenine deamination activity.

[0025] In one embodiment, in the methods provided herein, the effector domain with cytidine deamination activity is a wild-type or engineered AID, CDA, APOBEC1, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H or other APOBEC family enzyme. In one embodiment, the effector domain with adenine deamination activity is a wild-type or engineered ADA, ADAR, ADAR1 family enzyme, or tRNA adenosine deaminase TadA, TATA, TAD3.

[0026] In one embodiment, the effector protein further comprises a linker. Advantageously, the linker allows for greater control over the three-dimensional space of the ligand and thus the effector. In one embodiment, the effector protein further comprises a linker configured to link the ligand and the effector domain.

[0027] In one embodiment, the sequence targeting component and / or the effector protein comprises one or more nuclear localization signals (NLS). In one embodiment, the target nucleic acid is extrachromosomal DNA or genomic DNA on a chromosome. In an embodiment of the invention, a method is provided in which a sequence-targeting moiety comprising a sequence-targeting protein is fused to one or more uracil DNA glycosylase (UNG) inhibitor peptides (UGI).

[0028] In one embodiment, the sequence-targeting protein is a type II Cas protein or a type V Cas protein. In one embodiment, the sequence-targeting protein is a type II Cas protein that is nuclease-deficient or has nickase activity and / or comprises the sequence of dCas9 or nCas9 of a species selected from the group consisting of Streptococcus pyogenes, Streptococcus agalactiae, Staphylococcus aureus, Streptococcus thermophiles, Neisseria meningitides, and Treponema denticola. In one embodiment, the sequence-targeting protein is a type II Cas protein that comprises the sequence of dCas9 or nCas9 of Staphylococcus auriculalis.

[0029] In one embodiment, the sequence-targeting protein is a type V Cas protein. In one embodiment, the sequence-targeting protein is a type V Cas protein that is nuclease-deficient or has nickase activity, and / or comprises a dCas12 or nCas12 sequence of a species selected from the group consisting of Francisella cf.novicida Fx1, Deltaproteobacteria bacterium, Alicyclobacillus acidoterrestri, Planctomycetes bacterium, Oleiphilus sp., Bacterium CG09_39_24, Uncultured archaeon, Bacillus thuringiensis, Cyanothece sp., Rothia dentocariosa, Candidatus Micrarchaeota archaeon, Gordonia otitidis, Freshwater metagenome, Hypersaline lake sediment metagenome or Hot springs metagenome.

[0030] In one embodiment, the sequence-targeting protein is a type V Cas protein that is nuclease-deficient or has nickase activity, and / or comprises a dCas12 or nCas12 sequence of a species selected from the group consisting of Francisella cf. novicida Fx1, Deltaproteobacteria bacterium, Alicyclobacillus acidoterrestri, Planctomycetes bacterium, Oleiphilus sp., Bacterium CG09_39_24, Bacillus thuringiensis, Cyanothece sp., Rothia dentocariosa, Candidatus Micrarchaeota archaeon, Gordonia otitidis.

[0031] In one embodiment of the present invention, a method is provided in which a cell is base edited at a single genomic locus. In one embodiment, the cell is base edited at two or more genomic loci. In one embodiment, the cell is a eukaryotic cell or a prokaryotic cell. In one embodiment, the cell is a mammalian cell or a plant cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is an immune cell (e.g., a T cell, a B cell, a natural killer (NK cell), a CD34+ hematopoietic stem cell precursor cell (HSPC) or a macrophage), a stem cell or a cancer cell. In a preferred embodiment, the cell is a T cell.

[0032] In one embodiment of the present invention, a method is provided in which the genetic modification comprises at least two genetic modifications in the same locus. In one embodiment, the genetic modification is a point mutation. The point mutation introduces a premature stop codon, disrupts a start codon, disrupts a splice site, or corrects the genetic mutation.

[0033] In one embodiment of the present invention, a method is provided in which an RNA-ligand binding complex is introduced into a cell as a chemically synthesized RNA. In one embodiment, the RNA-ligand binding complex comprises one or more modifications. In one embodiment, the RNA-ligand binding complex may comprise one or more of the following modifications described herein. For example, the guide RNA is chemically engineered to comprise a 2'-(0-methyl phosphorothioate) modification on at least one 5' nucleotide and at least one 3' nucleotide of the guide RNA sequence. In one embodiment, the RNA-ligand binding complex is synthesized as a single component or two separate components. In one embodiment, the RNA-ligand binding complex is synthesized as two separate components, the first component comprising a) the crRNA and / or b) the ligand binding moiety, and the second component comprising c) the tracrRNA or scoutRNA and / or d) the ligand binding moiety. The two components are hybridized before being introduced into the cell. In one embodiment, the RNA-ligand binding complex is a synthetic RNA component.

[0034] In one embodiment of the invention, the RNA scaffold used in the methods herein is introduced into the cell as chemically synthesized RNA. In one embodiment, the RNA scaffold comprises one or more modifications. In one embodiment, the RNA scaffold comprises one or more modifications as described herein below. For example, the guide RNA is chemically engineered to comprise a 2'-(0-methyl phosphorothioate) modification on at least one 5' nucleotide and at least one 3' nucleotide of the guide RNA sequence. In one embodiment, the RNA scaffold is synthesized as a single component or two separate components. In one embodiment, the RNA scaffold is synthesized as two separate components, the first component comprising a) the crRNA and / or b) the ligand binding moiety, and the second component comprising c) the tracrRNA or scoutRNA and / or d) the ligand binding moiety. The two components are hybridized before being introduced into the cell. In one embodiment, the RNA scaffold is one or more synthetic RNA components.

[0035] In one embodiment of the present invention, the RNA-ligand binding complex comprises one or more modifications. In one embodiment of the present invention, the one or more modifications may increase the stability of the RNA-ligand binding complex. In one embodiment, the one or more modifications may increase the stability of the RNA-ligand binding complex by increasing the resistance of the complex to nuclease degradation. In one embodiment, the guide RNA is chemically modified to include at least one 2'-(0-methyl phosphorothioate) modification.

[0036] In one embodiment of the present invention, a method is provided in which the one or more ligand binding moieties are located at the 3' end, 5' end or within the guide RNA. In a preferred embodiment, the RNA-ligand binding complex (or RNA scaffold) may comprise one or more RNA motifs that can recruit one or more effectors. In a preferred embodiment, the RNA-ligand binding complex (or RNA scaffold) may comprise one or more RNA motifs that can recruit one or more effector proteins. In one embodiment, each RNA motif recruits either a single or multiple effector proteins. In an embodiment in which the RNA-ligand binding complex (RNA scaffold) comprises two or more RNA motifs, the RNA motifs are the same or different. In an embodiment in which two or more RNA motifs are present and are different, each RNA motif recruits either a single or multiple effector proteins. In an embodiment in which two or more RNA motifs are present and are different, at least one RNA motif recruits multiple effector proteins. In an embodiment in which multiple effector proteins are recruited, the functions of these proteins are the same or different. In any embodiment in which multiple effector proteins are recruited, the functions of the effector domains of the effector proteins may be the same or different.

[0037] In a preferred embodiment, at least one RNA motif is an MS2 aptamer, and optionally, said MS2 aptamer has an extended stem. In one embodiment, the extended stem comprises 2-24 nucleotides. In one embodiment, at least one RNA motif is a PP7 aptamer. In one embodiment, the MS2 aptamer and the PP7 aptamer each recruit a single effector protein or multiple effector proteins. In one embodiment, the MS2 aptamer and the PP7 aptamer recruit the same or different effector proteins.

[0038] In one embodiment, the sequence-targeting protein comprises a nCas9 with one or two UGIs, and the ligand-binding moiety is a single MS2 RNA motif or a PP7 RNA motif located at the 3' end of the RNA-ligand binding complex.

[0039] In one embodiment, the sequence-targeting protein comprises a nCas9 with one or two UGIs, and the ligand-binding moiety is a single MS2 RNA motif or a PP7 RNA motif located at the 3' end of the RNA-ligand binding complex.

[0040] In one embodiment of the invention, a method is provided in which the components are in the form of RNA or protein, e.g. the sequence targeting component, the RNA-ligand binding complex and the effector protein may be introduced into a cell as RNA or protein, meaning that the methods / systems provided herein may be used in vector-free systems if desired.

[0041] In a second aspect, the invention provides a population of genetically modified cells obtained by the method for genetic modification of cells by base editing, wherein at least 10% of the cells may comprise one or more genetic modifications.

[0042] In a third aspect, the present invention provides a kit for genetic modification of a cell by base editing, comprising: i) sequence-targeted proteins; ii) a) a guide RNA, and b) a ligand-binding moiety; and iii) c) a ligand capable of reversibly binding to said ligand binding moiety, and d) two or more effector proteins, each of which comprises an effector domain; Includes.

[0043] In one embodiment of the kit according to the third aspect of the invention, the effector protein further comprises a linker. In one embodiment of the kit according to the third aspect of the invention, the ligand binding moiety is an RNA motif and the ligand is an RNA binding domain. In a fourth aspect, the present invention provides a kit for genetic modification of a cell by base editing, comprising: i) sequence-targeted proteins; ii) a) a guide RNA, and b) a ligand-binding moiety; and iii) c) a ligand capable of reversibly binding to said ligand binding moiety, and d) two or more effector proteins, each of which comprises an effector domain; Includes. In one embodiment of the kit according to the fourth aspect of the invention, the guide RNA comprises a crRNA comprising a targeting sequence complementary to the target nucleic acid. In one embodiment of the kit according to the fourth aspect of the invention, the guide RNA further comprises a tracrRNA or a scoutRNA capable of binding to a sequence-targeting protein. In one embodiment of the kit according to the fourth aspect of the invention, the ligand binding moiety is an RNA motif and the ligand is an RNA binding domain.

[0044] In a fifth aspect, the present invention provides a method for genetic modification of a cell by base editing, comprising: i) a) a sequence-targeting protein comprising an SH3 domain, and b) an effector domain comprising an SH3 domain, wherein (a) and (b) are linked by an SH3 mediated bond; ii) c) a guide RNA, and d) a ligand-binding moiety; and iii) e) a ligand capable of reversibly binding to said ligand binding moiety, and f) two or more effector proteins, each of which comprises an effector domain; The method includes introducing into a cell and / or expressing in a cell. In one embodiment of the kit according to the fifth aspect of the invention, the guide RNA comprises a crRNA comprising a targeting sequence complementary to the target nucleic acid. In one embodiment of the kit according to the fifth aspect of the invention, the guide RNA further comprises a tracrRNA or a scoutRNA capable of binding to a sequence-targeting protein. In one embodiment of the kit according to the fifth aspect of the invention, the ligand binding moiety is an RNA motif and the ligand is an RNA binding domain.

[0045] In a sixth aspect, the present invention provides a method for genetic modification of a cell by base editing, comprising: i) a) a sequence-targeting protein, and b) an effector domain, including an array targeting moiety; ii) a) a guide RNA, and b) an RNA-ligand binding complex comprising a ligand-binding portion; iii) c) a ligand capable of reversibly binding to said ligand binding moiety, and d) two or more effector proteins, each of which comprises an effector domain; The method includes introducing into a cell and / or expressing in a cell. In one embodiment of the kit according to the sixth aspect of the invention, the guide RNA comprises a crRNA comprising a targeting sequence complementary to the target nucleic acid. In one embodiment of the kit according to the sixth aspect of the invention, the guide RNA further comprises a tracrRNA or a scoutRNA capable of binding to a sequence-targeting protein. In one embodiment of the kit according to the sixth aspect of the invention, the ligand binding moiety is an RNA motif and the ligand is an RNA binding domain.

[0046] In a seventh aspect, the present invention provides a cell line derived from at least one cell prepared by the methods disclosed herein. [Brief description of the drawings]

[0047] [Figure 1] FIG. 1 is a diagram of an exemplary gRNA molecule for use with Type II Cas enzymes, detailing the main features; lower stem, upper stem, repeat, anti-repeat, bulge, stem loop 1, stem loop 2, stem loop 3. [Diagram 2] 1 is a depiction of an exemplary gRNA sequence for use with a Cas12b protein showing the potential location of one or more modifications, including an aptamer sequence indicated by an asterisk. (A) A gRNA with an aptamer sequence 5' of the tracrRNA. (B) A gRNA with an aptamer sequence 3' of the tracrRNA. (C) A gRNA with an aptamer sequence 5' of the crRNA. (D) A gRNA with an aptamer sequence 5' of the crRNA and a second aptamer sequence 5' of the tracrRNA. (E) A sgRNA with an aptamer sequence at the 5' end of the sgRNA. (F) A sgRNA with an aptamer sequence in the extension of the tetraloop. (G) A sgRNA with an aptamer sequence positioned as an extension on the tetraloop and a second aptamer sequence at the 5' end of the sgRNA. (H) A sgRNA with an aptamer sequence in the extension to stem loop 2. An RNA-ligand binding complex comprising a gRNA (guide RNA) and an aptamer sequence(s), where the aptamer sequence(s) is an RNA motif, may also be referred to as an RNA scaffold. [Diagram 3]Depiction of an exemplary gRNA sequence for use with Cas12e enzyme showing potential locations of modification to include an aptamer sequence, indicated by an asterisk. (A) gRNA with an aptamer sequence 3' of the tracrRNA. (B) gRNA with an aptamer sequence 5' of the tracrRNA. (C) gRNA with an aptamer sequence 5' of the crRNA. (D) gRNA with an aptamer sequence 3' of the crRNA. (E) sgRNA with an aptamer sequence at the 5' end of the sgRNA. (F) sgRNA with an aptamer sequence at the extension of the tetraloop. (G) sgRNA with an aptamer sequence at the 3' end of the sgRNA. (H) sgRNA with an aptamer sequence at the extension to stem loop 2. An RNA-ligand binding complex comprising a gRNA (guide RNA) and an aptamer sequence(s), where the aptamer sequence(s) is an RNA motif, may also be referred to as an RNA scaffold. [Figure 4] Depictions of exemplary gRNA sequences for use with Cas12f enzyme showing potential locations of one or more modifications including aptamer sequences indicated by stars. (A) sgRNA with aptamer sequence at 3' of sgRNA. (B) sgRNA with aptamer sequence at tetraloop extension. (C) sgRNA with aptamer sequence at extension on tetraloop and second aptamer sequence at 5' end of sgRNA. (D) gRNA with aptamer sequence at 5' of tracrRNA. (E) gRNA with aptamer sequence at 3' end of tracrRNA. (F) gRNA with aptamer sequence at 5' of crRNA. An RNA-ligand binding complex comprising a gRNA (guide RNA) and aptamer sequence(s), where the aptamer sequence(s) is an RNA motif, may be referred to as an RNA scaffold. [Diagram 5]FIG. 1 is a diagram of an exemplary gRNA for use with Cas12a showing potential locations for including an aptamer sequence. The location of one or more modifications for including an aptamer sequence are indicated by an asterisk and are located at the 5' base of the direct repeat, in the loop of the direct repeat or at the 3' end of the gRNA. An RNA-ligand binding complex comprising a gRNA (guide RNA) and an aptamer sequence(s), where the aptamer sequence(s) is an RNA motif, may also be referred to as an RNA scaffold. [Figure 6] Diagram of an exemplary gRNA for use with the Cas12c and Cas12d systems showing potential locations for including an aptamer sequence. Locations of one or more modifications including aptamer sequences indicated with an asterisk: 5' of the scoutRNA, 3' of the scoutRNA, stem of the scoutRNA. An RNA-ligand binding complex comprising a gRNA (guide RNA) and an aptamer sequence(s), where the aptamer sequence(s) is an RNA motif, may also be referred to as an RNA scaffold. [Figure 7] Figure 13. Editing efficiency of the Pinpoint system with single aptamer recruitment, Apobec only, AID only, and Apobec and AID combined at the PCD1 locus. Editing efficiency of the Pinpoint system with single ligand binding moiety (RNA motif) recruitment, Apobec only, AID only, and Apobec and AID combined. The ratio of cytosine to thymine nucleobase transitions is shown. [Figure 8]Simultaneous single aptamer (ligand binding moiety) multi-deaminase mobilized base editing at multiple loci for novel base editing spacer profiles. Tetraplex gene KO base editing using synthetic sgRNA-aptamer (RNA-ligand binding complex) with cytosine to thymine base editing in primary human cells. A) Quantification of C to T conversion of targeted bases within the targeted guide sequence for gene KO of B2M. B) Quantification of C to T conversion of targeted bases within the targeted guide sequence for gene KO of CD52. C) Quantification of C to T conversion of targeted bases within the targeted guide sequence for gene KO of TRAC. D) Quantification of C to T conversion of targeted bases within the targeted guide sequence for gene KO of PDCD-1. Synthetic RNA-ligand binding complex and deaminase-based editor mRNA (effector protein) were co-delivered into CD3 positive T cells via electroporation and base conversion was determined by analysis of Sanger sequencing traces 5-7 days after delivery. To display the different profiles of each deaminase enzyme, the protospacer is reported in the whole figure. Included controls contain all three deaminases (AnoApo, RatApo, and HoAID) but with a non-targeted / scrambled guide. AnoApo=NLS-Anolis(P16A-E17A)-Apobec 1-linker-MCP. RatApo=NLS-Rattus-Apobec 1-linker-MCP, HoAID=NLS-Homo-AID-linker-MCP. The ratio of cytosine to thymine nucleobase transitions is shown. [Figure 9]Simultaneous single aptamer (ligand binding moiety) multi-deaminase mobilized base editing at multiple loci for novel base editing functionality in therapeutically relevant immune cell models. Tetraplex functional KO by base editing with synthetic sgRNA-aptamer (RNA-ligand binding complex) by cytosine to thymine base editing in primary human cells. Flow cytometry quantification of the proportion of live cells that do not contain any of the four surface markers: B2M, CD52, TCR (reduced as a result of TRAC editing), and PD1 (reduced as a result of PDCD-1 editing); results indicate successfully edited tetraplex edited cells in the live cell population. Included controls contain all three deaminases (AnoApo, RatApo, and HoAID) but with non-targeted / scrambled guide. AnoApo=NLS-Anolis(P16A-E17A)-Apobec 1-Linker-MCP. RatApo=NLS-Rattus-Apobec 1-linker-MCP, HoAID=NLS-Homo-AID-linker-MCP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] The present invention relates to a novel method for genetic modification of cells by base editing. The present invention is based on the use of an RNA-mediated base editing system in which two or more effectors are reversibly recruited to a target genomic locus. In one aspect, provided herein is a method for genetic modification of a cell by base editing, comprising introducing into and / or expressing in a cell: i) a sequence-targeting moiety (or sequence-targeting protein) comprising a sequence-targeting protein; ii) an RNA-ligand binding complex comprising a) a guide RNA, and b) a ligand-binding portion; and iii) two or more effector proteins comprising c) a ligand capable of reversibly binding to said ligand-binding portion, and d) an effector domain. The RNA-ligand binding complex guides the sequence-targeting protein and the effector protein to a target polynucleotide at a target site, and the effector domain of the effector protein modifies the sequence. As disclosed herein, the sequence-targeting protein, such as the Cas9 protein or the Cas12 protein, is engineered to eliminate its double-strand cleavage activity.

[0049] RNA-mediated base editing system The system of the present invention relates, at least in part, to prior RNA-mediated base editing systems (see Patent Documents 1 to 3). The RNA-mediated base editing used in the method provided herein takes a different approach from the prior art.More specifically, in the prior art RNA-mediated base editing, in the RNA-ligand binding complex of CRISPR / Cas complex, RNA motif comprises guide RNA molecule and serves as anchor for recruiting single effector.Therefore, RNA motif recruits single effector, such as base editing enzyme, fused with RNA binding domain (ligand). In the RNA-mediated base editing complex of the present invention, the recruitment of the effector fused to the RNA binding domain (ligand) is reversible, i.e., the effector functions to reversibly associate with its cognate RNA motif (ligand binding moiety). The reversible nature of this system allows the recruitment of two or more effectors to the desired target site induced by the CRISPR / Cas system. Thus, the same RNA-ligand binding complex and its associated Cas protein can be used with multiple different effectors when editing within the same genomic locus.

[0050] This system can be used to increase the efficiency of base editing, whereby multiple effector proteins with the same RNA binding domain and different effector domains performing the same function are recruited to a single genomic locus. By reversibly associating with the cognate RNA motif (present in the RNA-ligand binding complex) and its associated sequence targeting protein, multiple effector proteins can be transiently incorporated into the base editing complex until the effector protein with optimal activity associates and performs the appropriate editing. Thus, two, three, four, five or more effectors with the same function can be used in the methods or kits disclosed herein. Alternatively, multiple effector proteins with the same RNA binding domain and different effector domains with different functions can be guided to a single genomic locus. By reversibly associating with the cognate RNA motif (present in the RNA-ligand binding complex) and its associated sequence targeting protein, multiple effector proteins can be transiently incorporated into the base editing complex and perform appropriate editing. Thus, two, three, four, five or more effectors with different functions can be used in the methods or kits disclosed herein. Multiple RNA-ligand binding complexes with different guide RNA sequences can also be used to perform editing at multiple different genomic loci using the methods and kits disclosed herein. Multiple effector proteins can be used as described above to perform single or multiple edits at each genomic locus.

[0051] As demonstrated herein, the RNA-ligand binding complex-mediated base editing method and system provided herein, particularly the RNA scaffold-mediated base editing method and system, exhibit several important different features compared to the prior (first generation) systems described in US Pat. No. 5,999,233 and US Pat. No. 5,999,252 (incorporated herein by reference in their entirety). First, the method and system of the present invention substantially increases on-target efficacy compared to the first generation systems, while detectable off-target effects remain low or nonexistent. Second, the method and system of the present invention can utilize a wide variety of cytidine deaminases from different species and different deaminase families, many of which exhibit distinctly different activity windows and preferred locations than any prior first generation construct. Third, the ability to provide multiple different effector proteins (e.g., deaminases) at all sites where editing is to be performed provides the benefit of increasing editing efficiency without the time-consuming task of determining the optimal deaminase for each genomic locus. Fourth, by bringing both cytidine deaminase and adenosine deaminase into the same site at the same time, both CT editing and AG editing can occur simultaneously within the same genomic locus. The various components that make up the RNA-mediated base editing system of the present invention are now discussed in detail.

[0052] i) Sequence Targeting Moiety The sequence targeting component (also referred to as sequence targeting protein) of the methods and systems provided herein typically utilizes a Cas protein from a CRISPR-Cas system from a bacterial species as the sequence targeting protein. In one embodiment, the Cas protein is from a type II CRISPR-Cas system. In an alternative embodiment, the Cas protein is from a type V CRISPR-Cas system. Generally, Cas proteins contain at least one Cas RNA binding domain. The Cas RNA binding domain interacts with the guide RNA at the Cas association region. The Cas protein can be a wild-type Cas protein or a recombinant form lacking nuclease activity (dCas protein) or only single-strand nicking activity (nCas protein). Cas proteins can be engineered to increase nucleic acid binding affinity and / or specificity, engineer enzymatic activity, and / or change other properties of the protein. For example, the nuclease (i.e., DNase, RNase) domain of the protein can be modified, deleted, or inactivated. Alternatively, the protein can be truncated to remove domains that are not essential for the function of the protein. The protein can also be truncated or modified to optimize activity.

[0053] Cas proteins Various Cas proteins can be used in the present invention. Cas protein, CRISPR-associated protein, or CRISPR protein, which are used interchangeably, refer to proteins of or derived from CRISPR-Cas type I, type II, type III, or type V system with RNA-guided DNA binding activity. Generally, Cas protein comprises at least one RNA-binding domain, herein referred to as Cas RNA-binding domain. Cas RNA-binding domain interacts with guide RNA at Cas-associated region. Non-limiting examples of suitable Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas12a (or Cpf1), Cas12b (or C2c1), Cas12c, Cas12d (or CasY), Cas12e (or CasX), Cas12f, Cas12g, Cas12g1, Cas12h, Cas12i, Cas12j, Cas12k, CasF, These include CasG, Cash, Csy1, Csy2, Csy3, Cse1 (or CASA), Cse2 (or CasB), Cse3 (or CaSe), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966.For example, see Koonin and Makarova, 2019, origins and evolution of crispr cas systems, Review Philos Trans R Soc Lond B Biol Sci. 2019 May 13; 374 (1772); for Type-V, see Yan et al, Science, 363 pg 88-92, 2019; and for Miniature Cas14, see Harrington et al, 2018, Science, vol 362 pg 839-842, the contents of which are incorporated herein by reference in their entirety.

[0054] The Cas proteins (as well as other protein components described in the present invention) can be obtained as recombinant polypeptides. Alternatively, the proteins can be chemically synthesized (see, e.g., Creighton, "Proteins: Structures and Molecular Principles," W. H. Feeman & Co., NY, 1983) or produced by recombinant DNA techniques as described herein. For further guidance, the skilled artisan can refer to Frederick M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2003; and Sambrook et al., Molecular Cloning, A Laboratory Manual, "Cold Spring Harbor Press, Cold Spring Harbor, NY, 2001).

[0055] The Cas proteins according to the invention may be provided in purified or isolated form or may be part of a composition. Preferably, when in a composition, the protein is first purified to some degree, more preferably to a high level of purity (e.g., about 80%, 90%, 95%, or 99% or more). The composition according to the invention may be any type of composition desired, but is typically an aqueous composition suitable for use as or inclusion in a composition for RNA-guided targeting. Those skilled in the art will appreciate the variety of materials that may be included in such nuclease reaction compositions.

[0056] The Cas proteins described in the present invention can be directly fused to an effector protein such that, in addition to two or more effector proteins that are reversibly recruited to the base editing complex via an RNA-ligand binding complex (or RNA scaffold), the directly fused effector domain can also carry out its activity at the target site. Alternatively, the Cas proteins described in the present invention can be bound to an effector domain via a ligand such that in addition to two or more effector proteins that are reversibly recruited to the base editing complex via an RNA-ligand binding complex (or RNA scaffold), the ligand-bound effector domain can also carry out its activity at the target site, as described in Science 16 Sep 2016:Vol.353, Issue 6305. To carry out the methods for recombining target nucleic acids disclosed herein, proteins may be produced in target cells via mRNA, protein-RNA complexes (RNPs), or any suitable expression vector. Examples of expression vectors include chromosomal, non-chromosomal and synthetic DNA sequences, bacterial plasmids, minicircles, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, poxvirus and pseudorabies. Further details are provided in the Expression Systems and Methods section below.

[0057] Application to type II CRISPR-Cas systems In some embodiments, the Cas protein is derived from a type II CRISPR-Cas system. In an exemplary embodiment, the Cas protein is or is derived from a Cas9 protein. Cas9 protein is derived from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides,Bacillus selenitireducens,Exiguobacterium sibiricum,Lactobacillus delbrueckii,Lactobacillus salivarius,Microscilla marina,Burkholderiales bacterium,Polaromonas naphthalenivorans,Polaromonas sp.,Crocosphaera watsonii,Cyanothece sp.,Microcystis aeruginosa,Synechococcus sp.,Acetohalobium arabaticum,Ammonifex degensii,Caldicelulosiruptor becscii,Candidatus Desulforudis,Clostridium botulinum,Clostridium difficile,Finegoldia magna,Natranaerobius thermophilus,Pelotomaculum the rmopropionicum,Acidithiobacillus caldus,Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp.,Nitrosococcus halophilus,Nitrosococcus watsoni,Pseudoalteromonas haloplanktis,Ktedonobacter racemifer,Methanohalobium evestigatum,Anabaena variabilis,Nodularia spumigena,Nostoc sp.,Arthrospira maxima,Arthrospira platensis,Arthrospira sp.,Lyngbya sp.,Microcoleus It may be from chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina. .

[0058] In some embodiments, the Cas protein can be a mutant of a wild-type Cas9 protein or a fragment thereof. In other embodiments, the Cas protein can be derived from a mutant Cas9 protein. For example, the amino acid sequence of the Cas9 protein can be modified to alter one or more properties of the protein (e.g., nuclease activity, affinity, stability, etc.). Alternatively, domains of the Cas9 protein that are not involved in the RNA target can be removed from the protein such that the recombinant Cas9 protein is smaller than wild-type Cas9. In some embodiments, the system utilizes a Cas9 protein from S. pyogenes, either encoded in bacteria or codon-optimized for expression in mammalian cells.

[0059] A mutant Cas9 protein refers to a polypeptide derivative of a wild-type protein, such as a protein with one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof. The mutant has at least one of RNA-guided DNA binding activity or RNA-guided nuclease activity, or both. Generally, the recombinant is at least 50% (e.g., any number between 50% and 100%, including, for example, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, and 99%) identical to a wild-type Cas9 protein, such as SEQ ID NO: 44 below. As disclosed herein, nuclease-dead Cas9 (dCas9, e.g., from S. pyogenes D10A, H840A mutant protein, SEQ ID NO: 17), or nuclease-deficient nickase Cas9 (nCas9, e.g., from S. pyogenes D10A mutant protein, SEQ ID NO: 46) can be used. dCas9 or nCas9 can also be derived from various bacterial species. Table 1 lists a non-exhaustive list of examples of Cas9s and their corresponding PAM requirements. Synthetic Cas replacements such as those described in Rauch et al., Programmable RNA-Guided RNA Effector Proteins Built from Human Parts. Cell Volume 178, Issue 1, 27 June 2019, Pages 122-134.e12 can also be used.

[0060] Table 1. Non-exhaustive list of examples of Cas9 proteins and their corresponding PAM requirements.

[0061] [Table 1] Application to V-type CRISPR-Cas systems In certain embodiments, the Cas protein is derived from a type V CRISPR-Cas system (e.g., Wang, J., Zhang, C., and Feng, B. (2020). The rapidly advancing Class 2 CRISPR-Cas technologies: a customizable toolbox for molecular manipulations. J. Cell. Mol. Med. 24, 3256-3270. doi:10.1111 / jcmm.15039; and Makarova, K.S., Wolf, Y.I., Iranzo, J. et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol 18, 67-83 (2020); and Tong, B. et al. (2021) The versatile type V CRISPR effectors and their application prospects. Front. Cell Dev. Biol. 8, 1835. doi:10.3389 / fcell.2020.622103). The Cas12 protein can be derived from Francisella (novicida Fx1, Deltaproteobacteria, Alicyclobacillus acidoterrestri, Planctomycetes, Oleiphilus species, bacteria CG09_39_24, uncultured archaea, Bacillus thuringiensis, Cyanothes species, Rothia dentocaiosa, Candidatus Micrarchaeota archaea, Gordonia otitidis, freshwater metagenome, hypersaline lake sediment metagenome, or hot spring metagenome.The Cas protein can be Cas12a (or Cpf1), Cas12b (or C2c1), Cas12c, Cas12d (or CasY), Cas12e (or CasX), Cas12f, Cas12g, Cas12g1, Cas12h, Cas12i, Cas12j, or Cas12k.

[0062] In some embodiments, the Cas protein can be a mutant of a wild-type Cas12 protein (such as Cas12a from Acidaminococcus sp.) or a fragment thereof. In other embodiments, the Cas12 protein can be derived from a mutant Cas12 protein. For example, the amino acid sequence of the Cas12 protein can be modified to change one or more properties of the protein (e.g., nuclease activity, affinity, stability, etc.). Alternatively, domains of the Cas12 protein that are not involved in the RNA target can be removed from the protein such that the recombinant Cas12 protein is smaller than the wild-type Cas12 protein. In some embodiments, the system uses the Cas12a protein from Lachnospiraceae bacteria ND2006, Cas12d from bacteria of the Candidate Phyla Radiation group, or A. acidoterrestris, either encoded in bacteria or codon-optimized for expression in mammalian cells. Mutant Cas12 proteins refer to polypeptide derivatives of the wild-type protein, such as proteins with one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof. The mutants have at least one of RNA-guided DNA binding activity or RNA-guided nuclease activity, or both. Generally, the recombinant forms are at least 50% (e.g., any number between 50% and 100%, including, for example, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, and 99%) identical to wild-type Cas12, such as AsCas12a (SEQ ID NO: 48), AaCas12b (SEQ ID NO: 49), or Cas12d15 (SEQ ID NO: 50).

[0063] As disclosed herein, nuclease-dead AsCas12a (e.g., dAsCas12a from Acidaminococcus sp. D908A mutant protein) can be used, and dCas12 or nCas12 can also be derived from various bacterial species. Table 2 lists a non-exhaustive list of exemplary Cas12 proteins and their corresponding PAM requirements. The humanized nucleotide sequences of dCas12a (SEQ ID NO:54), dCas12b (SEQ ID NO:55), and dCas12d (SEQ ID NO:56) and the amino acid sequences of dAsCas12a (SEQ ID NO:57), dAaCas12b (SEQ ID NO:58), and dCas12d15 (SEQ ID NO:59) are provided. Table 2

[0064] [Table 2] PS=protospacer; N=any base; R=A / G; V=A / C / G.

[0065] U.G.I. In an embodiment of the present disclosure, the sequence targeting component comprises a fusion between (a) a sequence targeting protein and (b) a first uracil DNA glycosylase (UNG) inhibitor peptide (UGI). For example, the fusion protein can comprise a Cas protein, such as a type II or type V Cas protein, fused to UGI. Such a fusion protein can have increased nucleic acid editing efficiency compared to a fusion protein that does not include a UGI domain. In an embodiment, the UGI comprises a wild-type UGI sequence from Bacillus phage PBS2 or one having the following amino acid sequence (SEQ ID NO: 60):

[0066] In some embodiments, the UGI proteins provided herein include fragments or variants of UGI, and proteins homologous to UGI or UGI fragments. For example, in some embodiments, UGI comprises an amino acid sequence homologous to a fragment of SEQ ID NO: 60. In some embodiments, UGI comprises an amino acid sequence homologous to (SEQ ID NO: 60), or an amino acid sequence homologous to a fragment of (SEQ ID NO: 60). In some embodiments, a protein comprising UGI or a fragment of UGI, or a homolog of UGI or a UGI fragment, is referred to as a "UGI variant." A UGI variant shares homology with UGI or a fragment thereof. For example, a UGI variant is at least about 70% (e.g., at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%) identical to the wild-type UGI or UGI sequence (SEQ ID NO: 60).

[0067] In one embodiment, an active or inactivated Cas protein comprises a fusion with two or more UGI peptides or variants. A UGI peptide or variant of a UGI peptide can be linked to another UGI peptide or Cas protein directly or via a linker of 1-100 amino acid residues. Suitable UGI protein and nucleotide sequences are provided herein, and further suitable UGI sequences will be known to those of skill in the art, see, e.g., Wang et al., Uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 encodes a binding protein specific for uracil-DNA glycosylase. J Biol. Chem. 264:1163-1171 (1989); Lundquist et al., Site-directed mutagenesis and characterization of uracil-DNA glycosylase inhibitor protein. Role of specific carboxylic amino acids in complex formation with Escherichia coli uracil-DNA glycosylase. J Biol. Chem. 272:21408-21419 (1997); Ravishankar et al., X-ray analysis of a complex of Escherichia coli uracil DNA glycosylase (EcUDG) with a proteinaceous inhibitor. The structure elucidation of a prokaryotic See UDG. Nucleic Acids Res. 26:4880-4887 (1998); and Putnam et al., Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with Escherichia coli uracil-DNA glycosylase. J Mol. Biol. 287:331-346 (1999), the contents of which are incorporated by reference in their entireties.

[0068] ii) RNA-ligand binding complex The second component of the platform disclosed herein is an RNA-ligand binding complex, which performs the essential functions of sequence recognition and effector recruitment. The RNA-ligand binding complex comprises two subcomponents: (a) guide RNA and (b) ligand binding moiety. In a preferred embodiment, the ligand binding moiety is an RNA motif. The nucleotides in the RNA strand may be entirely ribonucleotides or a combination of ribonucleotides and other nucleotides such as deoxyribonucleotides. Each nucleotide may be unmodified or may be one or more nucleotides, where all nucleotides may be modified, for example, with one or more of the following modifications: 2'-0-methyl, 2'fluoro, or 2'aminopurine. In certain embodiments, there are consecutively modified nucleotides over one or more spans of 1-40 or 2-20 or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, or 36 nucleotides, or the modification pattern is such that every other or every third or every fourth nucleotide is modified at its 2' position, with all other nucleotides being unmodified. Additionally, or alternatively, there may be modified or unmodified internucleotide linkages between one or more or all pairs of consecutive nucleotides.

[0069] {a}Programmable guide RNA CRISPR-Cas system is simple and efficient, and has been used to perform genome editing in cells of various organisms. The specificity of this system is determined by the base pairing between the target DNA and the custom-designed guide RNA. By manipulating and adjusting the base pairing properties of the guide RNA, any sequence of interest can be targeted. For some CRISPR-Cas systems, the target sequence of interest must contain a PAM sequence that can be successfully targeted by the base editing complex, as detailed below. The guideRNA may comprise any one or more of crRNA, tracrRNA and / or scoutRNA. In embodiments where type II Cas proteins are utilized, the guide RNA may include crRNA and tracrRNA. In embodiments where type V Cas proteins are utilized, the guide RNA may include crRNA, crRNA and tracrRNA, or crRNA and scoutRNA.

[0070] Type II CRISPR-Cas systems: crRNA:tracrRNA guide Type II CRISPR-Cas systems utilize a guide RNA (type II crRNA: tracrRNA guide) that includes a crRNA and a tracrRNA. The crRNA comprises a sequence complementary to a target nucleic acid. The guide RNA can at least hybridize with the crRNA over a stretch of at least three nucleotides and may further comprise a tracrRNA that can retain association with a type II Cas protein when hybridized over that region. The guide RNA can be either a single RNA molecule or a complex of multiple RNA molecules. Thus, in embodiments in which the sequence targeting component comprises a type II Cas protein, it is preferred that the RNA-ligand binding complex comprises a type II crRNA:tracrRNA guide.

[0071] Among the subcomponents of the RNA-ligand binding complex (or RNA scaffold) disclosed herein, the crRNA provides target specificity. The crRNA comprises a programmable spacer that is complementary to and capable of hybridizing to a preselected target site of interest. In various embodiments, the spacer can comprise from about 10 nucleotides to more than about 25 nucleotides. For example, the nucleotide length of the region of base pairing between the spacer and the corresponding target site sequence can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25. In exemplary embodiments, the spacer is about 17-20 nucleotides long, e.g., 20 nucleotides. The spacer is variable and may be selected based on the target sequence of interest where the Cas protein and / or effector induces base editing. The spacer does not hybridize to the tracrRNA and may be downstream of the Cas association region.

[0072] One requirement for selecting a suitable target nucleic acid for a type II CRISPR-Cas system is the presence of a PAM site / sequence. Each target sequence and its corresponding PAM site / sequence is referred to herein as a Cas target site. A type II CRISPR-Cas system, being a type II CRISPR-Cas, only requires a Cas9 protein and a guide RNA complementary to the target sequence to affect target cleavage. The type II CRISPR-Cas system of S. pyogenes uses a target site with N12-20NGG, where NGG represents the PAM site from S. pyogenes and N12-20 represents the 12-20 nucleotides directly 5' to the PAM site. Additional PAM site sequences from other species of bacteria include NGGNG, NNNNGATT, NAGAAA, NAGAAAW, and NAAAAC. For example, U.S. Patent Application No. 20140273233, International Publication No. 2013176772, Cong et al., (2012), Science 339(6121):819-823, Jinek et al., (2012), Science 337(6096):816-821, Mali et al., (2013), Science 339(6121):823-826, Gasiunas et al., (2012), Proc Natl Acad Sci USA.109(39):E2579-E2586, Cho et al., (2013) Nature Biotechnology 31,230-232, Hou et al., Proc Natl Acad Sci USA.2013 Sep 24;110(39):15644-9, Mojica et al., Microbiology. 2009 Mar;155(Pt3):733-40, and www.addgene.org / CRISPR / , the contents of which are incorporated herein by reference in their entirety. The PAM site / sequence is also a requirement for selecting a suitable target nucleic acid for the V-type CRISPR system.

[0073] The target nucleic acid strand can be either of the two strands on the genomic DNA in the host cell. Examples of such genomic dsDNA include, but are not limited to, host cell chromosomes, mitochondrial DNA, and stably maintained plasmids. However, it should be understood that the method of the present invention can be performed on other dsDNA present in the host cell (e.g., unstable plasmid DNA, viral DNA, and phagemid DNA), as long as the Cas target site is present, regardless of the nature of the host cell dsDNA. The method can also be performed on RNA. In addition to the spacer, the crRNA also comprises a repeat region. The repeat region hybridizes with the anti-repeat region of the tracrRNA described below to form a repeat:anti-repeat duplex. In various embodiments, the repeat region may comprise from about 10 nucleotides to more than about 25 nucleotides. For example, the nucleotide length of the repeat region may be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25 nucleotides. In an exemplary embodiment, the nucleotide length of the repeat region is about 21-25 nucleotides long, e.g., 23 nucleotides.

[0074] The repeat:anti-repeat duplex acts as a Cas association region and is designed on the Cas RNA binding domain of the Cas protein that is intended to associate. Not all nucleotides in the Cas association region may directly associate with the Cas protein. Not all nucleotides in the repeat:anti-repeat duplex hybridize. The repeat:anti-repeat duplex includes a lower stem, a bulge, and an upper stem. The bulge is essential for interaction with the Cas protein. In a non-limiting example, a repeat:anti-repeat duplex of a guide RNA that can interact with Cas9 from S. pyogenes may include a lower stem of about 6 nucleotides long, a bulge of about 6 nucleotides long, and an upper stem of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 nucleotides long. In some cases, the upper stem may not be present in the repeat:anti-repeat duplex. In addition to the crRNA, the guide RNA of the type II CRISPR-Cas system further comprises a trans-activating CRISPR RNA (tracrRNA). The tracrRNA sequence may comprise from about 40 nucleotides to more than about 100 nucleotides. For example, the nucleotide length of the tracrRNA may be about 40, 50, 60, 70, 80, 90 or more than 100. In an exemplary embodiment, the nucleotide length of the tracrRNA is about 88-90 nucleotides long, such as 89 nucleotides. The tracrRNA sequence comprises an anti-repeat region, a nexus, a stem-loop 2 and a stem-loop 3. The tracrRNA further comprises a distal region that does not hybridize to the crRNA, which may be upstream of the anti-repeat region. The anti-repeat region is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the crRNA repeat region over at least 7 consecutive nucleotides. Thus, the repeat region of the programmable crRNA and the anti-repeat region of the tracrRNA can hybridize to form a hybridization region, referred to herein as a repeat:anti-repeat duplex.

[0075] In one embodiment, the tracrRNA is derived from Streptococcus pyogenes. In some embodiments, the tracrRNA activity and the crRNA activity are part of a single continuous strand of nucleotides known as a single guide RNA (or sgRNA). In some embodiments, the crRNA may be immediately upstream of the tracrRNA or may be upstream of the tracrRNA with an intervening sequence or portion thereof between the tracrRNA and the crRNA. As used herein, when the tracrRNA and the crRNA are part of a continuous strand of nucleotides (sgRNA), there may be a loop region between the tracrRNA and the crRNA, e.g., 3-6 nucleotides, called a tetraloop. See, e.g., WO 2014 / 099750, U.S. Patent Application No. 2014 / 0179006, and U.S. Patent Application No. 2014 / 0273226, the contents of which are incorporated herein by reference in their entirety. Methods for generating crRNA:tracrRNA guide RNAs and sgRNAs are well known in the art. In one embodiment, the tracrRNA activity and the crRNA, including the guide RNA, are in two separate RNA molecules that together form part of a functional guide RNA and RNA-ligand binding complex (or RNA scaffold). In this case, the molecule with tracrRNA activity must be able to interact (usually by base pairing) with a molecule with crRNA activity to form a bipartite guide crRNA:tracrRNA.

[0076] An exemplary hybrid type II crRNA:tracrRNA guide RNA sequence (SEQ ID NO:61; Chen et al. Cell. 2013 Dec 19;155(7):1479-91) is shown below:

[0077] [ka] Various tracrRNA and crRNA sequences are known in the art, and non-limiting examples of crRNA and tracrRNA that may be used in the context of the present invention are provided below (SEQ ID NO:1, SEQ ID NO:2). As used herein, an active portion of a tracrRNA retains the ability to form a complex with a Cas protein, such as Cas9 or dCas9 or nCas9. See, e.g., WO 2014 / 144592.

[0078] [ka] Thus, in embodiments in which the sequence targeting component of the invention comprises a type II Cas protein, preferably the RNA-ligand binding complex comprises a type II crRNA:tracrRNA guide RNA.

[0079] Application to V-type CRISPR-Cas systems V-type CRISPR-Cas systems may utilize guide RNAs including crRNA (V-type crRNA-only guide), crRNA and tracrRNA (V-type crRNA:tracrRNA guide), or crRNA and scoutRNA (V-type crRNA:scoutRNA guide). Guide RNAs can be either single RNA molecules or complexes of multiple RNA molecules. Thus, in embodiments in which the sequence targeting component of the invention comprises a V-type Cas protein, it preferably comprises an RNA-ligand binding complex, a crRNA guide RNA, a crRNA:tracrRNA guide RNA or a crRNA:scoutRNA guide RNA.

[0080] V-type CRISPR-Cas systems: a crRNA-only guide V-type CRISPR-Cas systems, in which the sequence-targeting component is a Cas protein such as Cas12a (AsCas12a) from Acidaminococcus sp. BV3L6, require a guide RNA that contains only a crRNA molecule (V-type crRNA-only guide). The crRNA includes a spacer that provides targeting specificity and has a nucleotide sequence that is complementary to and capable of hybridizing to a preselected target site of interest. In various embodiments, the spacer can include from about 10 nucleotides to more than about 25 nucleotides. For example, the nucleotide length of the region of base pairing between the spacer and the corresponding target site sequence can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25. In exemplary embodiments, the targeting sequence is about 18-25 nucleotides long, e.g., 24 nucleotides. The spacer is variable and can be selected based on where it is desired for the Cas protein and / or effector to edit bases. One requirement for selecting a suitable target nucleic acid for some V-type CRISPR-Cas systems (detailed in Table 2) is the presence of a PAM site / sequence. Some V-type CRISPR-Cas systems, such as Cas12g1, do not require a PAM site / sequence for target recognition. Each target sequence and its corresponding PAM site / sequence (if necessary) are referred to herein as Cas targeting sites. The target nucleic acid strand can be either of the two strands on the genomic DNA in the host cell. Examples of such genomic dsDNA include, but are not limited to, host cell chromosomes, mitochondrial DNA, and stably maintained plasmids. However, it should be understood that the method of the present invention can be performed on other dsDNA present in the host cell (e.g., unstable plasmid DNA, viral DNA, and phagemid DNA), so long as the Cas target site is present, regardless of the nature of the host cell dsDNA. The method can also be performed on RNA. In addition to the spacer, the crRNA in V-type crRNA-only guides contains direct repeats that form a pseudoknotted hairpin secondary structure, which makes critical contacts with the Cas enzyme.

[0081] Shown below are non-limiting examples of V-type crRNA-only guides.

[0082] [ka] Thus, in embodiments in which the sequence targeting component comprises a V-type Cas protein, such as AsCas12a, it is preferred that the RNA-ligand binding complex comprises a guide RNA that is only a V-type crRNA.

[0083] V-type CRISPR-Cas system: crRNA:tracrRNA guide V-type CRISPR-Cas systems (wherein the sequence targeting component is a Cas protein such as Cas12b from Alicylo- bacillus acidophilus (AaCas12b)) require a guide RNA (V-type crRNA: tracrRNA guide) that includes crRNA and tracrRNA. The crRNA includes a programmable spacer that provides targeting specificity and has a nucleotide sequence that is complementary to and can hybridize to a preselected target site of interest. In various embodiments, the spacer can include from about 10 nucleotides to more than about 25 nucleotides. For example, the nucleotide length of the region of base pairing between the spacer and the corresponding target site sequence can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25. In an exemplary embodiment, the spacer is about 19-22 nucleotides long, e.g., 20 nucleotides. The spacer is variable and can be selected based on the target sequence of interest where the Cas protein and / or effector induces base editing. The spacer does not hybridize to the tracrRNA. One requirement for selecting a suitable target nucleic acid for some V-type CRISPR-Cas systems (detailed in Table 2) is the presence of a PAM site / sequence. Some V-type CRISPR-Cas systems, such as Cas12g1, do not require a PAM site / sequence for target recognition. Each target sequence and its corresponding PAM site / sequence (if necessary) are referred to herein as Cas targeting sites. The target nucleic acid strand can be either of the two strands on the genomic DNA in the host cell. Examples of such genomic dsDNA include, but are not limited to, host cell chromosomes, mitochondrial DNA, and stably maintained plasmids. However, it should be understood that the method of the present invention can be performed on other dsDNA present in the host cell (e.g., unstable plasmid DNA, viral DNA, and phagemid DNA), as long as the Cas target site is present, regardless of the nature of the host cell dsDNA. The method can also be performed on RNA. In addition to the spacer, the crRNA also contains a repeat region. The repeat region hybridizes with the anti-repeat region of the tracrRNA described below to form a repeat:anti-repeat duplex. In various embodiments, the repeat region can comprise from about 20 nucleotides to more than about 35 nucleotides. In an exemplary embodiment, the repeat region is about 28-33 nucleotides in length, e.g., 31 nucleotides.

[0084] In addition to the crRNAs described above, the V-type crRNA:tracrRNA guide includes a trans-activating CRISPR RNA (tracrRNA). The tracrRNA sequence may include from about 40 nucleotides to more than about 100 nucleotides. For example, the nucleotide length of the tracrRNA may be more than about 40, 50, 60, 70, 80, 90, or 100. In an exemplary embodiment, the tracrRNA is about 98-100 nucleotides long, e.g., 100 nucleotides. The tracrRNA sequence includes an anti-repeat region and a stem loop. Various tracrRNA sequences are known in the art. As used herein, an active portion of a tracrRNA retains the ability to form a complex with a V-type Cas protein, such as Cas12b or dCas12b or nCas12b. In one embodiment, the tracrRNA is derived from A. acidoterrestris.

[0085] In some embodiments, the tracrRNA activity and the crRNA activity are part of a single continuous strand of nucleotides known as a single guide RNA (or sgRNA). In some embodiments, the crRNA may be immediately downstream of the tracrRNA or downstream of the tracrRNA with an intervening sequence or portion between the tracrRNA and the crRNA. As used herein, when the tracrRNA and the crRNA are part of a continuous strand of nucleotides (sgRNA), there may be a loop region between the tracrRNA and the crRNA, e.g., 3-6 nucleotides, called a tetraloop. Methods for generating V-type crRNA:tracrRNA guideRNAs and sgRNAs are known in the art. A non-limiting exemplary V-type crRNA:tracrRNA sgRNA is shown below (SEQ ID NO: 4).

[0086] [ka] In one embodiment, the tracrRNA activity and the crRNA containing guide RNA are two separate RNA molecules that together form part of a functional guide RNA and RNA-ligand binding complex (or RNA scaffold). In this case, the molecule with tracrRNA activity should be able to interact (usually by base pairing) with the molecule with crRNA activity to form a two-part V-type crRNA:tracrRNA guide. Thus, in embodiments in which the sequence targeting component comprises a V-type Cas, such as Cas12b from Alicylobacillus acidophilus (AaCas12b), it is preferred that the RNA-ligand binding complex comprises a V-type crRNA:tracrRNA guide.

[0087] V-type CRISPR-Cas systems: crRNA: scoutRNA guide Type V CRISPR-Cas systems, in which the sequence targeting component is a Cas protein such as Cas12d15, require guide RNAs including crRNA and scoutRNA (crRNA: scoutRNA guide RNA). The crRNA includes a programmable spacer that provides targeting specificity and has a nucleotide sequence that is complementary to and can hybridize to a preselected target site of interest. In various embodiments, the spacer can include from about 10 nucleotides to more than about 25 nucleotides. For example, the nucleotide length of the region of base pairing between the spacer and the corresponding target site sequence can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25. In an exemplary embodiment, the spacer is about 19-22 nucleotides long, e.g., 20 nucleotides. The spacer is variable and can be selected based on the target sequence of interest where the Cas protein and / or effector induces base editing. The spacer does not hybridize to the tracrRNA. One requirement for selecting a suitable target nucleic acid for some V-type CRISPR-Cas systems (detailed in Table 2) is the presence of a PAM site / sequence. Some V-type CRISPR-Cas systems, such as Cas12g1, do not require a PAM site / sequence for target recognition. Each target sequence and its corresponding PAM site / sequence (if necessary) are referred to herein as Cas targeting sites. The target nucleic acid strand can be either of the two strands on the genomic DNA in the host cell. Examples of such genomic dsDNA include, but are not limited to, host cell chromosomes, mitochondrial DNA, and stably maintained plasmids. However, it should be understood that the method of the present invention can be performed on other dsDNA present in the host cell (e.g., unstable plasmid DNA, viral DNA, and phagemid DNA), as long as the Cas target site is present, regardless of the nature of the host cell dsDNA. The method can also be performed on RNA. In addition to the spacer, the crRNA also contains a 5' direct repeat region. This region contains a conserved 5' nt sequence that hybridizes to the complementary sequence of the scoutRNA described below. In various embodiments, the repeat region can contain from about 20 nucleotides to more than about 35 nucleotides. In an exemplary embodiment, the repeat region is about 28-33 nucleotides in length, e.g., 31 nucleotides.

[0088] In addition to the crRNAs described above, the V-type crRNA:scoutRNA guides of the present invention contain short complementary non-coding RNAs (scoutRNAs). The crRNAs and ScoutRNAs are required for the activity of certain V-type CRISPR systems, such as Cas 12 d catalytic activity. The scoutRNAs have a different secondary structure than the preceding tracrRNAs used by CRISPR-Cas9 and some Cas 12 enzymes, and in Cas 12 d-containing systems, the scoutRNAs contain a conserved five-nucleotide sequence that is essential for hybridization to the crRNA and subsequent enzymatic activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish the scoutRNA as an essential cofactor for Cas 12 c catalytic pre-crRNA maturation. The scoutRNA can be 40-100 nucleotides in length. The scoutRNA sequence comprises a crRNA complementary region, an upstream region upstream of the crRNA complementary region, and a downstream region downstream of the crRNA complementary region. Preferably, the crRNA complementary region is 5 nucleotides in length. The crRNA complementary region can be located at or near the 5' end of the scoutRNA, or at or near the 3' end of the scoutRNA, or between consecutive nucleotides within the scoutRNA that are neither at the 5' nor the 3' end of the scoutRNA. In some naturally occurring scoutRNAs, the self-complementary region may contain one or more self-hybridization regions, loops, and bulges, and in some cases, 5' ssRNA overhangs and / or no overhangs. In certain embodiments, any bulge(s) of naturally occurring scoutRNAs are preserved even when a ligand-binding moiety is attached.

[0089] In an embodiment, the anti-repeat region is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the Cas-associated region over at least 5 consecutive nucleotides. Thus, the Cas-associated region of the programmable crRNA and the anti-repeat region of the scoutRNA can hybridize to form a hybridization region. When the scoutRNA self-hybridizes to form one or more hairpin regions, in an embodiment, the anti-repeat region can form a bulge. When the anti-repeat region of the scoutRNA and the Cas-association region of the crRNA hybridize to form a hybridization region, the RNA containing both the scoutRNA and the crRNA can retain association with the Cas RNA-binding domain of the V-type Cas protein.

[0090] In an embodiment, the scoutRNA activity and the crRNA activity are part of a single continuous strand of nucleotides. In an embodiment, the crRNA may be immediately downstream of the scoutRNA or downstream of the scoutRNA with an intervening sequence or moiety between the scoutRNA and the crRNA. The intervening sequence or moiety may be a ligand binding moiety, or a nucleotide or non-nucleotide loop region, or an ethylene glycol spacer such as 18S, 9S or C3. From the 5' to 3' direction, the crRNA:scoutRNA guide RNA may comprise, consist essentially of, or consist of a first region of the scoutRNA, an anti-repeat region of the scoutRNA, a second region of the scoutRNA, a loop between the scoutRNAs, a Cas-associated region, and a targeting region. In one embodiment, the crRNA with scoutRNA activity and guide RNA are two separate RNA molecules that together form a functional guide RNA and part of an RNA-ligand binding complex (or RNA scaffold). In this case, the molecule with scoutRNA activity should be able to interact (usually by base pairing) with a molecule with a targeting sequence (crRNA) to form a bipartite guide crRNA:scoutRNA.

[0091] Non-limiting examples of scoutRNAs and crRNAs that can be used in the V-type CRISPR-Cas system in the context of the present invention are provided below.

[0092] [ka] Thus, in embodiments in which the sequence targeting component comprises a V-type Cas protein such as Cas12d15 (Fu, BXH, Smith, JD, Fuchs, RT et al. Target-dependent nickase activities of the CRISPR Cas nucleases Cpf1 and Cas9. Nat Microbiol 4, 888-897 (2019). https: / / doi.org / 10.1038 / s41564-019-0382-0), the RNA-ligand binding complex preferably comprises a V-type crRNA:scoutRNA guide.

[0093] {b} Ligand-binding moiety In any embodiment, the RNA-ligand binding complex further comprises a ligand binding moiety capable of associating with a cognate ligand that actually recruits two or more effector proteins to the target DNA. As disclosed herein, the guide RNA and Cas protein together form a CRIS PR / Cas-based module for sequence targeting and recognition, while the ligand binding moiety recruits one or more effector proteins, such as base editing enzymes, that perform one or more genetic modifications. This linkage is critical to the base editing systems and methods disclosed herein.

[0094] In certain embodiments, the ligand binding moiety is selected from the group consisting of: MS2 coat protein (MCP), Ku, PP7 coat protein (PCP), ComRNA binding protein or binding domain thereof, SfMu, Sm7, Tat, CSY4, Qbeta, COM, pumilio, lambda N22, a moiety that associates with a ligand. A non-exhaustive list of examples of ligand binding moieties / ligands that can be used in the methods and systems provided herein is summarized in Table 3.

[0095] Table 3 - Non-exhaustive examples of ligand binding moiety-ligand pairs that may be used in various embodiments of the present invention. Both unmodified and chemically modified forms of ligand binding moieties and ligands are within the scope of the present invention.

[0096] [Table 3] In a preferred embodiment, the ligand-binding moiety is an RNA motif (which may be referred to as an RNA aptamer) that can associate with a cognate RNA-binding domain (which may be referred to as an RNA aptamer ligand) that actually recruits an effector protein (a base-editing enzyme) to the target DNA. In an embodiment in which the ligand-binding moiety is an RNA motif, the RNA-ligand binding complex may be referred to as an RNA scaffold. As disclosed herein, the guide RNA and the Cas protein together form a CRIS PR / Cas-based module for sequence targeting and recognition, while the RNA motif recruits, via the RNA-binding domain, two or more effector proteins, such as base-editing enzymes, that perform one or more genetic modifications. Thus, the RNA scaffold reversibly connects the effector module (e.g., a base-editing enzyme) to the sequence recognition module (e.g., a type II Cas protein or a type V Cas protein). For this purpose, the RNA scaffold is designed such that an RNA motif (e.g., an MS2 operator motif) that specifically binds to an RNA-binding protein (e.g., MS2 coat protein, MCP) is attached to the guide RNA. As a result, this RNA scaffold component of the platform disclosed herein is a designed RNA molecule that contains not only a guide RNA for specific DNA / RNA sequence recognition and Cas protein binding, but also an RNA motif (also known as an RNA aptamer) for effector recruitment. In this way, effector proteins can be recruited to target sites by their ability to bind to the RNA motif via associated or fused RNA binding domains. Due to the flexibility of RNA scaffold-mediated recruitment, functional monomers, as well as dimers, trimers, tetramers, or oligomers, can be relatively easily formed near target DNA or RNA sequences. Preferably, a dimeric RNA binding domain is recruited to a single RNA motif. These pairs of RNA motifs / binding proteins can be derived from naturally occurring sources (e.g., RNA phages, or yeast telomerase) or can be artificially designed (e.g., RNA aptamers and their corresponding binding protein ligands). A non-exhaustive list of examples of recruiting RNA motif / RNA binding protein pairs that can be used in the methods and systems provided herein is summarized in Table 4. As will be apparent to one of skill in the art, chemical modifications and / or sequence variants of aptamers and their binding partners may also be utilized. Table 4. Examples of recruiting RNA motifs that can be used in the present invention and their paired RNA-binding proteins / protein domains

[0097] [Table 4] *In the present invention, the RNA binding domain is fused to an effector protein (see, for example, Table 5).

[0098] The sequences of the above binding pairs are listed below. 1. Telomerase Ku-binding motif / Ku heterodimer a. Ku-binding hairpin (SEQ ID NO: 7)

[0099] [ka] b. Ku heterodimer (SEQ ID NO:8)

[0100] [ka] 2. Telomerase Sm7 binding motif / Sm7 homoheptamer a. Sm consensus site (single stranded) (SEQ ID NO: 9)

[0101] [ka] b. Monomeric Sm-like protein (Archaea) (SEQ ID NO: 10)

[0102] [ka] 3. MS2 phage operator stem loop / MS2 coat protein a. MS2 phage operator stem loop (SEQ ID NO: 11)

[0103] [ka] b. MS2 coat protein (SEQ ID NO: 12)

[0104] [ka] 4. PP7 phage operator stem loop / PP7 coat protein a. PP7 phage operator stem loop (SEQ ID NO: 13)

[0105] [ka] b. PP7 coat protein (PCP) (SEQ ID NO: 14)

[0106] [ka] 5. SfMuCom stem loop / SfMuCom binding protein a. SfMu cone stem loop (SEQ ID NO: 15)

[0107] [ka] b. SfMuCom binding protein (SEQ ID NO: 16)

[0108] [ka] Further recruitment RNA motifs and their RNA-binding proteins / protein domains are the Qbeta phage operator stem loop (SEQ ID NO: 68) and Qbeta coat protein [Q65H] (SEQ ID NO: 69), BoxB (SEQ ID NO: 70) and lambda bacteriophage protein N (SEQ ID NO: 71), the Csy4 binding motif (SEQ ID NO: 72) and Csy4[H29A] (SEQ ID NO: 73). The RNA-ligand binding complex may include one or more ligand binding moieties located at the 3' end, 5' end, or within the guide RNA of the RNA-ligand binding complex. In a preferred embodiment, the RNA-ligand binding complex may include one or more RNA motifs that allow for the recruitment of one or more effectors. In any embodiment in which the RNA-ligand binding complex includes two or more RNA motifs, the RNA motifs may be the same or different. In embodiments in which the RNA motifs are different, each motif may recruit either a single or multiple effector proteins. In embodiments in which multiple effector proteins are recruited, these proteins may have the same or different functions. In all embodiments, at least one of the RNA motifs is used to recruit two or more effector proteins. In a preferred embodiment, the at least one RNA motif may be an MS2 aptamer, and in some cases, the MS2 aptamer has an extended stem. The extended stem may comprise 2-24 nucleotides. In one embodiment, the at least one RNA motif may be a PP7 aptamer. The MS2 aptamer and the PP7 aptamer may each recruit a single effector protein or multiple effector proteins. The MS2 aptamer and the PP7 aptamer may recruit the same or different effector proteins. The RNA ligand binding complex (or RNA scaffold) can be either a single RNA molecule or a complex of multiple RNA molecules. For example, the guide RNA (including either crRNA, crRNA and tracrRNA, or crRNA and scoutRNA) and the ligand binding portion (or recruiting RNA motif) can be three segments of one long single RNA. Alternatively, the various components can be on separate molecules. In the latter case, two or more components can be bound together via covalent or non-covalent bonds or bonds (including, for example, Watson-Crick base pairing) to form the scaffold.

[0109] In one example, the RNA scaffold can comprise two separate RNA molecules. The first RNA molecule can comprise a region capable of forming a stem duplex structure with the programmable crRNA and a complementary region. The second RNA molecule can comprise a complementary region in addition to the tracrRNA and one or more RNA motifs. Through this stem duplex structure, the first and second RNA molecules form the RNA scaffold of the present invention. In one embodiment, the first and second RNA molecules each comprise a sequence (of about 6 to about 30 nucleotides) that base pairs with another sequence. Similarly, the tracrRNA and the RNA motifs can be on different RNA molecules and can be combined with other stem duplex structures. In another example, the RNA scaffold can comprise two separate RNA molecules. The first RNA molecule can comprise a programmable crRNA and a complementary region. The second RNA can comprise a cognate complementary region in addition to the scoutRNA and one or more RNA motifs. Through hybridization of the complementary regions, the first and second RNA molecules form the RNA scaffold of the present invention. In one embodiment, the first and second RNA molecules each comprise a sequence (about 4-10 nucleotides, preferably 5 nucleotides) that base pairs with the other sequence. Similarly, the scout RNA and the RNA motif can be on different RNA molecules and can be with other stem duplex structures.

[0110] The RNAs and related scaffolds of the present invention can be produced by a variety of methods known in the art, including cell-based expression, in vitro transcription, and chemical synthesis. The ability to chemically synthesize relatively long RNAs (200-mers or longer) using TC-RNA chemistry (see, for example, U.S. Pat. No. 8,202,983) allows the generation of RNAs with special characteristics that outperform the four basic ribonucleotides (A, C, G, and U). Cas protein-RNA ligand binding complexes can be produced by recombinant techniques using host cell systems or in vitro translation-transcription systems known in the art. Details of such systems and techniques can be found, for example, in WO 2014 / 144761, WO 2014 / 144592, WO 2013 / 176772, US 20140273226, and US 2014 / 0273233, the contents of which are incorporated herein by reference in their entirety. The complexes can be isolated or purified, at least to some extent, from the cellular material of the cells in which they are produced or from the in vitro translation-transcription system.

[0111] Positioning of the ligand-binding moiety The ligand binding moiety can be located at various positions within the RNA-ligand binding complex. The ligand binding moiety can be directly (e.g., via a covalent bond) attached to the guide RNA, or via a linker that is associated with each of the guide RNA and the ligand binding moiety via one or more covalent bonds. The attachment of the ligand binding moiety to the guide RNA, whether directly via a covalent bond or via a linker, can be at any of a number of positions. As non-limiting examples, the ligand-binding moiety may be attached directly (e.g., via a covalent bond) or via a linker to the 3'-end of the guide RNA, or to the 5'-end of the guide RNA if the guide RNA is single stranded. Alternatively, the ligand-binding moiety may be attached directly (e.g., via a covalent bond) or via a linker to the 3'-end or 5'-end of the components that make up the guide RNA, i.e., crRNA, tracrRNA or scoutRNA. If the ligand binding moiety is a nucleotide sequence, e.g. an RNA motif, it can be inserted into the guide RNA. More than one ligand binding moiety may be incorporated into the RNA-ligand binding complex at any one of the positions outlined in the non-limiting examples below. When multiple ligand binding moieties are incorporated, they may be adjacent to each other or may be attached at separate positions.

[0112] When the RNA-ligand binding complex is an RNA scaffold, the RNA motif can be placed at various positions on the RNA scaffold, as described in Example 1. The RNA motif, for example, the MS2 aptamer, can be located at the 3' end of the crRNA, the tetraloop of the sgRNA, the stem loop 2 of the tracrRNA, or the stem loop 3 of the tracrRNA. The positioning of the MS2 aptamer is important due to steric hindrance that may arise from bulky loops. In a preferred embodiment, the MS2 aptamer is at the 3' end of the tracrRNA. In another embodiment, the MS2 aptamer is at the 5' end of the scoutRNA. In another embodiment, the MS2 aptamer is at the 5' end of the tracrRNA. Advantageously, the placement of the MS2 aptamer at the 3' end of the tracrRNA or the 5' end of the scoutRNA is in space, thus reducing steric hindrance with other bulky loops of the RNA scaffold.

[0113] Positioning the ligand-binding moiety: Type II crRNA: tracrRNA guide If the guide RNA is a type II crRNA:tracrRNA guide, the ligand binding moiety can be attached to the 3' end of the guide RNA, or to the 5' end of the guide RNA if the guide RNA is single stranded (corresponding to the 3' end of the tracrRNA and the 5' end of the crRNA in the sgRNA), or to the 3' end of the crRNA if they are separate strands, to the 3' end of the tracrRNA, to the 5' end of the tracrRNA, or to the 5' end of the crRNA, either directly or via a linker. Thus, the ligand binding moiety can be attached to the first or last nucleotide in the sgRNA, crRNA or tracrRNA. Alternatively, the ligand binding moiety can be attached to a nucleotide that is not the first or last nucleotide in the sgRNA, tracrRNA or crRNA. Alternatively, the association of the ligand binding moiety with the guide RNA can be at the tracrRNA in either the anti-repeat or distal region, or at the crRNA adjacent to the spacer or in the repeat region, or at the tetraloop between the tracrRNA and the crRNA, if present.

[0114] Positioning the ligand-binding moiety: V-type crRNA: tracrRNA guide If the guide RNA is a V-type crRNA:tracrRNA guide, the ligand binding moiety can be attached to the 3' end of the guide RNA, or to the 5' end of the guide RNA if the guide RNA is single stranded (corresponding to the 3' end of the crRNA in the sgRNA and the 5' end of the tracrRNA), or to the 3' end of the crRNA if they are separate strands, to the 3' end of the tracrRNA, to the 5' end of the tracrRNA, or to the 5' end of the crRNA, either directly or via a linker. Thus, the ligand binding moiety can be attached to the first or last nucleotide in the sgRNA, crRNA or tracrRNA. Alternatively, the ligand binding moiety can be attached to a nucleotide that is not the first or last nucleotide in the sgRNA, tracrRNA or crRNA. Alternatively, the association of the ligand binding moiety with the guide RNA can be at the tracrRNA in either the anti-repeat or distal region, or at the crRNA adjacent to the spacer or in the repeat region, or at the tetraloop between the tracrRNA and the crRNA, if present. Non-limiting exemplary locations of the ligand binding moiety can be seen in Figures 2, 3 and 4.

[0115] Positioning of ligand-binding moieties: V-type crRNA: scoutRNA guide In the case where the guide RNA is a crRNA:scoutRNA guide, the ligand binding moiety can be attached to the 3' end of the crRNA, the 3' end of the scoutRNA, the 5' end of the scoutRNA, or the 5' end of the crRNA, either directly or via a linker. Thus, the ligand binding moiety can be attached to the first or last nucleotide in the guide RNA, crRNA or scoutRNA. Alternatively, the ligand binding moiety can be attached to a nucleotide that is not the first or last nucleotide in the guide RNA, scoutRNA or crRNA. Alternatively, the association of the ligand binding moiety with the guide RNA can be the anti-repeat region or the upstream or downstream region of the scoutRNA, or the crRNA adjacent to the spacer or the repeat region. Non-limiting exemplary locations of the ligand binding moiety can be seen in Figure 6.

[0116] Positioning the ligand-binding moiety: A guide only for V-type crRNA When the guide RNA is a V-type crRNA-only guide, the ligand-binding moiety can be attached directly or via a linker to the 3' end of the guide RNA or the 5' end of the guide RNA. Thus, the ligand-binding moiety can be attached to the first or last nucleotide in the guide RNA. Alternatively, the ligand-binding moiety may be attached to the gRNA at a position other than the 5' or 3' end. Non-limiting exemplary positions of the ligand-binding moiety can be seen in Figure 5.

[0117] Modification example The RNA-ligand binding complex (or RNA scaffold) disclosed herein may contain one or more modifications. Such modifications may include the inclusion of at least one non-naturally occurring or modified nucleotide, or an analog thereof. Examples of such modifications include, but are not limited to, the addition of nucleotides to extend the sequence, the substitution of nucleotides, the addition of linker sequences, and the reconfiguration of the various components of the RNA-ligand binding complex (or RNA scaffold). Nucleotides may be modified at the ribose, phosphate linkage, and / or base moiety. Modified nucleotides may include 2'-0-methyl analogs, 2'-fluoro analogs, or 2'-deoxy analogs or 2'-ribose analogs. The nucleic acid backbone may be modified, for example, a phosphorothioate backbone may be used. Locked nucleic acids (LNA) or bridged nucleic acids (BNA) may also be used. Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, 5-methylcytidine, 5-methoxyuridine, pseudouridine, inosine, 7-methylguanosine. These modifications may be applied to any component of the RNA-ligand binding complex (or RNA scaffold). These modifications may be applied to any component of the CRISPR system. In a preferred embodiment, these modifications are performed on the RNA component (e.g., the guide RNA sequence). In certain embodiments, the above-described RNA-ligand binding complexes (or RNA scaffolds) or subsections thereof can contain one or more modifications, e.g., base modifications, backbone modifications, etc., that provide new or enhanced characteristics (e.g., improved stability) to the nucleic acid.

[0118] Modified backbones and modified internucleoside linkages Examples of suitable nucleic acids containing modifications include nucleic acids containing modified backbones, bases, sugars, or non-natural internucleoside linkages. Nucleic acids with modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.

[0119] Suitable modified oligonucleotide backbones which contain a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-amino phosphoramidates and aminoalkyl phosphoramidates), phosphorodiamidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, selenophosphates and boranophosphates (with normal 3'-5' linkages), 2'-5' linked analogs thereof, as well as those in which one or more internucleotide linkages are of reverse polarity, being 3'→3', 5'→5' or 2'→2' linkages. Suitable oligonucleotides that are reverse polarity contain a single 3' to 3' linkage at the 3'-most internucleotide linkage, i.e., a single reverse nucleoside residue that may be basic (either the nucleobase is missing or has a hydroxyl group in its place).Various salts (e.g., potassium or sodium), mixed salts and free acid forms are also included.

[0120] In certain embodiments, the subject nucleic acid contains one or more phosphorothioate and / or heteroatom internucleoside linkages, particularly -CH 2 -NH-O-CH 2 -, -CH2 -N(CH 3 )-O-CH 2 - (known as the methylene (methylimino) or MMI backbone), -CH 2 -ON(CH 3 )-CH 2 -, -CH 2 -N(CH 3 )-N(CH 3 )-CH 2 - and -ON(CH 3 )-CH 2 -CH 2 - (wherein the natural phosphodiester internucleotide linkage is -OP(=O)(OH)-O-CH 2 -). MMI type internucleoside linkages are disclosed in the above-referenced U.S. Patent No. 5,489,677. Suitable amide internucleoside linkages are disclosed in U.S. Patent No. 5,602,240. Nucleic acids with morpholino backbone structures, such as those described in U.S. Patent No. 5,034,506, are also suitable. For example, in some embodiments, the subject nucleic acids contain a six-membered morpholino ring instead of a ribose ring. In some of these embodiments, phosphorodiamidate or other non-phosphodiester internucleoside linkages replace the phosphodiester linkage. Suitable modified polynucleotide backbones that do not contain phosphorus atoms therein include backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages, those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, riboacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and mixed N, O, S and CH 2 Contains other things that have component parts.

[0121] modified sugar moiety The nucleic acids of the invention may also contain one or more substituted sugar moieties. Preferred polynucleotides contain sugar substituents selected from: OH; H; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-C0-alkyl, where the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Particularly preferred are O((CH 2 )nO)mCH 3 , O(CH 2 )nOCH 3 , O(CH 2 )nNH 2 , O(CH 2 )nCH 3 , O(CH 2 )nONH 2 , and O(CH 2 )nON((CH 2 )nCH 3 ) 2 where n and m are from 1 to about 10. Other suitable polynucleotides contain sugar substituents selected from the following: C1-C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, 0-alkaryl or 0-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , S.O. 2 CH 3 , O.N.O. 2 , NO 2 , N 3 , N.H. 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Suitable modifications include 2'-methoxyethoxy (2'-O-CH 2 CH2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al. Chim. Acta, 1995, 78, 486-504), i.e., an alkoxyalkoxy group. Further suitable modifications include 2'-dimethylaminooxyethoxy, i.e., O(CH 2 ) 2 ON(CH 3 ) groups (also known as 2'-DMAOE) (described in the Examples below), and 2'-dimethylaminoethoxyethoxy (also known as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH 2 -O-CH 2 -N(CH 3 ) 2 Examples include:

[0122] Other suitable sugar substituents include methoxy (-O-CH 3 ), aminopropoxy (-OCH 2 CH 2 CH 2 NH2), allyl (-CH 2 -CH=CH 2 ), -0-AllylCH 2 -CH=CH 2 ) and fluoro (F). The 2'-sugar substituent may be at the arabino (up) or ribo (down) position. A suitable 2'-arabino modification is 2'-F. Similar modifications can also be made at other positions on the oligomeric compound, particularly the 3' position of the sugar on the 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligomeric compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0123] Base Modifications and Substitutions A subject nucleic acid may also contain nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "non-recombinant" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one).

[0124] Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles (e.g., 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone). Additional nucleobases include those disclosed in U.S. Pat. No. 3,687,808, The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are useful for increasing the binding affinity of oligomeric compounds. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi et al., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are suitable base substitutions when combined with, for example, 2'-0-methoxyethyl sugar modifications.

[0125] The modifications disclosed herein can be incorporated into the tetraloop of the sgRNA, the 3' end of the tracrRNA or scoutRNA, and the RNA motif in stem loop 1, 2 loop, the 3' end of the tracrRNA or scoutRNA, and stem loop 1, 2 or 3. The modifications disclosed herein include extension of the repeat / anti-repeat of the sgRNA, positioning of the RNA motif at the 3' end of the tracrRNA or scoutRNA, linker sequences connecting the RNA motif to the tracrRNA or scoutRNA, modification of nucleotides of the RNA motif, and extension of the RNA motif.

[0126] Modification of nucleotides in RNA motifs When the RNA-ligand binding complex is an RNA scaffold, modifications can be made to the RNA motif, for example, the aptamer sequence. For example, suitable modifications are to the C-5 and F-5 aptamer variants. In a preferred embodiment, the modification to the aptamer is the substitution of 2-aminopurine (2-AP) for the adenine at position 10. Advantageously, the substitution induces a conformational change that results in greater affinity. The affinity of the F-5 variant to the MS2 coat protein was about three times higher than the parent F5 sequence. Without wishing to be bound by any theory, it is believed that the conformational change induced by 2-AP results in hydrogen bond formation between the exocyclic amino group of the 2-AP nucleotide at position 10 and the carbonyl of B59 of the backbone. It is believed that the substitution of the MS2 hairpin sequence with the higher affinity MS2 sequence enhances base editing efficiency, since the amino acid substitution helps to arrange the RNA stem loop into a conformation that is better recognized by the coat protein. Enumerated above are 2'deoxy-2-aminopurine, 2'ribose-2-aminopurine, phosphorothioate modifications, 2'-O-methyl modifications, 2'-O-methyl modifications, 2'-Fluro modifications and LNA modifications etc. Advantageously, the modifications provide increased stability and help promote stronger binding / folding of the desired hairpin.

[0127] RNA motif extension Where the RNA ligand binding complex is an RNA scaffold, the RNA motif can be extended. The length of the RNA motif extension can be variable. The extension to the RNA motif can range from 2 to 24 nucleotides. The extension to the RNA motif can be more than 24 nucleotides. Advantageously, the extension of the RNA motif increases the flexibility of the motif. The extension to the RNA motif can be a double-stranded extension or a single-stranded extension. A double-stranded extension provides a more stable RNA scaffold.

[0128] Repeat expansion: anti-repeat upper stem Guide RNAs, including crRNA and tracrRNA, can be provided as sgRNAs. The two components are linked via a repeat:anti-repeat. The repeat:anti-repeat upper stem can be extended to increase flexibility, proper folding and stability of the loop. The repeat:anti-repeat can be extended by 2, 3, 4, 5, 6, 7 bp or more on either side of the tetraloop.

[0129] Combination of Modifications The RNA scaffold may have one or more of the above modifications. The one or more modifications may be on different components of the RNA backbone (e.g., an extension of the repeat:anti-repeat of the sgRNA and an extension of the RNA motif) or on the same component of the RNA backbone (e.g., an extension of the RNA motif and a substitution of a nucleotide in the RNA motif). The modifications may be two or more, three or more, four or more, or five or more. In one embodiment, the modification may be an extension of the RNA motif and / or a substitution of one or more nucleotides. An example of an RNA motif used herein is the MS2 aptamer. The MS2 motif specifically binds to the MS2 bacteriophage coat protein (MCP). A series of aptamer families was obtained by repeating the in vitro selection process. Two of the aptamer family members include the MS2 C-5 mutant and the MS2 F-5 mutant. One significant difference between wild-type MS2 and the C-5 and F-5 mutants is the substitution of a uracil nucleotide to a cytosine at the 5th position of the aptamer loop. The F-5 mutant has been reported to have a higher affinity for the coat protein compared to the wild-type and other members of the aptamer family. Suitably, both the C-5 mutant and the F-5 mutant are used as aptamers in the present invention. In one embodiment, the MS2 aptamer is wild-type MS2, mutant MS2 or a mutant thereof. In another embodiment, the MS2 aptamer includes the C-5 and / or F-5 mutations. The MS2 protein bound to the tracrRNA can be a single copy (i.e., one MS2 loop) or a double copy (i.e., two MS2 loops). In a preferred embodiment, the RNA motif is a single copy. In other embodiments, the RNA motif is two or more copies.

[0130] Linker sequence The linker, if present, can be a species that connects the ligand binding moiety to the guide RNA. In embodiments where the ligand binding moiety is an RNA motif, the linker can be a species that connects the RNA motif to the guide RNA. The linker can be attached to each of the RNA motif and the guide RNA at one position, or to either or both of the RNA motif and the guide RNA at multiple positions. Attachment at multiple positions allows for greater control in the three-dimensional space of the RNA motif, which is then used by the effector. The RNA motif can be attached to the tracrRNA via a linker sequence. The linker sequence can be 2, 3, 4, 5, 6, 7 or more than 7 nucleotides. Advantageously, the linker sequence provides flexibility to the RNA-ligand binding complex (or RNA scaffold). The linker sequence can be a GC-rich sequence.

[0131] In certain embodiments, the linker comprises, consists essentially of, or consists of an oligonucleotide sequence, and optionally the linker comprises at least one or more 2' modifications, e.g., all nucleotides are 2' modified nucleotides in the linker. The nucleotide sequence may be random or may be purposely designed to be undesirably non-complementary to sequences in the ligand binding moiety, [RNA] or DNA target site. In certain embodiments, the linker comprises, consists essentially of, or consists of at least one phosphorothioate bond. In certain embodiments, the linker comprises, consists essentially of, or consists of a levulinyl moiety. In certain embodiments, the linker comprises, consists essentially of, or consists of an ethylene glycol moiety. In certain embodiments, the linker comprises or is selected from the group consisting of 18S, 9S, or C3.

[0132] In some embodiments, the linker, or each linker when more than one linker is present, is a nucleotide sequence that is 1-60, or 1-24, or 2-20, or 5-15 nucleotides in length. Furthermore, in some embodiments, the linker is GC-rich, e.g., has at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% GC nucleotides. When the linker comprises nucleotides, it can be, for example, single-stranded or double-stranded, or partially single-stranded and partially double-stranded. Furthermore, when the linker is an oligonucleotide, the linker can be exclusively RNA, exclusively DNA, or a combination thereof.

[0133] iii) Effector Proteins The third component of the platform disclosed in the present invention is a non-nuclease effector bound to a ligand. The effector protein may also be referred to as an "effector component." In embodiments where an RNA scaffold is used, the ligand is an RNA binding domain. The effector is not a nuclease and does not have any nuclease activity, but may have other types of DNA modifying enzymes, such as base editing activity. Examples of enzyme activities include, but are not limited to, deamination activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, dismutase activity, nickase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity. In some embodiments, the effectors have cytidine deaminase (e.g., AID, APOBEC3G), adenosine deaminase (e.g., ADA), DNA methyltransferase, and DNA demethylase activities. In some embodiments, the effectors can be derived from different vertebrate species, have different properties, or be novel chimeric proteins assembled in silico to bring together desired attributes and activities. In a preferred embodiment, this third component is a conjugate or fusion protein with an RNA-binding domain and an effector domain. These two domains may be joined via a linker.

[0134] In some embodiments, effectors are not required in some cell types (e.g., cancer lines that overexpress deaminases). In those cases, endogenous effectors (e.g., APOBEC, AID, etc.) can be genetically edited to include the recruitment module, so no exogenous editor is required. This is applicable to cell types that express the editor of interest (e.g., lymphatic system cells (B+T cells) and certain cancer cells).

[0135] {c} Ligand In any embodiment, the ligands of the invention are selected such that they can associate with the ligand-binding portion of the RNA-ligand binding complex and thus recruit an effector to the base editing complex. Suitable ligands and their cognate ligand-binding portions are shown in Table 3. Alternative ligands and their cognate ligand-binding portions are known in the art. When the RNA ligand binding complex is an RNA scaffold, the ligand is an RNA binding domain. Although various RNA binding domains can be used as ligands in the present invention, the RNA binding domain of Cas protein (such as Cas9) or its mutants (such as dCas9) should not be used. As mentioned above, direct fusion to dCas9, which is fixed to DNA in a defined structure, does not form a functional oligomeric enzyme complex at the correct position. Instead, the present invention utilizes various other RNA motif-RNA binding protein binding pairs. Examples include those listed in Table 4. In this way, effector proteins can be recruited to target sites through the function of the RNA binding domain binding to the recruiting RNA motif. Due to the flexibility of RNA scaffold-mediated recruitment, functional monomers, as well as dimers, tetramers, or oligomers, can be formed relatively easily near target DNA or RNA sequences. In each case, the monomer, dimer, tetramer, or oligomer can be recruited to a single RNA motif. In a preferred embodiment, an effector protein comprising a dimeric RNA binding protein can be recruited to a single RNA motif. In such an embodiment, each of the two RNA binding domain monomers is associated with an individual effector domain. The effector domains are the same or different. In an alternative embodiment, only one of the RNA binding domains that compose the dimer is associated with an effector domain.

[0136] {d} Effector domain The effector component comprises an active portion, i.e., an effector domain. An effector domain may also be referred to as an "effector." In some embodiments, the effector domain comprises a naturally occurring active portion of a non-nuclease protein (e.g., a deaminase). In other embodiments, the effector domain comprises a recombinant amino acid sequence (e.g., a substitution, deletion, insertion) of a naturally occurring active portion of a non-nuclease protein. The effector domain has an enzymatic activity. Examples of such activities include deamination activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, DNA methylation, histone acetylation activity, or histone methylation activity. Some modifications in non-nuclease proteins (e.g., deaminases) reduce off-target effects. For example, as described below, mutating Ser38 in AID to Ala (SEQ ID NO: 26) reduces the recruitment of AID to off-target sites. A non-exhaustive list of examples of DNA / RNA modifying enzymes that can be used as effector domains is detailed in Table 5.

[0137] Table 5. Examples of effectors (or effector domains) that can be used in the present invention

[0138] [Table 5] Effector (Effector Domain) Full Name: AID: Activation-induced cytidine deaminase, also known as AICDA APOBEC1: Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 1 APOBEC3A: Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3A APOBEC3B: Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3B APOBEC3C: Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3C APOBEC3D: Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3D APOBEC3F: Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3F APOBEC3G: Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G APOBEC3H: Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3H ADA: Adenosine deaminase ADAR1: Adenosine deaminase acting on RNA 1 ADAR2: Adenosine deaminase acting on RNA 2 ADAR3: adenosine deaminase acting on RNA 3 CDA: cytidine deaminase DnMt1: DNA (cytosine-5-)-methyltransferase 1 DnMt3a: DNA (cytosine-5-)-methyltransferase 3 alpha. DnMt3b: DNA (cytosine-5-)-methyltransferase 3 beta Tet1:10-11 translocation 1 Tet2:10-11 translocation 2 Tdg: thymine DNA glycosylase In alternative embodiments, the effector domain may be a reporter, a fluorescent tag, an active drug or an HDR enhancer.

[0139] Other domains The effector protein may include other domains. In certain embodiments, the effector protein may include at least one nuclear localization signal (NLS). Generally, an NLS includes a stretch of basic amino acids. Nuclear localization signals are known in the art (see, for example, Lange et al., J.Biol.Chem., 2007, 282:5101-5105). The NLS may be located at the N-terminus, C-terminus, or internal position of the fusion protein. The NLS helps to optimize on-target base editing, resulting in more efficient base editing. In some embodiments, the fusion protein may contain at least one cell-penetrating domain, which may facilitate the delivery of the protein to a target cell. In one embodiment, the cell-penetrating domain may be a cell-penetrating peptide sequence. Various cell-penetrating peptide sequences are known in the art, including, for example, the sequences of HIV-1 TAT protein, TLM of human HBV, Pep-1, VP22, and polyarginine peptide sequences.

[0140] In one embodiment, AID was used as an example to show how the system works. AID is a cytidine deaminase that can catalyze the deamination reaction of cytidine in the context of DNA or RNA. When delivered to the target site, AID changes the C base to a U base. In dividing cells, this can result in a C to T point mutation. Alternatively, the C to U change triggers cellular DNA repair pathways, primarily, to remove the mismatched UG base pair and replace it with a TA, AT, C-Ga-T, CG, or GC pair, resulting in a point mutation at the target CG site. Since excision repair pathways are present in most, if not all, somatic cells, recruitment of AID to the target site can correct the CG base pair relative to the others. In that case, if the CG base pair is the genetic mutation that induces the underlying disease in somatic tissues / cells, the above approach can be used to correct the mutation, thereby treating the disease. Similarly, if the underlying disease-inducing genetic mutation is an AT base pair at a particular site, the same approach can be used to recruit adenosine deaminase to the particular site, which can correct the AT base pair to another base pair (see, for example, US10113163 by David Liu). Other effector enzymes are expected to produce other types of changes in base pairing.

[0141] Linker The two domains, as well as other domains disclosed herein, can be linked by any means known to those of skill in the art, including but not limited to chemical modifications, peptide linkers, chemical linkers, covalent or non-covalent bonds, or protein fusions. The linkage can be permanent or reversible. See, for example, U.S. Pat. Nos. 4,625,014, 5,057,301, and 5,514,363, U.S. Patent Application Nos. 20150182596 and 20100063258, and WO 2012 / 142515, the contents of which are incorporated herein by reference in their entireties. In some embodiments, several linkers can be included to take advantage of the desired properties of each linker and each protein domain in the conjugate. For example, flexible linkers and linkers that increase the solubility of the conjugate are contemplated for use alone or with other linkers. Peptide linkers can be linked to one or more protein domains in the conjugate by expressing DNA encoding the linker. The linker can be an acid cleavable linker, a photocleavable linker, or a heat sensitive linker. Methods for attachment are well known to those skilled in the art and are encompassed for use in the present invention.

[0142] In some embodiments, the RNA binding domain and the effector domain can be linked by a peptide linker. The peptide linker can be linked by expressing a nucleic acid that encodes the two domains and the linker in frame. In some cases, the linker peptide can be linked at either or both of the amino and carboxy termini of the domains. In some examples, the linker is an immunoglobulin hinge region linker as disclosed in U.S. Patent Nos. 6,165,476, 5,856,456, U.S. Application Nos. 20150182596 and 2010 / 0063258, and International Application No. 2012 / 142515, each of which is incorporated herein by reference in its entirety.

[0143] The above three specific components ((i) sequence-targeting component (or sequence-targeting protein), (ii) RNA-ligand binding complex and (iii) effector protein) constitute a technology platform. Each component could be selected from Tables 1-5 to achieve a specific therapeutic / useful goal. A person skilled in the art would understand that the guide RNA must be suitable for the sequence targeting the selected protein. It would also be understood by a person skilled in the art that the ligand binding moiety must be suitable for the selected ligand and vice versa.

[0144] Non-limiting examples of RNA scaffold-mediated mobilization systems constructed using (i) dCas9 from S. pyogenes as the sequence-targeting protein, (ii) an RNA scaffold comprising crRNA, tracrRNA, and MS2 operator motifs, and (iii) effector fusions comprising human AID fused to the MS2 operator-binding protein MCP, and effector fusions containing PmCDA1 fused to MCP are shown below. The sequences of the components are listed below.

[0145] Amino acid sequence of Streptococcus pyogenes dCas9 (D10A, H840A active site mutant shown in bold and underlined) (SEQ ID NO: 17)

[0146] [ka] (Underlined residues: D10A, H840A active site mutant).

[0147] Cas9 D10A amino acid sequence (mutated residues underlined: D10A, SEQ ID NO: 18).

[0148] [ka] Humanized Cas9 D10A protein nucleotide sequence (SEQ ID NO:19)

[0149] [ka]

[0150] [ka]

[0151] [ka] RNA scaffold expression cassette (S. pyogenes) containing a 20 nucleotide programmable sequence, a CRISPR RNA motif, and an MS2 aptamer motif (SEQ ID NO: 20):

[0152] [ka] (N 20 : Programmable sequence. Underlined: CRISPR RNA motif; Bold: MS2 motif; Italics: Terminus).

[0153] The above RNA scaffold contains one MS2 loop (1xMS2). Shown below is an RNA scaffold containing two MS2 loops (2xMS2), with the MS2 scaffold underlined (SEQ ID NO: 21).

[0154] [ka] Effector AID-MCP fusion (SEQ ID NO:22):

[0155] [ka] (NH2)-AID-Linker-MCP-(COOH) Similar to the above Cas proteins, non-nuclease effectors can also be obtained as recombinant polypeptides. Techniques for producing recombinant polypeptides are known in the art. See, for example, Creighton, "Proteins: Structures and Molecular Principles," WH Freeman & Co., NY, 1983; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2003; and Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 2001). As described herein, the recruitment of AID to off-target sites can be reduced by mutating Ser38 to Ala in AID. The sequences of both wild-type AID and AID_S38A (phosphorylation null, pnAID) are listed below. wtAID cDNA (bold underlined Ser38 codon, SEQ ID NO:23):

[0156] [ka] wtAID protein (bold underlined Ser38, SEQ ID NO:24):

[0157] [ka] AID_S38A cDNA (bold underlined S38A mutation, SEQ ID NO: 25)

[0158] [ka] AID_S38A protein (bold underlined S38A mutation, SEQ ID NO: 26)

[0159] [ka] Humanized nucleotide sequence PmCDA1 (Petromyzon marinus) (SEQ ID NO: 27)

[0160] [ka] Amino acid sequence PmCDA1 (SEQ ID NO:28)

[0161] [ka] pMCDA1-Linker-MCP (SEQ ID NO:29)

[0162] [ka] Key: pMCDA 1-Linker-MCP Below are some non-limiting exemplary RNA sequences of guide RNA constructs that can be used with the Type II Cas proteins outlined above, each containing from the 3' to 5' end a customizable target, a gRNA scaffold, and one or two copies of the MS2 aptamer.

[0163] Sequence of gRNA_MS2 construct (SEQ ID NO:30):

[0164] [ka] Key: Customizable target-gRNA scaffold-MS 2 aptamer Sequence of gRNA_2xMS2 construct (SEQ ID NO:31):

[0165] [ka] Key: Customizable target-gRNA scaffold-MS 2 aptamer The above three components of the platform / system disclosed herein can be expressed using one, two, or three expression vectors. The system can be programmed to target virtually any DNA or RNA sequence. In addition to the above second generation base editors, the modular components of the system, including any suitable Cas orthologs, deaminase orthologs, and other DNA modifying enzymes, can be varied to generate similar second generation base editors.

[0166] binding affinity The most widely used value in the literature for RNA-protein binding affinity is the equilibrium dissociation constant (K d ) which is a ratio defined as:

[0167]

number

[0168]

number

[0169]

number

[0170] In one embodiment, ligand-binding moieties and / or ligands are selected that have a binding affinity with a dissociation constant (KD) of less than 100 nM. In a preferred embodiment, an RNA motif and / or an RNA-binding domain is selected that has a dissociation constant between 10 and 100 nM. In a preferred embodiment, a ligand-binding moiety and / or ligand is selected that has a dissociation constant between 1 and 10 nM. In a most preferred embodiment, a ligand-binding moiety and / or ligand is selected that has a dissociation constant less than 1 nM. Low binding affinities of ligand-binding moieties and / or ligands are not suitable for this system because they have dissociation constants greater than 100 nM. In one embodiment, an RNA motif and / or an RNA binding domain is selected that has a binding affinity with a dissociation constant (KD) of less than 150 nM, less than 140 nM, less than 130 nM, less than 120 nM, less than 110 nM, or less than 100 nM. In a preferred embodiment, an RNA motif and / or an RNA binding domain is selected that has a dissociation constant of 10-150 nM. In a preferred embodiment, an RNA motif and / or an RNA binding domain is selected that has a dissociation constant of 1-10 nM. In one embodiment, an RNA motif and / or an RNA binding domain is selected that has a dissociation constant of less than 1 nM. Low binding affinities of RNA motifs and / or RNA binding domains are not suitable for this system because they have dissociation constants of more than 150 nM. Methods for measuring dissociation constants at equilibrium are well known to those skilled in the art.Preferentially, surface plasmon resonance should be used, and details of such methods are described in Jing, M. & Bowser, MT (2011) Methods for measuring aptamer-protein equilibria: A review.Analytica Chimica Acta.686(1-2),9-18.

[0171] Cell Types and Therapeutic Uses Immune cells, particularly primary immune cells, naturally occurring in a host animal or patient or derived from induced pluripotent stem cells (iPSCs) can be genetically modified using the methods and systems provided herein. Immune cells include pluripotent cells that are immune cell precursors, such as hematopoietic stem cells (HSCs), which can differentiate into T cells, natural killer (NK) cells, B cells, myeloblasts, erythroblasts, and all blood and immune cells. Hematopoietic stem cells (HSCs) arise from hemangioblasts, which differentiate into hemangioblasts, vascular smooth muscle cells, and vascular endothelial cells, which can give rise to HSCs. HSCs can give rise to common myeloid and common lymphoid progenitor cells, which give rise to T cells, natural killer (NK) cells, B cells, myeloblasts, erythroblasts, and other cells involved in the production of cells of the blood, bone marrow, spleen, lymph nodes, and thymus.

[0172] Also provided herein are methods for genome engineering (e.g., for altering or manipulating the expression of one or more genes or one or more gene products) in prokaryotic or eukaryotic cells in vitro, in vivo, or ex vivo. In particular, the methods provided herein are useful for targeted base editing disruptions in mammalian cells, including primary human T cells, natural killer (NK) cells, CD34+ hematopoietic stem and progenitor cells (HSPCs), such as HSPCs isolated from umbilical cord blood or bone marrow, and cells differentiated therefrom. Also provided herein are genetically engineered cells derived from hematopoietic stem cells, such as T cells, that have been modified by the methods described herein.

[0173] In some cases, the methods are configured to produce engineered T cells derived from HSCs or iPSCs that are suitable as "universally accepted" cells for therapeutic applications. Such methods can also be applied to natural killer (NK) cells, CD34+ hematopoietic stem and progenitor cells (HSPCs), such as HSPCs isolated from umbilical cord blood or bone marrow, and cells differentiated therefrom.

[0174] In another aspect, methods of targeting diseases for base editing correction are provided herein. The target sequence can be any disease-associated polynucleotide or gene established in the art. Examples of useful applications of mutation or "correction" of endogenous gene sequences include recombination of disease-associated gene mutations, recombination of sequences encoding splice sites, recombination of regulatory sequences, recombination of sequences inducing gain-of-function mutations, and / or recombination of sequences inducing loss-of-function mutations, and targeted recombination of sequences encoding structural features of proteins. In some cases, it may be advantageous to genetically engineer cells using the methods described herein so that they express a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR). A "chimeric antigen receptor (CAR)" may also be referred to as a "chimeric receptor", "T body", or "chimeric immune receptor (CIR)". As used herein, the term "chimeric antigen receptor (CAR)" refers to an artificially constructed hybrid protein or polypeptide that includes an extracellular antigen-binding domain of an antibody (e.g., a single chain variable fragment (scFv)) operably linked to a transmembrane domain and at least one intracellular domain. In general, the antigen-binding domain of a CAR has specificity for a particular antigen expressed on the surface of a target cell of interest. For example, T cells can be engineered to express a CAR specific for CD19 on B cell lymphoma. For allogeneic antitumor cell therapy not limited by donor matching, cells can be engineered to knock in the nucleic acid encoding the CAR but also knock out the genes involved in donor matching (TCR and HLA markers).

[0175] As used herein, the terms "genetically modified" and "genetically engineered" are used interchangeably and refer to a prokaryotic or eukaryotic cell that contains an exogenous polynucleotide, regardless of the method used for insertion. In some cases, an effector cell is engineered to contain a non-naturally occurring nucleic acid molecule that has been created or engineered by the hand of man (e.g., using recombinant DNA technology) or derived from such a molecule (e.g., by transcription, translation, etc.). An effector cell that contains an exogenous, recombinant, synthetic, and / or otherwise modified polynucleotide is considered to be an engineered cell. In another aspect, the present disclosure provides a host cell or cell line comprising the above-mentioned system or a progeny thereof. In another aspect, the cell line may be derived from at least one cell prepared according to the method provided herein. Methods for preparing cell lines are well known in the art.

[0176] Cellular and Ex Vivo Therapies Various embodiments of the invention also provide cells produced or used according to any of the other embodiments of the invention for use in therapy. In one embodiment, the invention is directed to a method for generating therapeutic cells, such as T cells engineered to express a chimeric antigen receptor (CAR-T) or a T cell receptor (TCR-T). CAR-T / TCR-T cells may be derived from primary T cells or may be differentiated from stem cells. Suitable stem cells include, but are not limited to, mammalian stem cells, such as human stem cells, including, but not limited to, hematopoietic, neural, embryonic, induced pluripotent stem cells (iPSC), mesenchymal, mesodermal, hepatic, pancreatic, muscle, and retinal stem cells. Other stem cells include, but are not limited to, mammalian stem cells, such as mouse stem cells, e.g., mouse embryonic stem cells.

[0177] In various embodiments, the present invention may be used to knock out, engineer or enhance expression of a single gene or multiple genes in various cells or cell lines, including but not limited to cells of mammalian origin. The present systems and methods can be applied to multiple gene engineering, including engineering multiple genes or multiple targets within the same gene, as known in the art. This technology can be used for many applications, including but not limited to knocking out genes to make non-host cells non-immunogenic to the host, thereby preventing graft-versus-host disease, or knocking out genes to make non-host cells resistant to attack by the host, thereby preventing host-versus-graft disease. The approach is also relevant to generating allogeneic (off-the-shelf) or autologous (patient-specific) cell-based therapeutics.Such genes include genes involved in T cell receptors (TRAC, TRBC1, TRBC2, TRDC, TRGC1, TRGC2), major histocompatibility complex (MHC class I and class II) genes, including, for example, B2M, coreceptors (HLA-F, HLA-G), genes involved in the innate immune response (MICA, MICB, HCP5, STING, DDX41, Toll-like receptors (TLRs)), inflammation (NKBBiL, LTA, TNF, LTB, LST1, NCR3, AIF1), heat shock proteins (HSPA1L, HSPA1A, HSPA1B), complement cascade, regulatory receptors (NOTCH family members), antigen processing (TAP, HLA-DM, HLA-DO), PD-1, CTLA-4, other members of the B7 family of confirmation point proteins, etc.), genes involved in immunosuppressive immune cells (FOXP3 and interleukin-3). Examples of genes that may be useful include, but are not limited to, genes involved in the interaction of T cells with the tumor microenvironment (including but not limited to, receptors for cytokines such as TGFB, IL-4, IL-7, IL-2, IL-15, IL-12, IL-18, IFNγ, etc.), genes involved in cytokine release syndrome (including but not limited to, IL-6, IFNγ, IL-8 (CXCL8), IL-10, GM-CSF, MIP-1α / β, MCP-1 (CCL2), CXCL9, and CXCL10 (IP-10)), genes that encode the antigen targeted by the CAR / TCR (e.g., endogenous CS1 if the CAR is designed against CS1), or other genes found to be beneficial in CAR-T / TCR-T (e.g., TET2), or other cell-based therapeutics, including but not limited to, CAR-NK, CAR-B, etc. See, e.g., DeRenzo et al., Genetic Modification Strategies to Enhance CAR T Cell Persistence for Patients With Solid Tumors. Front. Immunol., 15 February 2019. This technique can also be used to knock down or modify genes involved in the destruction of immune cells (e.g., T cells and NK cells), or genes that alert the patient's or animal's immune system that foreign cells, particles or molecules have entered the patient's or animal's body, or genes that code for proteins that are current therapeutic targets used to reduce or boost the immune response (e.g., CD52 and PD1, respectively).

[0178] One application of the methods and systems provided herein is to recombine HLA alleles in bone marrow cells or bone marrow cells differentiated from iPS cells to recombine haplotype hla alleles. The recombined cells can be used in bone marrow transplants to treat leukemia. Another application is to recombine the negative regulatory element of the fetal hemoglobin gene in hematopoietic stem cells to treat sickle cell anemia and β-thalassemia. When the negative regulatory element is mutated, expression of the fetal hemoglobin gene is reactivated in the hematopoietic stem cells, compensating for loss of function due to mutation of the adult alpha or beta hemoglobin gene. A further application is to recombine iPS cells to generate allogeneic therapeutic cells for various degenerative diseases, including Parkinson's disease (neuronal cell loss), type 1 diabetes (pancreatic β cell loss). Other exemplary uses include engineering T cells resistant to HIV infection by inactivating the CCR5 gene and other genes that encode receptors required for HIV cell entry; removing a premature stop codon in the DMD gene to re-establish expression of dystrophin; and correcting cancer driver mutations such as p53 Y163C.

[0179] Type of genetic modification Thus, provided herein are methods for the targeted disruption of transcription or translation of a target gene. In particular, the methods include the targeted disruption of transcription or translation of a target gene by disruption of a start codon, introduction of a premature stop codon, and / or targeted disruption of an intron / exon splice site. The methods described herein can be used to knock in and / or knock out one or more genes of interest in primary cells with improved efficiency and reduced rates of off-target indel formation. In a preferred embodiment, the methods are used for multiplexed base editing, including gene knock-ins, gene knock-outs, and missense mutations. As described in the following paragraphs and examples, our streamlined approach to genome engineering uses base editors for applications including targeted gene disruption by knockout and missense mutations, as well as gene correction. The methods described herein are well suited for studying immune cell biology and gene function, modeling diseases such as primary immune deficiencies, and correcting disease-causing point mutations, and generating novel cell products (e.g., T cell products) for therapeutic applications. Disruptions at splice acceptor-splice donor (SA-SD) sites are also useful because they can knock out coding sequences and non-coding RNAs (ncRNAs) without stop codon readthrough.

[0180] Delivery of components to cells Suitable methods for delivering base editing components to immune cells, such as hematopoietic cells, are provided in the Examples herein below. In the embodiments provided herein, the guide RNA molecule can be delivered to the target cell via various methods, including but not limited to the following: First, by direct introduction of synthetic RNA molecules (sgRNA, crRNA, tracrRNA, and any of their modifications) into the cell of interest, via electroporation, nucleofection, transfection, via nanoparticles, via viral-mediated RNA delivery, via non-viral-mediated delivery, via extracellular vesicles (e.g., exosomes and microvesicles), eukaryotic cell transfer (e.g., recombinant yeast), and other methods that can package RNA molecules and deliver them to the target living cell without altering the genomic landscape. Other methods for introduction of guide RNA include non-integrative transient introduction of polynucleotides containing relevant sequences for protein recruitment such that they can be transcribed into target guide RNA, including, but not limited to, DNA-only vehicles (e.g., plasmids, MiniCircles, MiniVectors, MiniStrings, protelomerase-generated DNA molecules (e.g., Doggybones), artificial chromosomes (e.g., HACs), cosmids), nanoparticle-based DNA vehicles, extracellular vesicles (e.g., exosomes and microvesicles), eukaryotic cell transfer (e.g., recombinant yeast), transient viral transfer with AAV, non-integrative viral particles (e.g., lentivirus- and retrovirus-based systems), cell-penetrating peptides, and other techniques that allow DNA to be introduced into cells without directly integrating into the genomic landscape. Other methods for the introduction of guide RNA include the use of integrative gene transfer techniques to stably introduce the machinery for guide RNA transcription into the genome of the target cell, which can be controlled via constitutive or promoter-inducible systems to attenuate the expression of the guide RNA, and can also be designed to allow the system to be removed after fulfilling its usefulness (e.g., by introducing a Cre-Lox recombination system). Such techniques for stable gene transfer include, but are not limited to, integration of viral particles (e.g., lentivirus, adenovirus, retrovirus-based systems), transposase-mediated transfer (e.g., Sleeping Beauty and Piggybac), use of non-homologous repair pathways introduced by DNA breaks (e.g., using CRISPR and TALEN), surrogate DNA molecules, and other techniques that facilitate integration of the target DNA into the cells of interest.

[0181] Although the methods for delivering deaminase effector fusion proteins and CRISPR sequencing targeting components are often mediated by the same technology, in some situations there are advantages to mediating delivery by different methods. First, mRNA and protein molecules are directly introduced into the cells of interest by electroporation, nucleofection, transfection, via nanoparticles, via virus-mediated packaged delivery, via extracellular vesicles (e.g., exosomes and microvesicles), eukaryotic cell transfer (e.g., by recombinant yeast), and other methods that can package macromolecules and deliver them to target live cells without integrating into the genomic landscape. Other methods for introducing molecules include, but are not limited to, non-integrative transient introduction of the molecule or a polynucleotide containing relevant sequences for protein recruitment such that the molecule can be transcribed and translated into a target protein molecule, via DNA-only vehicles (e.g., plasmids, MiniCircles, MiniVectors, MiniStrings, protelomerase-generated DNA molecules (e.g., Doggybones), artificial chromosomes (e.g., HACs), cosmids), nanoparticle-based DNA vehicles, extracellular vesicles (e.g., exosomes and microvesicles), eukaryotic cell transfer (e.g., via recombinant yeast), transient viral transfer via AAV, non-integrative viral particles (e.g., lentivirus and retrovirus-based systems), and via other technologies that can mediate DNA introduction into cells without directly integrating into the genomic landscape. An alternative method for introducing the deaminase effector fusion protein and CRISPR sequencing targeting components includes the use of integrative gene transfer techniques to stably introduce the machinery for transcription and translation into the genome of the target cell, which can be controlled via constitutive or inducible promoter systems to attenuate the expression of the molecule or molecules, which can also be designed to allow the system to be removed after its usefulness has been fulfilled (e.g., ). Such techniques for stable gene transfer include, but are not limited to, integration of viral particles (e.g., lentivirus, adenovirus, retrovirus-based systems), transposase-mediated transfer (e.g., Sleeping Beauty and Piggybac), the use of non-homologous repair pathways introduced by DNA breaks (e.g., using CRISPR and TALEN) techniques, surrogate DNA molecules, and other techniques that facilitate integration of the target DNA into the cells of interest.

[0182] Expression system To use the above platforms, it may be desirable to express from nucleic acids encoding one or more of the proteins and RNA components. This can be done in a variety of ways. For example, the nucleic acid encoding the RNA scaffold or protein can be cloned into one or more intermediate vectors for introduction into prokaryotic or eukaryotic cells for replication and / or transcription. The intermediate vectors are usually prokaryotic vectors, such as plasmids, or shuttle vectors, or insect vectors, for storage or recombination of the nucleic acid encoding the RNA scaffold or protein for production of the RNA scaffold or protein. The nucleic acid can also be cloned into one or more expression vectors for administration to plant cells, animal cells, preferably mammalian or human cells, fungal cells, bacterial cells, or protozoan cells. Thus, the present invention provides nucleic acids encoding any of the above RNA scaffolds or proteins. Preferably, the nucleic acid is isolated and / or purified.

[0183] The present invention also provides recombinant constructs or vectors that contain sequences encoding one or more of the above RNA scaffolds or proteins. Examples of constructs include vectors (e.g., plasmids or viral vectors) into which the nucleic acid sequences of the present invention are inserted in a forward or reverse orientation. In a preferred embodiment, the construct further comprises a regulatory sequence (including a promoter) operably linked to the sequence. Many suitable vectors and promoters are known to those of skill in the art and are commercially available. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts are also described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press).

[0184] A vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. A vector may replicate autonomously or integrate into a host DNA. Vectors include plasmids, cosmids, or viral vectors. A vector of the invention comprises a nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably, the vector comprises one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed. "Regulatory sequences" include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence as well as inducible regulatory sequences. The design of an expression vector may depend on factors such as the choice of the host cell to be transformed, transfected, or transduced, the level of expression of RNA or protein desired, and the like. Examples of expression vectors include chromosomal, non-chromosomal and synthetic DNA sequences, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, poxvirus and pseudorabies. However, any other vector may be used provided it is replicable and viable in the host. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, a nucleic acid sequence encoding one of the above RNAs or proteins may be inserted into an appropriate restriction endonuclease site by procedures known in the art. Such procedures and associated subcloning procedures are within the skill of the art.

[0185] The vector may contain appropriate sequences for amplifying expression. In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells (e.g., dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline resistance or ampicillin resistance in E. coli). The vector for expressing RNA may be an RNA Pol III promoter, such as the H1, U6 or 7SK promoter, to drive the expression of RNA. These human promoters may express RNA in mammalian cells after plasmid transfection. Alternatively, the T7 promoter may be used, for example, for in vitro transcription, and the RNA may be transcribed in vitro and purified.

[0186] A suitable host may be transformed, transfected, or infected with a vector containing an appropriate nucleic acid sequence as described above and an appropriate promoter or control sequence, such that the host expresses the RNA or protein. Examples of suitable expression hosts include bacterial cells (e.g., E. coli, Streptomyces, Salmonella typhimurium), fungal cells (yeast), insect cells (e.g., Drosophila and Spodoptera frugiperda (Sf9)), animal cells (e.g., CHO, COS, and HEK 293), adenovirus, and plant cells. The selection of a suitable host is within the skill of the art. In one embodiment, the present invention provides a method for producing the RNA or protein by transforming, transfecting, or infecting a host cell with an expression vector having a nucleotide sequence encoding one of the RNA, or polypeptide, or protein. The host cell is then cultured under suitable conditions to allow expression of the RNA or protein. Any procedure known in the art for introducing foreign nucleotide sequences into host cells may be used, including, for example, calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors (both episomal and integrative), and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into host cells.

[0187] Cell culture The method further includes maintaining the cell under suitable conditions such that the guide RNA guides the effector protein to a target site in the target sequence and the effector domain modifies the target sequence. Generally, cells can be maintained under conditions suitable for cell growth and / or maintenance. Suitable cell culture conditions are well known in the art and are described, for example, in Current Protocols in Molecular Biology "Ausubel et al., John Wiley & Sons, New York, 2003 or "Molecular Cloning: A Laboratory Manual" Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001), Santiago et al. (2008) PNAS 105:5809-5814; Moehle et al. (2007) PNAS 104:3055-3060; Urnov et al. (2005) Nature 435:646-651; and Lombardo et al. (2007) Nat. Biotechnology 25:1298-1306. Methods for culturing cells are known in the art and the skilled artisan will appreciate that they can and will vary depending on the cell type. In all cases, routine optimization can be used to determine the best technique for a particular cell type. Cells useful for the methods provided herein can be freshly isolated primary cells or can be obtained from frozen aliquots of primary cell cultures. In some cases, cells are electroporated for incorporation of gRNA and base-editing fusion proteins. As described in the Examples below, electroporation conditions for some assays (e.g., T cells) can be 1600 volts, 10 millisecond pulse width, 3 pulses. After electroporation, electroporated T cells are allowed to recover in cell culture medium and then cultured in T cell growth medium. In some cases, electroporated cells are allowed to recover in cell culture medium for about 5 to about 30 minutes (e.g., about 5, 10, 15, 20, 25, 30 minutes). Preferably, the recovery cell culture medium does not contain antibiotics or other selection agents. In some cases, the T cell growth medium is Immunocult XT T cell growth medium.

[0188] kit Other aspects of the technology described herein relate to kits for use in generating genetically modified cells. Described herein are kit components that may be included in one or more of the kits. In some embodiments, the components described herein can be provided alone or in any combination as a kit, including the components described herein, such as a sequence-targeting protein, an RNA-ligand binding complex (or RNA scaffold), and two or more effector proteins. In certain embodiments, the components of the kit may be provided as mRNA, plasmid, ribonucleoprotein (RNP), or a combination of two or more of these formats. For example, in one embodiment, the sequence targeting component and the effector protein may be provided as separate mRNA molecules, while the partially synthetic sgRNA and associated RNA motif (RNA scaffold) are provided as RNA. Alternatively, in other embodiments, the sequence targeting component and the effector protein may be provided as separate mRNA molecules, while the RNA scaffold is provided as one or more separate components that can hybridize to form a single RNA-ligand binding complex. In other embodiments, the sequence targeting component and the effector protein may be provided as separate RNPs, while the partially synthetic sgRNA and associated RNA motif (RNA scaffold) are provided as RNA. Alternatively, in other embodiments, the sequence targeting component and the effector protein may be provided as separate RNPs, while the RNA scaffold is provided as one or more separate components that can hybridize to form a single RNA-ligand binding complex.

[0189] In certain embodiments, the Cas proteins may be provided using stably expressing cell lines. In some embodiments, the compounds in the kit can be provided in a water-tight or air-tight container, which in some embodiments is substantially free of other components of the kit. In some embodiments, the components can be provided in a single container. It is preferred that the component(s) described herein are substantially pure and / or sterile. The kit is typically provided with its various elements contained within a single package, e.g., a fiber-based, e.g., cardboard, or polymeric, e.g., Styrofoam, box. The enclosure can be configured to maintain a temperature differential between the interior and exterior, e.g., to provide insulating properties to maintain reagents at a preselected temperature for a preselected period of time.

[0190] definition Nucleic acid or polynucleotide refers to a DNA molecule (such as, but not limited to, cDNA or genomic DNA) or an RNA molecule (such as, but not limited to, mRNA), including DNA or RNA analogs. DNA or RNA analogs can be synthesized from nucleotide analogs. DNA or RNA molecules can contain non-naturally occurring moieties, such as modified bases, modified backbones, deoxyribonucleotides in RNA, etc. Nucleic acid molecules can be single-stranded or double-stranded. The term "isolated" when referring to a nucleic acid molecule or polypeptide means that the nucleic acid molecule or polypeptide is substantially free from at least one other component with which it is associated or found in nature.

[0191] The term "guide RNA" as used herein generally refers to an RNA molecule (or collectively a group of RNA molecules) that can bind to a CRISPR protein and target the CRISPR protein to a specific location within a target DNA. A guide RNA may comprise two segments: a DNA-targeting guide segment and a protein-binding segment. The DNA-targeting segment comprises a nucleotide sequence that is complementary to (or can hybridize under at least stringent conditions to) a target sequence. The protein-binding segment interacts with a CRISPR protein, such as Cas9 or a Cas9-related polypeptide, or Cas12 or a Cas12-related polypeptide. These two segments may be located in the same RNA molecule or in two or more separate RNA molecules. The components that comprise the guide RNA are appropriately selected and / or designed based on the sequence targeting the protein for which the guide RNA is intended to be used. For example, when the sequence targeting protein is Cas9 or a Cas9-related polypeptide, the guide RNA comprises a crRNA and a tracrRNA. When the sequence-targeting protein is derived from a type V CRISPR-Cas system, the guide RNA may be a cr-only guide RNA, a crRNA:tracrRNA guide RNA, or a crRNA:scoutRNA guide RNA.

[0192] As used herein, the term "target nucleic acid" or "target" refers to a nucleic acid containing a target nucleic acid sequence. A target nucleic acid can be single-stranded or double-stranded, and is often double-stranded DNA. As used herein, "target nucleic acid sequence," "target sequence," or "target region" refers to a specific sequence or its complement that is desired to be bound or modified using a CRISPR system. The target sequence can be in a nucleic acid in vitro or in vivo, in the genome of a cell, which can be in any form of single-stranded or double-stranded nucleic acid. "Target nucleic acid strand" refers to the strand of the target nucleic acid that is subjected to base pairing with the guide RNA disclosed herein. That is, the strand of the target nucleic acid that hybridizes with the crRNA and the target sequence is referred to as the "target nucleic acid strand". The other strand of the target nucleic acid that is not complementary to the target sequence is referred to as the "non-complementary strand". In the case of a double-stranded target nucleic acid (e.g., DNA), each strand may be a "target nucleic acid strand" for designing the crRNA and guide RNA, and may be used to carry out the method of the present invention as long as a suitable PAM site is present. It should be noted that some V-type Cas proteins can base pair with the target nucleic acid strand without the need for a PAM site. As used herein, the term "RNA scaffold" refers to an RNA-ligand binding complex in which the ligand-binding moiety is an RNA motif (or RNA aptamer).

[0193] As used herein, the term "derived" refers to a process of isolating, deriving, or creating a different second component (e.g., a second molecule that is different from the first component) using a first component (e.g., a first molecule) or information from the first component. For example, a mammalian codon-optimized Cas9 polynucleotide is derived from a wild-type Cas9 protein amino acid sequence. Also, mutant mammalian codon-optimized Cas9 polynucleotides, including Cas9 single mutant nickase (nCas9, e.g., nCas9D10A) and Cas9 double mutant null nuclease (dCas9, e.g., dCas9 D10A H840A), are derived from a polynucleotide encoding a wild-type mammalian codon-optimized Cas9 protein.

[0194] The term "wild-type" as used herein is an art term understood by those of skill in the art and refers to the normal form of a naturally occurring organism, strain, gene or characteristic, as distinguished from mutant or recombinant forms.

[0195] The term "variant" as used herein refers to a first composition (e.g., a first molecule) relative to a second composition (e.g., a second molecule, also referred to as a "parent" molecule). A mutant molecule can be derived from, isolated from, based on, or homologous to a parent molecule. For example, mutants of mammalian codon-optimized Cas9 (hspCas9), including a Cas9 single mutant nickase and a Cas9 double mutant null nuclease, are mutants of mammalian codon-optimized wild-type Cas9 (hspCas9). The term "variant" can be used to describe either a polynucleotide polynucleotide or a polypeptide. As applied to polynucleotides, recombinant molecules can be completely identical to the nucleotide sequence of the original parent molecule, or can be less than 100% identical to the nucleotide sequence of the parent molecule. For example, a variant of a gene nucleotide sequence can be a second nucleotide sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical in nucleotide sequence compared to the original nucleotide sequence. Polynucleotide variants also include polynucleotides that include the entire parent polynucleotide and further include additional fused nucleotide sequences. Polynucleotide recombinants also include polynucleotides that are partial or subsequences of the parent polynucleotides, for example, unique subsequences of the polynucleotides disclosed herein (e.g., as determined by standard sequence comparison and alignment techniques) are also included in the present invention.

[0196] In other aspects, polynucleotide recombinants include nucleotide sequences that contain minor, trivial, or insignificant changes relative to the parent nucleotide sequence. For example, minor, trivial, or insignificant changes include changes to nucleotide sequences that (i) do not change the amino acid sequence of the corresponding polypeptide, (ii) occur outside the protein-coding open reading frame of the polynucleotide, (iii) result in deletions or insertions that may affect the corresponding amino acid sequence but have little or no effect on the biological activity of the polypeptide, and (iv) result in nucleotide changes that result in the replacement of an amino acid with a chemically similar amino acid. If the polynucleotide does not encode a protein (e.g., tRNA or crRNA or tracrRNA), variants of the polynucleotide may contain nucleotide changes that do not result in loss of function of the polynucleotide. In other aspects, conservative recombinants of the disclosed nucleotide sequences that result in functionally identical nucleotide sequences are included in the present invention. One of skill in the art will appreciate that many recombinants of the disclosed nucleotide sequences are included in the present invention.

[0197] When applied to proteins, a recombinant polypeptide can be completely identical to the amino acid sequence of the original parent polypeptide or can be less than 100% identical to the amino acid sequence of the parent protein. For example, an amino acid sequence variant can be a second amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical in amino acid sequence compared to the original amino acid sequence. Polypeptide variants include those that contain the entire parent polypeptide and further contain additional fused amino acid sequences. Polypeptide recombinants also include polypeptides that are portions or subsequences of the parent polypeptide, for example, unique subsequences of the polypeptides disclosed herein (e.g., as determined by standard sequence comparison and alignment techniques) are also included in the present invention.

[0198] In other aspects, polypeptide variants include polypeptides that contain minor, trivial, or inconsequential changes relative to the parent amino acid sequence. For example, minor, trivial, or insignificant changes include amino acid changes (including substitutions, deletions, and insertions) that have little or no effect on the biological activity of the polypeptide and result in a functionally identical polypeptide, including the addition of non-functional peptide sequences. In other aspects, variant polypeptides of the invention are mutant variants of Cas9 polypeptides that have altered biological activity of the parent molecule, e.g., modified or lost nuclease activity. One of skill in the art will appreciate that many recombinant versions of the disclosed polypeptides are encompassed by the invention.

[0199] In certain aspects, recombinant polynucleotides or polypeptides of the invention can include recombinant molecules that have recombinations, additions or deletions at a low percentage of nucleotide or amino acid positions (e.g., typically less than about 10%, less than about 5%, less than 4%, less than 2%, or less than 1%).

[0200] The term "conservative substitution" as used herein in a nucleotide or amino acid sequence refers to a change in a nucleotide sequence that either (i) does not result in any corresponding change in the amino acid sequence due to the redundancy of triplet codon codes, or (ii) results in the replacement of the original parent amino acid with an amino acid that has a chemically similar structure. Conservative substitution tables providing functionally similar amino acids are well known in the art, where one amino acid residue is replaced with another amino acid residue that has similar chemical properties (e.g., aromatic side chain or positively charged side chain), thus not substantially changing the functional properties of the resulting polypeptide molecule.

[0201] Below are groupings of natural amino acids with similar chemical properties, where substitutions within a group are "conservative" amino acid substitutions. The groups shown below are not fixed, as these natural amino acids may be placed into different groups when different functional properties are considered. Amino acids with non-polar and / or aliphatic side chains include glycine, alanine, valine, leucine, isoleucine, and proline. Amino acids with polar, uncharged side chains include serine, threonine, cysteine, methionine, asparagine, and glutamine. Amino acids with aromatic side chains include phenylalanine, tyrosine, and tryptophan. Amino acids with positively charged side chains include lysine, arginine, and histidine. Amino acids with negatively charged side chains include aspartic acid and glutamic acid.

[0202] "Cas9 mutant" or "Cas9 variant" refers to a protein or polypeptide derivative of a wild-type Cas9 protein, such as the S. pyogenes Cas9 protein (SEQ ID NO: 44), e.g., a protein with one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof, that substantially retains the RNA targeting activity of the Cas9 protein. The protein or polypeptide can comprise, consist of, or consist essentially of a fragment of SEQ ID NO: 44. Generally, the mutant / variant is at least 50% (e.g., including any number between 50% and 100%) identical to SEQ ID NO: 44. The mutant / variant can bind to an RNA molecule and target a specific DNA sequence via the RNA molecule, and may further have nuclease activity. Examples of these domains include the RuvC-like motif (aa. 7-22, 759-766, and 982-989) and the HNH motif (aa 837-863). See Gasiunas et al., Proc Natl Acad Sci USA. 2012 September 25;109(39):E2579 E2586 and WO 2013 / 176772. "Cas12 mutant" or "Cas12 variant" refers to a protein or polypeptide derivative of a wild-type Cas protein from a type V CRISPR-Cas system, such as Alicylobacillus acidophilus Cas12b (SEQ ID NO: 49), e.g., a protein with one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof. A "Cas12 mutant" or "Cas12 variant" substantially retains the RNA targeting activity of the Cas12 protein from which it is derived. As non-limiting examples, the protein or polypeptide can comprise, consist of, or consist essentially of a fragment of SEQ ID NO: 48, 49, or 50. The mutant / variant can bind to and target specific DNA sequences via RNA molecules, and may further have nuclease activity.

[0203] "Complementarity" refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence, either by conventional Watson-Crick base pairing or other non-conventional methods. Percentage complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Fully complementary" means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. As used herein, the term "substantially complementary" refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or to two nucleic acids that hybridize under stringent conditions.

[0204] The term "stringent conditions" as used herein for hybridization refers to conditions under which a nucleic acid complementary to a target sequence will predominantly hybridize to the target sequence and will not substantially hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and will vary depending on many factors. In general, the longer the sequence, the higher the temperature at which it will specifically hybridize to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes Part I, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, NY. "Hybridization" or "hybridizing" refers to the process by which fully or partially complementary nucleic acid strands come together under specific hybridization conditions to form a double-stranded structure or region in which the two constituent strands are bound by hydrogen bonds. Hydrogen bonds are usually formed between adenine and thymine or uracil (A and T or U) or cytidine and guanine (C and G), although other base pairs may be formed (e.g., Adams et al., The Biochemistry of the Nucleic Acids, 11th ed., 1992).

[0205] As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as the "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0206] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. The term also encompasses recombinant amino acid polymers; e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or any other manipulation (e.g., conjugation with a labeling component). As used herein, the term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, as well as amino acid analogs and peptidomimetics. The term "fusion polypeptide" or "fusion protein" as used herein refers to a protein made by linking two or more polypeptide sequences together. Fusion polypeptides encompassed by the present invention include the translation product of a chimeric gene construct in which a nucleic acid sequence encoding a first polypeptide (e.g., an RNA binding domain) is linked to a nucleic acid sequence encoding a second polypeptide (e.g., an effector domain) to form a single open reading frame. In other words, a "fusion polypeptide" or "fusion protein" is a recombinant protein of two or more proteins linked by peptide bonds or through several peptides. A fusion protein may also include a peptide linker between the two domains.

[0207] The term "linker" refers to any means, entity or moiety used to link two or more entities. A linker can be a covalent linker or a non-covalent linker. Examples of covalent linkers include a covalent bond or linker moiety covalently attached to one or more of the proteins or domains to be linked. A linker can also be a non-covalent bond (e.g., an organometallic bond through a metal center such as a platinum atom). For covalent attachment, a variety of functional groups can be used (e.g., amide groups including carboxylic acid derivatives, esters including ethers, organic esters and inorganic esters, amino, urethane, urea, etc.). To provide the attachment, the domains can be modified by oxidation, hydroxylation, substitution, reduction, etc. to provide sites for coupling. Methods for attachment are well known to those of skill in the art and are encompassed for use in the present invention. Linker moieties include, but are not limited to, chemical linker moieties, or, for example, peptide linker moieties (linker sequences). It is understood that modifications that do not significantly reduce the function of the RNA binding domain and the effector domain are preferred.

[0208] As used herein, the terms "conjugate" or "conjugation" or "linked" refer to the joining of two or more entities to form one entity. Conjugates include both peptide-small molecule conjugates as well as peptide-protein / peptide conjugates.

[0209] The terms "subject" 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. In some embodiments, the subject may be an invertebrate, such as an insect or nematode, and in other embodiments, the subject may be a plant or a fungus.

[0210] As used herein, the terms "treatment" or "treating" or "alleviating" or "ameliorating" are used interchangeably. These terms refer to an approach to obtain beneficial or desired results, including but not limited to therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms being treated. For prophylactic benefit, the composition may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject who reports one or more of the physiological symptoms of a disease, even if the disease, condition, or symptom has not yet manifested.

[0211] As used herein, the term "contacting" when used with respect to any set of components includes any process in which the components being contacted are mixed into the same mixture (e.g., added to the same compartment or solution) and does not necessarily require actual physical contact between the listed components. The listed components can be contacted in any order or in any combination (or subcombination) and can include situations in which one or some of the listed components are subsequently removed from the mixture, possibly prior to the addition of other listed components. For example, "contacting A with B and C" includes any and all of the following situations: (i) mixing A with C and then adding B to the mixture; (ii) mixing A and B into a mixture; removing B from the mixture and then adding C to the mixture; and (iii) adding A to a mixture of B and C. "Contacting" a target nucleic acid or cell with one or more reaction components, such as a Cas protein or guide RNA, includes any or all of the following situations: (i) contacting the target or cell with a first component of a reaction mixture to create a mixture; then adding other components of the reaction mixture to the mixture in any order or combination; and (ii) forming the reaction mixture completely before mixing with the target or cell.

[0212] The term "mixture" as used herein refers to a combination of elements that are interspersed without any particular order. A mixture is heterogeneous and its different components cannot be separated spatially. Examples of mixtures of elements include many different elements dissolved in the same aqueous solution or many different elements bound to a solid support randomly or in no particular order where the different elements are not spatially distinct. In other words, a mixture is not addressable.

[0213] As disclosed herein, several ranges of values ​​are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of the range is also specifically disclosed, unless the context clearly dictates otherwise. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the range, and each range in which either limit is included in the smaller range, neither is included, or both limits are included in the smaller range is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. The term "about" generally refers to plus or minus 10% of the indicated number. For example, "about 10%" may indicate a range of 9% to 11%, and "about 20" may mean 18 to 22. Other meanings of "about" may be apparent from the context, such as rounding, so for example, "about 1" may also mean from 0.5 to 1.4. Various illustrative embodiments of the compositions and methods according to the present invention will now be illustrated by the following non-limiting examples. [Example] In the following examples, we demonstrate that eukaryotic cells can be used to actively recruit multiple effectors to the same genomic locus using multiple strategies to obtain novel functionalities with surprising efficiency and outcomes. A surprising observation is that a single aptamer can transiently recruit effectors when presented with a pool of effectors that provide multifunctional activity at a single locus by a combined CRISPR-Cas system. Examples 1 and 8 are non-limiting examples. Examples 2-7 are prophetic examples. EXAMPLES

[0214] Locus-directed multi-effectors via a single aptamer recruitment system for novel base editing applications in human cells In this example, we demonstrated that multiple enzymes can be actively recruited to the same locus using a single aptamer recruitment system in primary human lymphocytes. Multiple deaminases (Apobec1 and AID) are recruited to the site via a single aptamer (RNA motif) on the sgRNA. Pan T cells were activated with anti-CD3 and anti-CD28, then cells were electroporated with mRNA components for either Apobec1-MCP, AID-MCP or both deaminases, nCas9-UGI-UGI components, tracrRNA-MS2 aptamer and crRNA. Cells were then incubated for an additional 24 hours and then stimulated with anti-CD3 and anti-CD28 for 48 hours, after which base editing was confirmed by targeted PCR amplification and Sanger sequencing.

[0215] Materials and Methods Guides were designed for specific base editing windows calculated at set distances from the PAM motif (NGG). crRNA and tracrRNA were synthesized by Horizon Discovery (formerly Dharmacon).

[0216] Synthetic crRNA sequence (SEQ ID NO:32):

[0217] [ka] 2'OMe(m) and phosphorothioate(*) modified residues Synthetic tracrRNA-MS2 aptamer sequence (SEQ ID NO:33):

[0218] [ka] 2'OMe(m) and phosphorothioate(*) modified residues Generation of mRNA components: Messenger RNA molecules were custom generated by Trilink utilizing modified nucleotides: pseudouridine and 5-methyl-cytosine. The mRNA components were translated into the following proteins: Deaminase AID-MCP = NLS-hAID-linker-MCP, Deaminase Apobec1-MCP = NLS-rApobec 1-linker-MCP, and Cas9-UGI-UGI = NLS-nCas9-UGI-UGI-NLS. T cell culture: PBMCs were isolated from a fresh blood source using Lymphoprep (STEMCELL Technologies) gradient centrifugation followed by total T cell isolation via negative selection (STEMCELL Technologies). T cells were cultured in Immunocult XT medium (STEMCELL Technologies) containing 1x penicillin / streptomycin (Thermofisher). T cell electroporation: After 48-72 activations, T cells were electroporated using a Neon Electroporator (Thermofisher) or 4D Nucleofector (Lonza). Neon electroporation conditions were 1600v / 10ms / 3 pulses in a 10μl tip with 250k cells, 1-5μg total mRNA amount for both deaminase-MCP and nCas9-UGI-UGI, and 0.2-1.8μmol complexed crRNA:tracrRNA or sgRNA, where applicable. 4D Nucleofector conditions were EO-115 with 20ul cuvettes with a combined total of 500k with 1-5ug mRNA amount for both single or dual deaminase-MCP and nCas9-UGI-UGI (synthesized by Trilink), and 0.2-1.8umol complexed crRNA:tracrRNA or sgRNA (Horizon Discovery). After electroporation, the cells were transferred to Immunocult XT medium containing 100 U IL-2, 100 U IL-7, and 100 U IL-15 (STEMCELL Technologies) and cultured at 37°C and 5% CO2 for 24 to 72 hours. CD3+ T cell activation: T cells were activated by using Dynabeads Human T Activator CD3 / CD28 beads (Thermofisher) at a 1:1 bead:cell ratio cultured in Immunocult XT medium (STEMCELL Technologies) in the presence of 100U / ml IL-2 (STEMCELL Technologies) and 1× penicillin / streptomycin (Thermofisher) for 48 hours at 37° C. and 5% CO2. After activation, the beads were removed by placing on a magnet and the cells were returned to culture. Genomic DNA analysis: Genomic DNA was released from lysed cells 48-72 h after electroporation. Loci of interest were amplified by PCR and the products were then sent for Sanger sequencing (Genewiz). Data were analyzed by proprietary in-house software.

[0219] The data demonstrate that multiple enzymes can be recruited via the same aptamer (RNA motif) on the same guide in addition to the target enzymatic activity of the guidance protein (nickase). Figure 7 demonstrates that by including both enzymes, there is a complementary synergy with the two enzymes, resulting in the best of both enzymes at the target locus. This surprising result indicates that the recruitment strategy is a dynamic process that allows on-site recruitment of multiple effectors to the same locus via the same aptamer (RNA motif), which offers many practical therapeutic advantages and opens opportunities for new technologies being developed in the field of genome editing. EXAMPLES

[0220] A locus-directed multiprotein multiaptamer recruitment system for novel base editing applications in human cells In this example, using primary human lymphocytes, we show that multiple effector proteins can be actively recruited to the same locus using multiple aptamers (RNA motifs) on the same single sgRNA that deliver proteins of interest to a guidance system (e.g., nCas9-UGI-UGI, which cleaves DNA open) and multiple deaminases (rat Apobec1 and human AID) that are specifically recruited to the site via different aptamers on the same guide. Pan T cells are activated using anti-CD3 and anti-CD28, then the cells are electroporated with either Apobec1-MCP, AID-PCP or both deaminases mRNA components, nCas9-UGI-UGI components, sgRNA-MS2-P7 aptamer, then the cells are incubated for an additional 24 hours and then stimulated with anti-CD3 and anti-CD28 for 48 hours, after which surface KO is confirmed by flow cytometry and base editing is confirmed by targeted PCR amplification and Sanger sequencing. The data show that in addition to the targeted enzymatic activity of the guidance protein (nickase), multiple different enzymes can be recruited to the same locus using multiple different aptamers on the same sgRNA. EXAMPLES

[0221] A locus-directed system for novel base editing applications in human cells via single-aptamer recruitment of multiple effectors to nickase In this example, using primary human lymphocytes, we show that multiple enzymes can be actively recruited to the same locus using aptamer-based recruitment (e.g., via AID-MCP and Apobec1-MCP), and that the aptamer is attached to the C-terminus of a nickase induction system (e.g., MS2-nCas9-UGI-UGI), with the benefit of the induction system (nickase activity) and multiple effectors recruited by the same aptamer at the same locus, which has the advantage of single-site-specific multigene effector action. Pan T cells are activated using anti-CD3 and anti-CD28, then the cells are electroporated with the mRNA component for AID-MCP, MS2-nCas9-UGI-UGI component, sgRNA, then the cells are incubated for an additional 24 hours, then stimulated with anti-CD3 and anti-CD28 for 48 hours, then confirmed for surface KO by flow cytometry and base editing by targeted PCR amplification and Sanger sequencing. The data show that multiple base editing techniques can be combined for improved functionality, and that both fusion and aptamer recruitment strategies are complementary for improved functionality. Editing efficiency was improved without the need to optimize two or more systems that deliver effectors to a single locus. EXAMPLES

[0222] A locus-directed system for novel base editing applications in human cells via multiaptamer recruitment of multiple effectors to nickases In this example, using primary human lymphocytes, we show that aptamer-based recruitment, in which multiple aptamers are coupled to a nickase induction system (e.g., MS2-MS2-nCas9-UGI-UGI), can be used to actively recruit multiple enzymes to the same locus (e.g., via AID-MCP and Apobec1-MCP), taking advantage of the induction system (nickase activity) and having multiple effectors recruited by the same aptamer in various orientations at the same locus. This has the advantage of single-site-specific multigene effector action. Pan T cells are activated using anti-CD3 and anti-CD28, then the cells are electroporated with the mRNA component for AID-MCP, MS2-MS2-nCas9-UGI-UGI component, sgRNA, then the cells are incubated for an additional 24 hours and then stimulated with anti-CD3 and anti-CD28 for 48 hours, then surface KO is confirmed by flow cytometry and base editing is confirmed by targeted PCR amplification and Sanger sequencing. The data illustrate that multiple base editing techniques can be combined for enhanced functionality and that both fusion and aptamer recruitment strategies are complementary for improved functionality. EXAMPLES

[0223] Locus-directed protein aptamer and direct fusion recruitment systems for novel base editing applications in human cells In this example, using primary human lymphocytes, we show that multiple enzymes can be actively recruited to the same locus using aptamer-based recruitment (e.g., via AID-MCP and sgRNA-MS2 aptamers) and via direct enzyme fusion to a guidance system (e.g., Apobec1-nCas9-UGI-UGI), allowing one to benefit from the guidance system (e.g., guidance and nickase activity), the direct fusion partner (e.g., Apobec1), and benefit from multiple base editing techniques and improved editing efficiency via protein molecules recruited by aptamers (e.g., AID-MCP). Pan T cells are activated using anti-CD3 and anti-CD28, then cells are electroporated with the mRNA component for AID-MCP, Apobec1-nCas9-UGI-UGI component, sgRNA, then cells are incubated for an additional 24 hours and then stimulated with anti-CD3 and anti-CD28 for 48 hours, then surface KO is confirmed by flow cytometry and base editing is confirmed by targeted PCR amplification and Sanger sequencing. The data illustrate that multiple base editing techniques can be combined for enhanced functionality and that both fusion and aptamer recruitment strategies are complementary for improved functionality. EXAMPLES

[0224] Locus-directed multiprotein via single aptamer and direct fusion recruitment systems for novel base editing applications in human cells In this example, we use primary human lymphocytes to demonstrate that multiple enzymes can be actively recruited to the same locus using aptamer-based recruitment of multiple proteins with the same aptamer (e.g., Apobec-MCP, AID-MCP, and sgRNA-MS2 aptamer), and simultaneously via direct enzyme fusion to a guidance system (e.g., TadA-TadA*-nCas9). To gain the benefits of the guidance system (e.g., guidance and nickase activity), we combine for an enhancer via multiple protein molecules recruited by a direct fusion partner (e.g., Apobec1), as well as an aptamer (e.g., AID-MCP and Apobec1-MCP) that simultaneously benefits from multiple base editing technologies. Pan T cells are activated with anti-CD3 and anti-CD28, then the cells are electroporated with Apobec1-MCP, AID-MCP, Apobec1-MCP, TadA-TadA*-nCas9-UGI-UGI components, and mRNA components for sgRNA. The cells are then incubated for an additional 24 hours, then stimulated with anti-CD3 and anti-CD28 for 48 hours, then surface KO is confirmed by flow cytometry, and base editing is confirmed by targeted PCR amplification and Sanger sequencing. The data illustrate that multiple base editing techniques can be combined for enhanced functionality and that both fusion and aptamer recruitment strategies are complementary for improved functionality. EXAMPLES

[0225] Locus-directed multiprotein via multiple aptamer and direct fusion recruitment systems for novel base editing applications in human cells In this example, using primary human lymphocytes, we demonstrate that aptamer-based recruitment of multiple proteins with multiple different aptamers (e.g., Apobec-MCP, AID-PCP, and sgRNA-MS2-P7 aptamer) can be used to actively recruit multiple enzymes to the same locus via direct enzyme fusion to a guidance system (e.g., TadA-TadA*-nCas9) to obtain the benefits of the guidance system (e.g., guidance and nickase activity), a direct fusion partner (e.g., TadA-TadA*), and that multiple base editing technologies can be benefited from and combined into a reinforcer via multiple protein molecules recruited by aptamers (e.g., AID-MCP and Apobec1-PCP). Pan T cells are activated using anti-CD3 and anti-CD28, then cells are electroporated with Apobec1-MCP, AID-MCP, Apobec1-MCP, TadA-TadA*-nCas9-UGI-UGI components, and mRNA components for sgRNA. Cells are then incubated for an additional 24 hours and then stimulated with anti-CD3 and anti-CD28 for 48 hours, after which surface KO is confirmed by flow cytometry and base editing is confirmed by targeted PCR amplification and Sanger sequencing. The data illustrate that multiple base editing techniques, in combination, improve functionality and that fusion and aptamer recruitment strategies are complementary as they both contribute to improved functionality. EXAMPLES

[0226] Locus-directed multiprotein via a single aptamer recruitment system for novel multiplexed base editing applications in human cells In this example, primary human lymphocytes were used to demonstrate that multiple different effector proteins can be actively recruited to the same locus using a single aptamer recruitment system (RNA-ligand binding complex). In this example, each of the three effector proteins contained an effector domain with deaminase activity and a ligand, MCP. By sharing a common ligand-binding moiety, the three effector proteins could be recruited by the same RNA-ligand binding complex containing a guide RNA and an MS2 RNA motif. Thereby, they are recruited to the target site and deliver the benefits of both the sequence targeting component on the guide system (e.g., nCas9-UGI-UGI, which opens and cleaves DNA) and multiple effector proteins (ligands fused or bound to different effector proteins, e.g., Lizard Apobec1 (Anolis), Rat Apobec1 (Rattus), and / or human AID (Homo)) via a single ligand-binding moiety on the sgRNA (RNA-ligand binding complex). Furthermore, it was further demonstrated that by providing multiple RNA-ligand binding complexes, each containing a different guide RNA but the same ligand binding moiety (MS2 RNA motif), the system described in the present invention can be applied to target multiple loci in a genome, allowing for simultaneous multiplexed base editing. Pan T cells were activated using anti-CD3 and anti-CD28, then cells were electroporated with mRNA components for either Anolis Apobec1-MCP, Rattus Apobec1-MCP, Homo AID-MCP or both deaminases, nCas9-UGI-UGI components, and sgRNA. Cells were then incubated for an additional 96-120 hours before generating stimulated T cell populations for flow cytometry and unstimulated populations for gDNA collection, which were then incubated for an additional 24-48 hours. Base editing was confirmed by targeted PCR amplification and Sanger sequencing (Figure 8), and surface KO was confirmed by flow cytometry with a multiplex staining panel including (B2M, CD52, TRAC, and PD-1) which was used to confirm multiplex KO in the population (Figure 9). The data demonstrate that not only one but multiple effector proteins can be recruited through the same RNA-ligand binding complex and can be utilized simultaneously across many loci in the genome. The data also show that different combinations of deaminases can lead to different functional outcomes, with certain mixtures leading to preferential editing profiles and functional outcomes compared to using a single deaminase. Figure 9 shows a surprising improvement in functional tetraplex editing compared to a single deaminase in a therapeutically relevant immune cell system. This supports other data in that the modularity of aptamer-mobilized base editing can facilitate novel and practical therapeutic as well as non-therapeutic applications (e.g., screening), opening up the possibility of novel methodologies in the field of genome editing.

[0227] Materials and Methods Guides were designed for specific base editing windows calculated at set distances from the PAM motif (NGG). sgRNAs were synthesized by Agilent Technologies. Synthetic sgRNA sequence: B2M sgRNA (SEQ ID NO:34)

[0228] [ka] CD52 sgRNA (SEQ ID NO:35):

[0229] [ka] TRAC sgRNA (SEQ ID NO:36):

[0230] [ka] PDCD-1 sgRNA (SEQ ID NO:37):

[0231] [ka] Scrambled sgRNA (SEQ ID NO:38):

[0232] [ka] 2'OMe(m) and phosphorothioate(*) modified residues Generation of mRNA components: Most of the messenger RNA molecules were custom generated by Trilink utilizing modified nucleotides: pseudouridine and 5-methyl-cytosine. The mRNA components were translated into the following proteins: Deaminase AID-MCP = NLS-homo-AID-linker-MCP (SEQ ID NO: 41), Deaminase Apobec1-MCP = NLS-Rattus-Apobec 1-linker-MCP (SEQ ID NO: 40), and Cas9-UGI-UGI = NLS-nCas9-UGI-UGI-NLS (SEQ ID NO: 42). One mRNA component was synthesized in-house using unmodified ribonucleotides, which was translated into the following protein: NLS-Anolis(P16A-E17A)-Apobec 1-linker-MCP (SEQ ID NO: 39). NLS-Anolis(P16A-E17A)-Apobec 1-Linker-MCP (SEQ ID NO: 39):

[0233] [ka] NLS-Rattus-Apobec 1-Linker-MCP (SEQ ID NO: 40):

[0234] [ka] NLS-Homo-AID-L 25-MCP (SEQ ID NO: 41):

[0235] [ka] NLS-nCas9(D10A)-UGI-UGI-NLS (SEQ ID NO: 42):

[0236] [ka] T cell culture: PBMCs were isolated from a fresh blood source using Lymphoprep (STEMCELL Technologies) gradient centrifugation followed by total T cell isolation via negative selection (STEMCELL Technologies). T cells were cultured in Immunocult XT medium (STEMCELL Technologies) containing 1x penicillin / streptomycin (Thermofisher). CD3+ T cell activation: T cells were activated by using Dynabeads Human T Activator CD3 / CD28 beads (Thermofisher) at a 1:1 bead:cell ratio cultured in Immunocult XT medium (STEMCELL Technologies) in the presence of 100U / ml IL-2 (STEMCELL Technologies) and 1× penicillin / streptomycin (Thermofisher) for 48 hours at 37° C. and 5% CO2. After activation, the beads were removed by placing on a magnet and the cells were returned to culture. T cell electroporation: After 48–72 activations, T cells were electroporated using a Neon Electroporator (Thermofisher) or 4D Nucleofector (Lonza). Neon electroporation conditions were 1600v / 10ms / 3 pulses using a 10μl tip containing 250k cells, 1–2μg total mRNA amount for both deaminase-MCP and nCas9-UGI-UGI, and 0.2–1.8μmol of each sgRNA. Cells after electroporation were transferred to Immunocult XT medium containing 100U IL-2, 100U IL-7, and 100U IL-15 (STEMCELL Technologies) and cultured at 37°C and 5% CO2 for 96–120 hours. The cell culture wells were then split in two, one half was unstimulated and the other was stimulated with 50 ng / ml phorbol myristate acetate and 250 ng / ml ionomycin, and incubated for a further 24-48 hours. Genomic DNA analysis: Genomic DNA was released from lysed cells 48-72 h after electroporation. Loci of interest were amplified by PCR and the products were then sent for Sanger sequencing (Genewiz). Data were analyzed by proprietary in-house software. array Humanized nucleotide sequence WT Streptococcus pyogenes Cas9 (SEQ ID NO: 43)

[0237] [ka]

[0238] [ka] Humanized amino acid sequence WT Streptococcus pyogenes Cas9 (SEQ ID NO: 44)

[0239] [ka] Humanized nucleotide sequence of Streptococcus pyogenes nCas9 (bold and underlined A29C mutation) (SEQ ID NO: 45)

[0240] [ka]

[0241] [ka]

[0242] [ka] Humanized amino acid sequence of Streptococcus pyogenes nCas9 (D10A residue change in bold and underlined) (SEQ ID NO: 46)

[0243] [ka] Humanized nucleotide sequence of Streptococcus pyogenes dCas9 (A29C, C2518G, and A2519C base changes are shown in bold and underlined) (SEQ ID NO:47)

[0244] [ka]

[0245] [ka]

[0246] [ka] Amino acid sequence WT AsCas12a (SEQ ID NO: 48)

[0247] [ka] Amino acid WT AaCas12b (SEQ ID NO: 49)

[0248] [ka] Amino acid sequence WT Cas12d15 (SEQ ID NO:50)

[0249] [ka] Humanized nucleotide sequence WT AsCas12a (SEQ ID NO:51)

[0250] [ka]

[0251] [ka] Humanized nucleotide sequence WT AaCas12b (SEQ ID NO:52)

[0252] [ka]

[0253] [ka] Humanized nucleotide sequence WT Cas12d15 (SEQ ID NO:53)

[0254] [ka]

[0255] [ka] The substituted amino acids are shown in bold and underlined.

[0256] Humanized nucleotide sequence dCas12a (bold and underlined A2723C / T2724C mutations) (SEQ ID NO:54)

[0257] [ka]

[0258] [ka] Humanized nucleotide sequence dCas12b (bold underlined A1709C mutation) (SEQ ID NO:55)

[0259] [ka]

[0260] [ka] Humanized nucleotide sequence dCas12d (bold and underlined A2624C / A2912C / G2913C / A3593C mutations) (SEQ ID NO:56)

[0261] [ka]

[0262] [ka] Amino acid dAsCas12a sequence (bold underlined D908A mutation) (SEQ ID NO:57)

[0263] [ka] Catalytically inactive AsCas12a is described in Chem. Commun., 2020, 56, 2123-2126.

[0264] Amino acid dAaCas12b (bold and underlined D570A mutation) (SEQ ID NO: 58)

[0265] [ka] Catalytic death AaCas12b described in CRISPR Cas 12 b enables efficient plant genome engineering. See Ming M, Ren Q, Pan C, He Y, Zhang Y, Liu S, Zhong Z, Wang J, Maltsan AA, Wu J, Zheng X, Zhang Y, and Qi Y. Nature Plants (2020) 10.1038 / s41477-020-0614-6.

[0266] Amino acid sequence dCas12d15 (bold and underlined D875A / E971A / D1198A mutations) (SEQ ID NO:59)

[0267] [ka] NOTE: This is a putative catalytically dead Cas12d that has not yet been experimentally validated.

[0268] Bacillus phage PBS2 uracil-DNA glycosylase inhibitor (SEQ ID NO:60)

[0269] [ka] Hybrid type II crRNA:tracrRNA guide RNA sequence: 5'-(20 nt guide) (SEQ ID NO:61)

[0270] [ka] RNA scaffold expression cassette (S. pyogenes) (SEQ ID NO:62) containing a 20 nucleotide programmable sequence, a CRISPR RNA motif, and an MS2 operator motif:

[0271] [ka] (N 20: Programmable sequences. Underlined: CRISPR RNA motif; Bold: MS2 motif; Italics: terminators).

[0272] The above RNA scaffold contains one MS2 loop (1xMS2). Two MS2 loopsAn RNA scaffold containing two MS2 loops (2xMS2), the MS2 scaffold is underlined (SEQ ID NO: 63).

[0273] [ka] NLS-Anolis(P16A-E17A)-Apobec 1-Linker-MCP (SEQ ID NO:64)

[0274] [ka] NLS-Rattus-Apobec 1-Linker-MCP (SEQ ID NO: 65)

[0275] [ka] NLS-Homo-AID-L 25-MCP (SEQ ID NO: 66)

[0276] [ka]

[0277] [ka] NLS-nCas9(D10A)-UGI-UGI-NLS (SEQ ID NO: 67)

[0278] [ka]

[0279] [ka] Q beta phage operator stem loop (SEQ ID NO:68)

[0280] [ka] Q beta coat protein [Q65H] (SEQ ID NO: 69)

[0281] [ka] BoxB (SEQ ID NO: 70)

[0282] [ka] Lambda bacteriophage protein N (LambdaN-(1-22) (SEQ ID NO: 71)

[0283] [ka] Csy4 binding motif (SEQ ID NO: 72)

[0284] [ka] Csy4[H29A] (SEQ ID NO: 73)

[0285] [ka]

Claims

1. 1. A method for genetic modification of a cell by base editing, comprising: i) a sequence-targeting protein; ii) a) a guide RNA, and b) a ligand-binding moiety; and iii) c) a ligand capable of reversibly binding to said ligand binding moiety; and d) two or more effector proteins comprising an effector domain; A method comprising introducing into a cell and / or expressing in a cell.

2. The method of claim 1 , wherein the sequence-targeting protein is a Cas protein.

3. The method of claim 1, wherein the ligand binding moiety is an RNA motif and the ligand is an RNA binding domain that recognizes the RNA motif.

4. The method of any one of claims 1 to 3, wherein the cell is subjected to base editing at a single or multiple genomic loci.

5. The method of any one of claims 1 to 3, wherein the sequence-targeting protein is fused to one or more uracil DNA glycosylase (UNG) inhibitor peptides (UGI).

6. The method of any one of claims 1 to 3, wherein the sequence-targeting protein is a type II Cas protein or a type V Cas protein.

7. 4. The method of any one of claims 1 to 3, wherein the sequence-targeting protein is a type II Cas protein that is nuclease-deficient or has nickase activity, or a type V Cas protein that is nuclease-deficient or has nickase activity.

8. The RNA motif and RNA binding domain are as follows: telomerase Ku binding motif and Ku protein or an RNA binding section thereof; telomerase Sm7 binding motif and Sm7 protein or its RNA binding section; MS2 phage operator stem loop and MS2 coat protein (MCP) or an RNA-binding section thereof; PP7 phage operator stem loop and PP7 coat protein (PCP) or an RNA-binding section thereof; BoxB binding motif and lambda bacteriophage protein N (LambdaN-(1-22)) or an RNA-binding section thereof; A Csy4 binding motif and Csy4[H29A] or an RNA-binding section thereof; or Qbeta phage operator stem loop and Qbeta coat protein [Q65H] or an RNA-binding portion thereof; The method of claim 3, wherein the pair is selected from the group consisting of:

9. The method according to any one of claims 1 to 3, wherein the functions of the effector domains of two or more effector proteins are the same or different.

10. The method of claim 1, wherein the two or more effector proteins each comprise a different effector domain.

11. The method according to any one of claims 1 to 3, wherein the effector domain is an enzyme or a protein fragment.

12. The method of claim 11 , wherein the effector domain has cytidine or adenine deamination activity.

13. The method of any one of claims 1 to 3, wherein the genetic modification comprises at least two genetic mutations in the same locus.

14. The method according to any one of claims 1 to 3, wherein the RNA-ligand binding complex is synthesised as a single component or as two separate components.

15. The method of any one of claims 1 to 3, wherein the ligand-binding moiety is located at the 3' end of the guide RNA, at the 5' end of the guide RNA or within the guide RNA.

16. The method of any one of claims 1 to 3, wherein the RNA-ligand binding complex comprises one or more ligand binding moieties, and wherein said one or more ligand binding moieties are RNA motifs.

17. 4. The method of claim 3, wherein at least one RNA motif is an MS2 aptamer or a PP7 aptamer, and optionally, the MS2 aptamer has an extended stem.

18. 4. The method of any one of claims 1 to 3, wherein the sequence-targeting protein comprises nCas9 with one or two UGIs, the ligand binding moiety is a single MS2 RNA motif or a PP7 RNA motif, and the ligand binding moiety is located at the 3' end of the RNA-ligand binding complex.

19. A population of genetically modified cells, obtainable by the method of any one of claims 1 to 3, in which at least 10% of the cells contain one or more genetic modifications.

20. A kit for genetic modification of a cell by base editing, comprising: i) a sequence-targeting protein; ii) a) a guide RNA, and b) a ligand-binding moiety; and iii) c) a ligand capable of reversibly binding to said ligand binding moiety; and d) two or more effector proteins comprising an effector domain; A, including the kit.

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