Nuclease-independent targeted gene editing platform and uses thereof

Abstract

The present invention discloses a system for targeted gene editing and related uses.

Description

Noe.lease-Independent Targeted Gene Editing Platform and Uses Thereof CROSS REFERENCE TO RELATED APPLICATION This application claims priority to U.S. Provisional Application No, 62 / 192,876 5 filed on July 15, 2015. FIELD OF THE INVENTION This invention relates to a system for targeted gene editing and related uses. BACKGROUND OF THE INVENTION 15 Targeted gene editing is a powerful tool for genetic manipulation of eukaryotic cells, embryos, and animals. With that targeted genomic locations and / or specific chromosomal. sequences can be deleted, inactivated, or modified. Several. current methods rely on the use of engineered nuclease enzymes, such as zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases {TALENs). These chimeric nucleases 20 contain programmable, sequence-specific DNA-binding modules linked to a nonspecific DNA cleavage domain. Since each new genomic target requires the design of a new ZFN or T ALEN comprising a novel sequence-specific DNA-binding module, these custom designed nucleases tend to be costly and time-consuming to prepare. Moreover, the specificities of ZFNs and TALENS are such that they can mediate off-target cleavages. A 25 recently developed genome modification technology utilizes the bacterial clusters of regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9), an RNA-guided DNA endonuclease, to induce a specific double-stranded break (DSB) at DNA target sites. The RNA-Cas9 complex identifies and base pairs with. its cognate DNA target sequence, resulting in target cleavage to form a DSB. Date Re9ue / Date Received 2023-10-20 However, one major problem unsolved is how to correct genetic mutations in somatic ceUs:. Currently the common effectors for the existing technologies are nucleas:es,. which lead to DNA DSB, which in turn triggers activation of ceUular pathways such as homologous recombination and nonMhomologous end joining. The process bas a number 5, of major disadvan.tages. First, due to the unpredictable nature of the end-products by end joining, DSB leads to both in-frame and frame-shift mutations in a stochastic and unpredictabl,e mann,er, which limits its use for direct clinical application. Second. DSBs have the potential. of causing non~local mutagenic events, such as chromosome translocation, which is an undesirable outcome of the procedure. In vivo, these changes 10 could be potentlaUy del,et,erious. Third, the repair or correction usually requires DSBmediaited homologous recombination, the activity of which is low or even absent in most somatic tissueslcelLs, where therapeutics matter the most. Thus, the current nuclease-based technologies have limited applicability for g:ene editing and tlhere is a need for a targeted gene modification technology that does not rely 1:5 on nuclease ,activit:y tlhat causes double- strand break. SUMMARY OJ' INVENTION This invention addresses the above-mentioned need by providing a targeted gene editing system and r,dated uses. Accordingly, one aspect of the invention provides a system comprising: 0) a 20 sequence-targeting protein, or a polynucleotide encoding the same, (ii) an RNA scaffold, or a DNA polynucieotide encoding the same, and (iii) a non-nuclease effec.tor fusi.on protein, or a polynucleotide encoding the same. The RNA scaffold comprises. (a) a nudeic acid-targeting 1n.otif comprising a guide RNA sequence that is complementary 110 a target nucleic acid sequence, (b) a CRISPR motif capable of binding to the sequence-targeting 25 protein, and (c) a recmiting RNA motif. The non-nuclease effector fusion protein compdses (a), an RNA binding domain capable of binding to the recruiting RNA motif, (b) a linker sequence, and (c) an effector domain. The non-nucl.ease effector fusion protein has an enzymatic activity. For the above system, the sequence-targeting protein ,can be a CRISPR protei.n. 30 Preferaibly, the sequence-targeting protein does not have a nuclease activity. Examples of the sequence-'targ:eting pr,otein includes dCas9 of a species selected from the group consisting of Streptococcus pyogemu;, Streptococcus agalactiae, Staphylococ",:,s OJ1.reits, 2 Date Re9ue / Date Received 2022-07-19 Streptococcus thennophilus, Streptococcus thermophilus, Neisseria meningitid#,, and Trepont?ma denticola. [n th,e above mentioned RNA scaffold, the recruiting RNA motif and the RNA binding domain can be a pair sele(..1ed from the group consisting of (1) a telomerase Ku 5, bindin.g motif and Ku protein or a RNA-binding section thereof, (2) a tefomerase Sm7 binding motif and Sm7 protein or a RNA-binding section thereof, (3) a MS2 phage op,erato,r stem-loop and MS2 coat protein (MCP) or a RNA-binding section thereof'; (4) a PP7 phage operatm: stem-loop and .PP7 coat protein (PCP) or a RNA-binding section thereof., (5) a Sfl\llu phage Com stem-loop and Com RNA binding protein or a RNAl 0 binding section th,ereo(, and (6) a non-natural RNA aptamer and corresponding apta:mer l.igand or a RNA-binding section thereof. 1n the above mentioned non-nuclease effector fusion protein, the linker sequence can be O 10 100 (e .. g., 1-100, 5-80, 10-50, and 20-30) amino acid residues in length. The enzymatic activity ,can be dearnination activity, methyltransferase activity, dernethylase 1:5 activity, DNA repair activity, DNA damage activity,. dismutase activity, aJ.kylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, heli.case activity, photolyase activity or gl.ycosylase activity. fo some embodiments,. the enzymatic activity is dearninati.on activity (e .. g., a cytosine deamination 20 activity or adenosine d,eamin.ation activity), methyltransferase activity, or demethylase activity, The RNA !binding domain is not Cas9 nor its functional equivalent nor its RNAbinding domain. Also provided are an isolated nucleic acid encoding one or more of components (i)-(m) of the system des,cribed above, an expression vector comprisfog the nucleic acid, or 25 a host cell comprising the nucleic acid. In a se,cond aspect, the invention provides a method of site-specific modification of a target DNA The method Includes contacti.ng the target nucleic acid with co.mponen:ts (i)-(iii) of1he system described above. The target nucleic acid can be in a ,cell The target nucleic acid can be RNA, an. extrachromosomal DNA, or a genomic DNA on a 30 chromosome., Tbe ,cell can be selected from the group consisting of: an archaeal cell, a bacteria] cell, a euka.ryotic ceU, a eukaryotic single-cell organism, a somatic ceU, a genn cell, a stem ,ceU, a :p:lant c,ell, an algal cell, an. animal cell,, in invertebrate ceU,. a v,ertebrate 3 Date Re9ue / Date Received 2022-07-19 cell, a fish ceJJ, a frog ceU, a bird cell, a mammalian cell, a pig cell, a cow ceJJ, a goat cell, a sheep ceU, a. rodent cell,. a rat cell, a mouse cell, a non-human primate cell, and a human cell. The cell can be in or derived from a human or non-human s:ubje<,1. The .human or 5, 11.on-human :subject has a genetic mutation of a gene. In some embodiments:, the subject has a disorder caused by the genetic mutation or is at risk of having the disorder. In that case, the sit,e-specific modification corrects the genetic mutation or inacdvates, the expression of the gene. 1n other embodiments, the subje,ct has a pathogen or is at risk of exposing to the pathogen, and the site-specific modification inactivates a gene of the 10 pathogen. The i:nvendon further provides a kit containing the system described above or one or more components thereof. The system can further contain one or more components selected from th.e group consisting of a reagent for reconstitution and / or dilution and a reagent for introducing nucleic acid or polypeptide into a host cell. 1 :5 The details of one or more embodiments of the invention are set forth in the description below. Other features, objectives, and advantages of the inve11tiou wiU be apparent from the description and from the ,claims. BRIEF DESCRIPTION OF THE ORA WINGS FIGs ] A, lB, 1 C, ID, and IE are a set of schematic ntustrations of M exemplary 20 nuclease-independ,en:t CasRcure or CRC platform for in vivo targeted genetic editing. FIG. IA. Componenl:s ,of the pl.atfonn., from left to right: (l) a sequence targeting component dCas9, (2) a RNA scaffold containing a guide RNA motif (for sequence targeting}, a CRISPR motif (for dCas9 binding), and a recruiting RNA motif (for recruiting ,effectorRNA binding pmtein fusfon), and (3) an effector~RNA binding domain fusion protei.n. 25 The system can be programmed to target spedfic nucleotides on DNA or RNA molecules (right),. FIG. 1. B. If the effector protein functions as a monomer, the ;system can be targeted to a single site, up,:stream (Ieft) or downstream {right) of the target site. FIG. ]C. ff the effector protein fequiires dimerizati.on for proper catalytic function,. the system can be multip,!e&ed to ta:rget sequences upstr,eam a:od downstream of the target site 30 simultaneously, therefore allowing the effector proteins to dimerize (right), Alternatively, recruitment ,of effe,ctor protein to a single site may be sufficient to increase its affinity for neighboring effector protei.ns, promoting dimeriza:tion (right). FIG. JD. Examples of a 4 Date Re9ue / Date Received 2022-07-19 tetramer effector enzyme recruited and positioned at the target site, which can be achiev,ed by du.al (left) or si.ngle tar:geting (right). FIG. IR A system that can be used. to edit RNA targets (e.g. retrovirus inactivation). FIGs .. 2A,, 2B, 2C, 2D, 2E, 2F, and 20 show that ta~geted recruitment of AID is 5, able to introduce site .. specU'ic conversion of nucleotide conversion. FJG. 2A. Schematic of target region along RRDR Cluster] of E. coli's rpoB gene(SEQ ID Nos: 23 and 24). Shown are ,(top) DNA s.equence (SEQ ID No; 23), with PAMs (boxed) and mutable positions. (arrows) shown; (middle) bindi:ng sites of gRNAs used in these experiments~ ,aU gRNAs were programmed to target the template strand (TS,-);. (bottom) protein sequence 10 (SEQ ID No: 25) with criti,cal amino acids involved in rifampici.n resistance shown (a:rrows). FIG. 2B. coli MG1655 cells were treated with the indicated gRNAs ,and seJect:ed in plates containing l20 μM .rifampicin. FIG. 2C. Mutation frequency calculated from B top panel. FIG. 2D., Representative sequencing results from AIDCRC treatment with rpoB_TS-4 gRNA (top, SEQ ID No: 26) and untreated cells (middle, SEQ ID No: 27). 15 Cl592>T mutation results in S53lF change in protein sequen.ce (bottom, SEQ m Nos: 28 and 29),, a mutatio11. known to induce Rif (Petersen-Mahrt, et al., Nature 418,. 99-104 (2002), Xu, M., ,el al., Joumal of Bacteriology 187, 2783-2792, doi:10.1128 / JB.187.8.2783-2792,2005 (2005), and Zenkin,. N., et al,, AltlimicnJbi,al Agents and Chemotherapy 49, 1587-1590, doi:l0.1128 / AAC.49.4.1587-1590.2005 20 (2005)). The modified nucleotides and amino acid residues are shown in C and S (wild type) and T and F (muta:nt). FIG . .2E. Mutation distribution •Of treatments AIDC:RC with gRNAs .rpoB_}, rpoB_TS-4 and scramble (SEQ ID Nos: 30-41). FIG: 2F'. Data suggest that CRC actively deami11ates target cytosine residues located on the unpaired strand (protospacer), pref:erentially closer to the .5' end. 25 FIGs .. 3A and 3B show CRC system modularity: engineering of targeting module increases mutation frequency .. FIG. 3A. Changing the targeting module from dCas9 to nCas9omA increased the efficiency of the system in terms of survival frac,tion on rifampicin plates from 18 (AroCRC) to 43 fold (AIDCRCornA) over the control when targeted with rpoB TS-4 gRNA FIG. 3B. Mutation distribution of AIDCRC010A treatment 30 with rpoB_T8-4 as target (SEQ ID Nos: 30-32). C1592 was modified in 100° / o of the clones, 75% mutated C to T and 25% mutated C to A. 5 Date Re9ue / Date Received 2022-07-19 FIGs, 4A and 4B show CRC system modularity: engi.neering of effector module increases mutation freqm,11cy. FIG, 4A. APOBEC3G (APOJGCRCvmA) and APOBEC1 (ArmoCRCmoA) were tested as effector side by side with the prototype system, AIDCRC. Treatment with APOBECl increased the mutation ffequency over AIDCRComA when 5, targeted. with rp()B_ TS-4 gRNA. Al>o 3GCRCoiOA was less active than AJDCR.C. F'IG. 41R Mutation distribution (in%) of Apc.llCRComA treatment with rpoB_TS-4 as target (SEQ ID Nos: 30·32). C1592>T conversion was obseiver in lOO'r:o of the clones. In addition, 25% of analyzed! clones were double mutants, converting C1590'>T, without amino acid change. 10 FIGs. SA and SB show CRC system modularity: Increasing; the number of RNA recruitment scaffoMs enhances mutation frequency. FIG. SA. Increasing the number of recruiting scaffolds while targeting the same position increased the mutation efficiency from 50- (,;poB TS-4 lxMS2) to 140- (rpoB TS-4 2KMSZ) fold over their respective scramble gR.NA control. FIG. SB. Mutation distribution (in %) of AIDCRCvmA treatment 15 with rpt>B_TS-4_2XMS2 as target (SEQ ID Nos: 30-32). C1592 was modifi,ed i.n WO% of the clones, 62.5% mutated C to T and 375'% mutated C to A. FIGs. 6A, 6B, 6C, and 6D show that CRC system its able to modify target nucleotide i11 extrachromosomal DNA in mammalian cells. restoring protein fun.ction. FIG. 6A. Schemati.c representation of constructs used .in these experiments .. (Top) Protein 20 coding genes were cloned under the control of the human ubiquitin C promoter (UbC) as a multicistronfo oon:struct to en.:sure stoichiometric concentrations of the two, protein components. of the system. (Bottom) Chimeric gRNA_MS2 constructs were cloned under the control of a U6 or HI promoters, to express targets with 5' -G or 5' -A,. respectively. FIG. 6:S. Schemati,c of target r,egion around mEGFPY66c deficient .fluorophore .. Shown are 25 (top) binding sites of gRNAs used in these experiments., all gRNAs were programmed to target the non~template strand (NT, +); (middle) DNA sequence (SEQ ID Nos: 42 and 43), with P AMs (boxed) and mutable positions (arrow) shown;. (bottom) pr,otein sequence (SEQ ID No: 44) with mutant amino acid that abolishes EGFP fluorescence shown (arrow), FIG. 6C . .-EGFPY66 C targeting in. 293T cells, Treatment with l'!:,E'GF1'y66cNT-l, 30 a:nd with less ,efficiency with ,,l:COJ;7,rfific_NT-2 induced BGFP signal, whHe no signal was detected with sctamble gRNA In addition, the CRC platfonn was compared with a different gene editing :system (BE3), which requires a direct fusi.on of the cytidh:ae 6 Date Re9ue / Date Received 2022-07-19 demninase protein to Cas9 protein for recruitment and requires a co-expression of an inhibitor of uracil DNA glycosylase (UGI) to improve efficiency. BF. bright fiel.d. FIG, 6D. Quantltation of GFP positive cells (in ~lo) from treatments with AIDCRCumA and BE3 systems, usingi1.,EGFPY660NTM1 as targeting gRNA 5, FIGs.. 7 A and 7B show that treatment CRC system can lead to site-specific nucleotide conversion in endogenous gene in mammalian cells. FIG. 7A. Schematic of target region on exon 3 of the Chinese hamster HPR:T gene. Shown are (top) DNA sequence (SEQ ID Nos:45 and 46), with PAM (boxed) and mutable position (mow) shown; (miiddle) binding site of gRNA used in these experiments, the gRNA was l O programmed to targ:et tb,e template strand (TS, -); (botto.m) protei.n s.equence (SEQ ID No: 47) with a critical amino acid involved in HPRT protei.n instability (arrow) shown. FIG. 7B. Qwantitation of 6-TG resistant V79-4 cells after HPRT targeting with AIDC:RCuuiA, BE3 or without treatment. When compared to untreated cells,, the survival fraction in AIDCRCoioA treatment was 140-fold higher than untreated cells,. while BE3 was 40-fold 15 higher. DETAILED DESCRIPTION OF THE INVENTION Current gene~specific editing technologies are mostly based on nucleases-induced DNA DSB and resulting DS.B-induced homologous recombination.. As the activity o:f homologous recombination is low or absent in most somatic ceUs. these technologi.es have 20 Hmited use for therapeutk corrections of pathological genetic mutations in somatic tissues in. most diseas,es. As disclosed herein, this invention is based, at least in part, on a novel platform or system that aJlows DNA-sequence directed editing of a gene or :RNA transcript The system does not rely on nuclease activity) does not generate DSB, and does not rely on the 25 DSB-mediat,ed homologous recombination. Moreover, this design of the RNA scaffof d of the p.latform is modular, which allows ,extremely flexible and convenient way of targeti.ng any desirabllie DNA or RNA sequences, In essence, this approach enables one to guide a DNA or RNA editing enzyme to virtually any DNA or RNA sequence in somatic ceHs, i.ncludli.ng stem eel.ls. Through precise editing the target DNA or RNA sequence, the 30 enzyme can correct muta:ted genes in genetic disorders. inactivate a viral genome in virusinfected ceUs, eliminate expression of a disease--causing protein in neurnd~geneiative diseases,. or silence an oncogenic protein in cancers. In addition, this approach can be 7 Date Re9ue / Date Received 2022-07-19 used for cell-based therapy by editing the genome of a stem cell or progenitor cell ex vivo .. In adldition. to tberapeuth:: applicati.on, the system can be broadly applied to target,ed modification of genomes of any organism as a powerful research tool. Gene Editing Pl.atfo1m S One aspect of this invention provides a gene editing platform, which overcomes the above mentioned limitations of current nuclease and DSB dependent genomeengineering ,and gene-editing technologies. The platform, which is named the Casltcure system or CRC system, has three functional components: (I) a nuclease defective CRISPR / Ca:s-based module engineered for sequence targeting; (2) a RNA s:caffold-ba.sed lO module for guiding the platform to the target sequence as well as for recl'uitment of a correction module; and (3) a non-nuclease DNA / RNA modifying enzyme as an effector correcti.on modul,e, such as cytosine deaminases (e.g., activation .. :induced cytosine deaminase. AID). 'Together, the CasRcure system allows specific DNA / RNA s.e,quencmng anchoring, flexible and modular recruitment of effector DNA / RNA modifyi11g enzymes to [5 specific sequences,. and eliciting cellular pathways that are active in somatic ,cells. for correcting genetic information, in particular point mutation. mustrated in Figure 1 is a schematic of an ex.emplary CasRcure system. More sp,ecifical.ly, the system includes three structural and functional comp,onents summarized in Figure lA: (1) a :sequence targeting module (e.g., a dCas9 protein); (2) an RNA scaffold 20 for sequence .recognition. and for effector recruitment (an RNA molecule that contains a guide RNA motif, a CR[SPR RNA motif, and a recruiting RNA motif), and (3) an effector (a non-nuclease DNA modifying enzyme such as AID fused to a smaU protein that binds to, the recruiting RNA mot.if). The three components could be constructed .in a single expression vtdor or in two to three separate ex.pression vectors,. The totality and tb.e 25 combination of th,e three specific components constitute the enabling of the tedmologic p]atform. As disclosed herein., there are a number of clear distinctions between re1cruitme1:r1: mechanisms: RNA scaffbld mediated recruitment system (CRC) versus direct fusion of Cas9 to, effector protein (BE3).. The results shown in the examples. below indicate that 30 RNA scaffbld mediated recruitment is more efficient than direct fusion in both extracbromosomal targets. (Figs. 6C and 60) and endogenous genes (Fig, 7B). In addition, 8 Date Re9ue / Date Received 2022-07-19 the CRC system does not rely on UNO inhibition, a DNA repair enzyme, while .BE3 uses a pote11t. UNG inhibitor peptide (UGI).. Gfobal or local DNA repair inhibiti.on couJd lead to undesirable, uncontrollable, potentially deleterious outcomes. AJso,. the modular design of the CRC system aUows for flexible system engineering. Modules are interchangeable 5, and many combinations of different modules can be achi,eved with ease. Direct fusion,. on the other hand, always requires a new fusion process to engineer new modufos. Furthermore, RNA scaffold mediated recruitment likely facilitates ofigomeri.zation of e,:ffector proteins, while di.rect fusion would preclude the formation of oligomers due to steric hindrance. 1 O a. Seqi,ence-1"'argeting Module The sequence tal'geting component of the above system is based on CRISPR / Cas systems from bat'terial species. The original functional bacteria] CRISPR..Cas system requires. three components: the Cas protein which provides the nuclease activity and two short, non-coding RNA species referred to as CRISPR RNA (crRNAs) and trans-acting [5 RNA (tracrRNA). which two RNA species form a so-caUed guide RNA (gRNA). Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand break in four sequential steps. First, two non-coding RNAs~ a pre--crRNA and a tracrRNA, are transcribed from a CRISPR locus, Second,, the tracrRNA hybridizes to the repeat regions of the pre-c:rRNA molecules and mediates processing of pre--crRNA 20 molecules into mature crRNA molecules containing individual spacer sequences, 'Third,. a mature crRNA:tracrRNA complex: (i.e,, the so-called guide RNA) directs a Cas nuclease (such as Cas9) to target DNA via Watson-Crick base-pairing between the spacer sequence on the crRNA and the complement of the protospacer sequence on the target DNA,. whfoh comprises a 3-nucl.eotide (nt) protospacer adjacent motif (PAM), PAM sequences are 25 essential for Cas9 targeting. FinaUy, the Cas nuclease mediates cleavage of the target DNA to create a double-stranded break within the target site. In its native ,context, a CRISPR / Cas system ac,,1s as an adaptive immune system that protects bacteria from repeated viral infoctions,. and PAM sequences serve as self / non-self-recognition signals, and Cas9 protein bas nuc~ease activity. CRISPR / Cas systems have been shown to have 30 enormous: potential for gene editing, both in vitro and in vivo. 9 Date Re9ue / Date Received 2022-07-19 In the invention disclosed herein, the sequence recognition mechanism can lbe achieved in a similar manner. That is, a mutant Cas protein, for example, a dCas9 protein which contains mutations at its nuclease catalytic domains thus does not have nuclease activity, or a nCas9 protein which is partially mutated a:t one of the catalytic domains thus 5 does not have nuclease activity for generating DSB, specifically recognizes a non-coding RNA scaffold molecule containing a short spacer sequence, typically 20 nucleotides in length, which guides the Cas protein to its target DNA or RNA sequence.. The latter is flanked by a 3' IP AM. Various Cas proteins can be used in this invention, A CM protein. CRISPR- 1 O associated protein, or CRllSPR protein, used interchangeably, refers to a protein of or derived from a. CRISPR-Cas type I, type II, or type III system, which has an RNA-guided DNA-binding, Non-limiting examples of suitable CRISPR / Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, Cas]O, CaslOd., CasF, Caso, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or 15 CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csxl4, CsxlO, Csx16,. Csax, Csx3, Cszl, Csx15, Csfl, Csf'2, Csf3, Csf4,. and CUl966. See e.g., WO20l4144761 WO2014144592, WO2013176772, US20140213226, aod US20140273233. 20 In one embodiment, the Cas protein is derived from a type II CRISPR-Cas syst,em. In exemplary embodiments, the Cas protein is or is derived from a Cas9 protein. The Cas9 protein can be fmm Streptococcus pyogenes, Streptococcus thermophtluti,. Streptococcus sp;, Nocardiopsis dassonvillei, Streptomyces priirtinaespiralis,. Streptomyces 25 vtridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptospt)rangium roseum, Ahcyclobacillus acidocaldarius:, Bacillus pseudomycoides, Bacillus selenitireducens, Ex(guobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium,. Polaromonas naphthalenivor:am;, P:olaromonas sp., Crocosphaera watsontt, Cyanothece sp., 30 Microcyslis aeruginos.a, Synechococcus sp., Acetohalobium arabaticum,. Ammoni:fex degensit, Caldicelulo,~lruptor becscii, Candidatus Desu(j?,rudts, Clostrldium botulinum, Clostridium d!fficile, F'inegoldia magna, Natranaerobtus thermophilus, Pelotomaculum Date Re9ue / Date Received 2022-07-19 the rmoprof)ionicum, Acidithiobacillus caldus, Acidithiobacillus Jen·ooxidtu1s, Alloclrromatium 11int1s1m'1, Marinoba(:ter sp,, Nit.rosococciis halophiltts, Nitrosococcus watsoni, Psr.mdoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum,. Anabtiena variabilis, Nodular / a spumigena, Nostoc sp., Arthrospira .m,ixima, 5, Arthrospira platensis, Arthrm,pira sp., Lyngbya sp., Microcoleut; chtho,ioplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho qfricanus, or Acaryochloris marina. In general, a Cas protein includes at least one RNA binding domain. The RNA binding domain interacts with the guide RNA The Cas protein can be a wild type Cas protein or a modified version with no nuclease activity. The Cas protein can be modified 10 to increase nucUei,c acid binding a:ffini.ty a:nd / or specificity, a.lter an enzymatic activity, a:nd / or change another pr,operty of the protein. For example, nuclease (i.e .. , DNas,e, RNase) domains of the protein can be modified, deleted, or inactivated. Alternatively. the protein can be truncat.ed to remove domains that are not essential for the flu.notion of the protein. The protein ,can also be truncated or modified to optimize tile activity of the effector 1:5 domain. [n some embodiments, the Cas protein can be a nmtm1t of a. wild typ,e Cas protein (such as Cas9) or a fragment thereof In other embodiments, the Cas protein can be derived from a mutant Cas protein. For example, th,e amino acid seque.nce of the Cas9 protei.n ,can be modified to alter one or more properties (e.g., nucl.ease acti.vity, affinity, 20 stability, etc.) of the protein. Alternatively, domains of the Cas9 protein not involved in RNA targeting can lbe e1iminated from the protein such that the modified Cas9 protein is smaller than. the wild type Cas9 protein. In some embodiments, the present system utiUz,es the Cas9 prot.ein from S. pyogenes, either as encoded in bacteria or codon--optimiz.ed for e:Kpres:sfon in mammalian cells 25 A mutant Cas protein refers to a polypeptide derivative of the wild type protein, e.g .. a protein having one or more point mutations, insertions. deletions.,, truncations,,, a fusion protein, or a combination thereof. The mutant has at least one of the RNA-guided DNA binding activity, or RNA-guided nuclease activity, or both. In general, the modified version is ·at least 50% Ce.g;, any number between 50% and 100%, inclusive, e.. g:, 50%, 30 6Ql> / 4, 70 %, 75%, 80%, 85' / o, 90%, 95%, and 99%) identical to the wild type pmtein such as SEQ ID No. l below. 11 Date Re9ue / Date Received 2022-07-19 A Cas protein (as weII as other protein components described! in this invent.ion) ,can be obtained as a .recombinant polypeptide. To prepare a recombi.nant poly)P,eptide, a nuclefo acid encoding. it can be linked to another nucleic acid encoding a f1usion partner, e.g .• g:lutatbione-s-transferase (GST), 6x-His ephope tag, or Ml3 Gene 3 protein. The 5, resultant fusi.on nucleic acid expresses in suitable host cells a fusion p:mtein that can be itso1ated by methods known in the art. The isolated fusion protein can be further treated, e.g.. by enzymatic digestion. to remove the fusion partner and obtain the recombinant polypep,tide of tbi:s invention. Altematively. the proteins can be chemicaUy synthesiz1ed (see e.g., Creighton, "Proteins: Structures and Molecular Principles," W.H. Freeman & 10 Co., NY,. 1983)~ or produ,ood by recombinant DNA tecb.nolo,gy as described be:rein .. For additionail guidance, skill,ed artisans may consult Frederick M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2003;. and Sambrook et .c, / ., Molecular Cloning, A Laboratory Manual," Cold Spring Harbor Press. Cold Spring Harbor,. NY, 2001). 1:5 The Cas protein de,scribed in the invention can be provided in pul'ified or isolatied form, or can lbe part of a composition. Preferably, where in a composition, the proteins are first purified to some ext,ent, more preferably to a high level of purity (e.g., about 80%, 90%, 95%, or 99% or higber). Compositions according to the invention cru1 be an.y type of composition desired!, but typically are aqueous compositions suitable for use as, or 20 inclusion in, a composition for RNA-guided targeting. Those of skin in the art are well aware of the various substances that can be included in such nuclease reaction compositions. As disclosed here, one can use the nuclease dead Cas9 (dCas9, for example from S. pyogene,'i DlOA, H840A mutant protein, Figure IA), or the nuclease defective nickase 25 Cas9 (nCas9, for example from S. pyogenes Dl0A mutant protein, Fig. IA and Fig. 2F). dCas9 or nCas9 could also be derived from various bacte.rial species .. Table 1 Hsts a nonexhausting list of examples of dCas9, and their corresponding PAM requirements. Tablet. Species Streptococcus pyog,enes Streptococcus agalactiae Staphylococcus a,,reus Streptococcus thern,ophilrrs Date Re9ue / Date Received 2022-07-19 PAM NGO NGG NNGRRT NNAGAAW 12 Streptococcus thermaphilus Netsseria meningitidi.ir Treponema de.nticola Other Type H CRISPR / Cas9 systems from other bacterial species NGGNG NNNNGATT NAAAAC b. RNA Scqflold.for Sequence Recognition and £:ffector Recn,itment.: The second component ofthe platform disclosed herein is an RNA scaffold, which has three sub-components: a programmable guide RNA motif, a. CRISPR RNA m.otif, and a recruiting RNA motif This scaffold can be either a single RNA molec11de or a ,compl,ex S of multiple RNA molecules. As disclosed herein, the programmable guide RNA, CRISPR RNA and the Cas protein together form a CRISPR / Cas-based module for s.equence targetiing and recognition, while the recruiting RNA motif via an RNA-protein binding p,air recruits a protein effector, which carries out genetic correction. Aocordin.gly, this second component connects tbe correction module and sequence recognition module. l O P'rogrcmimahle Guide RNA One Ikey sub-component is the programmable guide RNA Due to its simplfoity and efficiency, the CRISPR-Cas system has been used to perform genome-editing in cells of various organisms. The specificity of this system is dictated by base-pairing lbetwe,en a target DNA and a custom-design.ed guide RNA. By engineering and adjusting the bas,e- 15 pairing properties of guide RNAs. one can target any sequences of interest provided that there is a PAM sequence in a target sequence. Among the sub-compon.ents of the RNA scaffold disclosed herein, the gu.ide sequence provides the targeting specificity. It includes a region that is complementary and capable of hybridization to a pre-selected target site of i.nterest In various embodiments, 20 this guide sequence can. comprise from about 10 nucleotides to more than about 25 nucleotides. For example, the region of base pairing between the guide sequence 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 nucleotides in length. In an exemplary embodiment, the guide sequence is about 17 M20 nucleoti.des in length, such as 20 nuc.leotides. 25 One requireme.nt for selecting a suitable target nucleic acid is that it has a 3' PAM she / sequence. Each target sequence and its corresponding PAM site / sequence are referred herein as a Cas-targeted site. Type II CRISPR system. one of the most wen ch.aracterized 13 Date Re9ue / Date Received 2022-07-19 systems. needs only Cas 9 p.rotein. and a guide RNA com,plementary to a target s,equence to affect target cleav,age., The typ,e II CRISPR system of S. pyogenes us,es target sites having Nl2-20NGG, where NGO represent the PAM site from S. pyogenes, and Nl2-20 represen1S the 12-20 nucleotides directly 5' to the PAM site. Additional PAM site 5 sequences from. 0th.er species of bacteria include NGGNG, NNNNGATT, NNAGAA, NNAGAAW, and NAAAAC. See, e.g.~ US 20140273233, WO 2013176772, Cong et aL, (2012), S,cience 339 (,6121): IH9-823, Jinek et al.,, (2012), Science 337 (6096): 816-821, Mali et al., (20B), Science 339 {6121): 823-826, Gasiunas et al., {2012), Proc Natl Acad Sci US A. 109 (3.9): E2579-E2586, Cho et al., (2013) Nature Biotechnology 31, 230-232, 10 Hou et al., Proc Nat] Acad Sci U S A 2013 Sep 24;110(39):15644-9, Mojica et Microbiology. 2009 Mar;l55(Pt 3):733-40, and www.addgene.org / CR1SPR / . T'.b.e target nucleic 1Cid strand can be either of the two strands on a genomic DNA in a host cell. Examples of such genomic dsDNA include, but are not necessarily limited 15 to, a,. host cell chromosome, mitocho.ndrial DNA and a stably maintmned plasmid. However, it is to be understood that the present method can be practiced on other dsDNA present in a ho,st ceU, such as non-stable plasmid DNA, viral DNA., and phagemid DNA, as long as there is Cas-targeted site regardless of the nature of the host cell dsDNA. The present method can be practiced on RNAs too. 20 CRJSPR.Motif 25 Besides the abo1Ve-descrlbed guide sequence, the RNA scaffold of this invendo.n indudes additional. active or non-active sub-components. In one example, the scaffold has a CRJSPR motif witb tracrRNA activity. For example, the scaffold can be a hybrid RNA molecule wher,e the above-described programm.a:ble guide RNA is fused to a tracrRNA to mimic the natural crRNA:tracrRNA duplex. Sho\vn below is an exemplary hybrid crRNA:tmcRNA, sgRNA sequence: 5'-(20nt guide),- GUUUAAGAOCUAUGCUGGAAACAG CAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC GAGUCOOU 30 GCUUUUUUU-31 (SEQ m No: 4; Chen et al. Cell 2013 Dec 19;155(7):1479""91). 35 Various tracrRNA sequences are known in the art and examples include the foUowing tmcrRNAs and active pmtions therl\lof. 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. See. ,e.g .• W02014144592. Methods for generating crRNA~tracrRNA hybrid RNAs are known. in 14 Date Re9ue / Date Received 2022-07-19 the art. See e.g.,,. W02014099750., US 20140179006, and US 20140273226. GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGIJCCGUUAUCAACUUGAAAA AGUGGCACCGAGUCGGUGC (SEQ ID No: 5); UAGCAAGUUAAAAU:AAGGCUAGUCCGUUAUCAACUOGAAAAAGUGGCACCGAGUCGGUGC 5 (SEQ ID No: 6); AGCAUAGCAAGUUAAMUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG GUGC (SEQ ID No: 7); CAAAACAGCAUAGCAAGUUAAAA.UAAGGCUAGUCCGUUAUCAACUUGAAAAA.GUGGCACC GAGUCGGUGC ( SEQ ID No: 8); lO UAGCAAGUUAAAAUAA.GGCUAGUCCGUUAUCAACUUGAAAAAGUG (SEQ ID No: 9); UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA (SEQ ID No: 10); and UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID No: 11). In :some embodiments~ the tracrRNA activity and the guide sequence are two separate RNA molecules, which together form the guide RNA and related scaffold .. In this 15 case,, the molecule with the tracrRNA activity should be able to interact with (usuallly by base pairing) the mole,cule having the guide sequence. Recruitlng RNA Motif The third sub-comp,onent of the RNA scaffold is the recruiting RNA motif, which links the correction modul,e and sequence recognition module. This Hnkage is critical for 20 the p]atform disclosed herein. 0.ne way to recruit effector / DNA editing enzymes to a target sequence is through a direct fusion of an effector protein to dCas9. The direct fusion of effector ,enzymes ("'correction module') to the proteins required for sequen.ce recognition (such as dCas9) has achieved suc,cess in sequence specific transcriptional activation or suppression, but the 25 protein-protein fusion design may render spatial hindrance, which is not ideal for enzymes that .need to form a multi.meric complex for th.eir activities. In fact, most n.ucleotide editing enzymes (such as AID or APOBEC3G) require formation of dimers, tetramers or high.er ord,er oligomers~ for their DNA editing catalytic activities. The direct fusion to dCas9, which anchors to DNA in a defined conformation, woul.d hinder the formadon of a 30 fwctional oligomeric ,enzyme complex at the right location. In contrast,. the pl.atform disclosed herein is based on RNA scaffold·mediated effector protein recruitment More specifically, the platform. takes advantages of vari,ous RNA motiO'.RNA binding protein binding pairs. To this end, a RNA scaffold is designed 15 Date Re9ue / Date Received 2022-07-19 such that a RNA motif (e.g., MS2 operator motif), whi.ch specificaHy binds t,o a RNA binding protein (e .. g:,, MS.2 coat protein, MCP), is lhiked to the gRNA..CRISPR :scaffold (Figure lA). As a r,esuh,. this RNA scaffold component of the platform disdos,ed herein is a 5, designed RNA molecule, which contains not only the gRNA motif for specifi.c DNAJRNA sequence recognition, the CRISPR RNA motif for dCas9 binding, but also the recruiting RNA motif for effector recruiting (Figure lA). In this way, recruited~effector protein fusions can be recruited to the target site through their ability to bind to the recruiting RNA motif. Due to the flexibility of RNA scaffold mediated recruitment, a functional l O monomer, as weU as dim,er, tetramer;, or oligomer could be reladvely easy to form near the target DNA or RNA sequence. Example configuration.s are mustra:ted fo Figure l B-E. These pairs of RNA recruiting motif7binding protein could be derived from naturally occurring sources (e.g., RNA phages, or yeast telomerase) or ,could be artificially designed (e.g., RNA aptamers and their corresponding binding protein ligands). A non-exhausting 1:5 list of examples of recruiting RNA motif / RNA binding protein pairs that could be used in the CasRcure system is summarized in Table 2. 20 Table 2. Example·s of .recruiting RNA motifs that ca11 be used i.n this hui,ention, as well as their pa.riag RNA binding protelus / protein domains. RNA motif Telomerase Ku binding motif Telomemse Sm7 binding motif MS2 phage operator stem~loop PP7 phage operator ,stem-loop SfMu phage Com stem-loop Non-natural RNA aptamer * Pairin.g. i nteracting protem Ku Sm? MS2 Coat Protein (MCP) .PP7 coat protein (PCP) Com RNA binding protein Corresponding aptamer ligand Recruited 11roleh1s are fused to effector prol.ei.as, for examples see Table l. 16 Organism Yea:st Yeast Phage Phage Phage Artifi ci ally designed! Date Re9ue / Date Received 2022-07-19 The sequences for the above binding pairs are listed below. 1~ Telo,mera:s:e Ku bi.di.ng motif / Ku beterodimer a. Ku bindiing hairpin 5'- S TTCTTGTCGT AcCTTATAGATCGCTACGTTATTTCAATTTTGAAAATCTGAGTCC T'GGGAGTGCGGA~l'(SEQ ID No: 12) b.. Ku lteterodimer MSGWESYYKTEGDEEAEEEQEENLEASGDYKYSGRDSLIFLVDASKAMFESQSED ELTPFDMSIQCIQSVYISKUSSDRDLLAVVFYGTEKDKNSVNFKNIYVLQELDNPG 10 AKRILELDQFKOQQGQKRFQDMMGHGSDYSLSEVLWVCANLFSDVQFKMSHKR IMLFTNEDNPHGNDSAKASRARTKAGDLRDTGIFLDLMHLKKPGGFDISLFYRDII SIAEDEDLRVHFEESSKLEDLLRKVRAKETRKRALSRLKLKLNKDIVISVGIYNL V QKALKPPPIKLYRETNEPVKTKTRTFNTSTGGLLLPSDTKRSQIYGSRQIILBKEETE ELKRFDDPGLMLMGf'KPLVLLKKHHYLRPSLFVYPEESLVIGSSTLFSALL[KCLEK 15 EVAALCRYTPRRNIPPYFVALVPQEEELDDQKIQVTPPGFQLVFLPFADDKRKMPF 20 TEKJMATPEQVGKMKAIVEKLRFTYRSDSFENPVLQQHFRNLEALALDLMEPEQA VDLTLPKVEAMNKRLGSLVDEFKELVYPPDYNPBGKVTKRKHDNEGSGSKRPKV EYSEEELKTHISKGTLGKFTVPMLKEACRAYGLKSGLKKQELLEALTKHFQD> (SEQ ID No: I3) MVRSGNKAAVVLCMDVGFTMSNSIPGIESPFEQAKKVITMFVQRQVFAENKDEIA LVLFGTDGTDNPLSGGDQYQNITVHRHLMLPDFDLLEDIESKIQPGSQQADFLDAL IVSMDVIQHETIGKKFEKRHIEIFTDLSSRFSKSQLDIIIHSLKKCDISERHSillWPCRL TIGSNLSIRIAAYKSILQERVKKTWTVVDAKTLKKEDIQKETVYCLNDDDETEVLK 25 EDIIQGFRYGSDIVPFSKVDEEQMKYKSEGKCFSVLGFCKSSQVQRRFFMGNQVL 30 KVFAARDDEAAAVALSSLIHALDDLDMVAIVRYAYDKRANPQVGVAFPHIKHNY ECLVYVQLPFMEDLRQYJ\IIFSSLKNSKKY APTEAQLNAVDALIDS.MSLAKKDEKT DTLEDLFPTTKIPNPRFQRLFQCLLHRALHPRE:PLPPIQQHIWNMLNPP AEVTTKSQ IPLS.KIKTLFPLIEAKKKDQVTAQEIFQDNHEDGPT AK (SEQ ID No; 14) 2. Telomerase Sm7 biding motif / Sm7 homobeptamer a. Sm ,consensus site (siugle stranded) 5'-AATTTTTGGA-3'(SEQ ID No: 15) b,. Mo.nomeric Sm - like protein (archaea) 35 GSVIDVSSQRVNVQRPLDALGNSLNSPVIJKLKGDREFRGVLKSFDLHMNLVLND AEELEDGEVTRRLGTVLIRGDNIVYISP(SEQ ID No: 16) 3. MS2 p,hage operator stem loop / MS2 coat protein a. MS,2 pha,ge operator stem loop 40 5'-GCGCACATGAGGATCACCCATGTGC-3' (SEQ ID No: 17) 45 b. MS2 coat protein MASNFTQFVL VDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQ NRKYTIKVEVPKGAWRSYLNMELTIJ>IFATNSDCEUVKAMQGLLKDGNl.,IPSAIA ANSGIY (SEQ ID· No: 18) 4. PP7 pha.ge oper1to1• stem loop / PP7 coat protein a. PP7 phage operator stem loop 17 Date Re9ue / Date Received 2022-07-19 -aTAAGGAGTTTATATOOAAACCCTTA-3' (SEQ ID No: 19) b,. PP7 coat protein (PCP) MSKTIVLSVGEA:TRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLRQNGAKTAYRV NLKLDQADVVDCSTSVCGELPKVRYTQVWSHDVTIV ANSTEASRKSLYDLTKSL 5 VATSQVEDLVVNLVPLGR. (SEQ ID No; 20) 5. SfMu Com :ste.m loop / Sf.Mu Com bind.ing protein a. SrMu Com stem loop 5'-CTGAATGCCTGCGAGCATC-3' (SEQ ID No: 21) 10 b, Smtu Com binding protein IWKSIRCKNCNKLLFKADSFDHIEIRCPRCKRHIIMLNACEHP1EKHCGKREKITHSD ETVRY (SEQ ID No: 22) The RNA sc,a:ffo]d can be either a single RNA molecule or a complex of multiple l5 RNA molecules. For example, the guide RNA, CRISPR mot.if, and recruiting RNA motif can be three segments of one, l.ong single RNA molecule, Alternatively, one, two or three of them can be on separate molecules, :In the latter case, the three components. can be linked together to form tile scaffold via covalent or non-covenant linkage or binding, including e.g., Watson-Crick base-pairing. 20 [none exmnple, the RNA scaffold can comprise two separate RNA molecul.es. The first RNA molecul,e can comprise tile programmable guide RNA and a region that can fonn a stem duplex struct11re with a complemetrtary region. The second RNA molecule can compri.se the compl.ementary region in addition to the CRISPR motif and the recruiting DNA motif. Via this stem duplex structure, the first and second RNA 25 molecules fonn a RNA scaffold of this invention. In on.e embodiment, the first and second RNA molecules each comprise a sequence (of about 6 to about 20 nucleotides) that base pairs to the other se,quence. By the same token. the CRISPR motif and the recruiting DNA motif can also be on different RNA molecule and be brought together with another stem duplex structure. 30 The RNAs and related scaffold of this invention can be made by various methods known in the art including cell-based expression, in vitro transcription., and chemical synthesis. The ability to chemica1ly synthesize relatively long RNAs (as fong as 200 mers or more) using 'TC-RNA chemistry (see. e.g., US Patent 8,.202,.983) allows one to produce RNAs with speciat features that outperform those enabled by the ba.5ic four 35 ribonucleotides (A, C. G and U), 18 Date Re9ue / Date Received 2022-07-19 The, Cas protein-guide RNA scaffold complexes can be made with recombinMt technolo1;:,"}' using a host cell system or an in. vitro transla:tion-transcriptio.n system kno'V\ln in the art. Details of :such systems and technology can be found in e.g., W00014144761 W02014144592, W02013l76772, US20140273226, and US20l40273233. 5 The complexes can be isolated or purifi.ed, at least to some extent, from cellular material of a ceU or an in vitro transtatiorll-trans,cription system in which they are produced The RNA scaffold may include one or more modifications. Such modifications may indude inclusion of at least one non-naturally occurring nucleotide. or a modifi,ed 10 nudeotide, or ainalogs. thereof: Modified nucleotides may be modified a:t the ribose, phosphate, and / or base moi,ety. Modified nucleotides may include 2'-0-methyl .analogs, 21-deoxy analogs,, or 21-fluoro analogs. The nucleic acid backbone may be modified, for examp]e, a phospho:rothioat,e backbone may be used. The use of locked nucleic acids (LNA) or bridged nucleic acids (BNA) may also be possible. Further examp[es of 15 modified bases include, but are n.ot limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine. 7-methylguanosine. These modifications may apply to any component of the CRISPR system . In a preferred embodiment these modifications are made to the :RNA components, e.g. the guide RNA sequence. c. li;{lectors: Non-Nuclease DNA Modifj1ing Enzymes 20 The third component of the platfonn disclosed in this invention is a non-nuclease effector. The effector is n,ot a nuclease and does not have any nuclease activity, but cain have the activity of other types of DNA modifying enzymes. Examples of the enzymatic activity include, but are not limited to, deamination activity. methyltransferase ,activity, demedlylase activity, DNA repair activity, DNA damage activity, dismutase, .activity, 25 alkybrtion activity, d,epurination activity, oxidation activity, pyrimidine dimer forming acHvity, inlegrase aclivily, transposase iicti vity, recornbinase acUvily, polymeras,e activity. ligase activity, helicase activity, photolyase activity or glycosylase, activity. In some embodiments. the effector has the activity of cytosine deammases (e.g., AID, APOBEC3G). adenosine d,eaminases (e .. g., ADA). DNA methyltmnsferases, and DNA 30 demethylases. 19 Date Re9ue / Date Received 2022-07-19 In pr,eferred embodiments, this third component is a conjugate or a fosion protein that has an RNA-binding domain. and an effector domain. These two domains ca111 be jioined via a linker. RNA~bindh1g Domain 5 Ahhough various RNA-binding domains can be used in this invention, the RNAbinding domain of Cas protein (such as Cas9) or its variant (such as dCas9) should not be used. As .mentioned above, the direct fusion to dCas9, which anchors 10 DNA in a defined conformation,. wou1d hinder the fonnati.on of a fi.J.nctional oligomeric enzyme comple1x. at the right location. Instead, the present invention takes advantages of various other RNA 10 motifMRNA binding protein binding pairs. Examples include those Hsted in Tabl,e 2. In this way, the ,effector protein can be recruited to the target site through RNAbinding domain's ability to bind to the recruiting RNA motif. Due to the flexibility of RNA sc1ffold mediated re,cruitment, a functional monomer, as well as dimer,. tetramer, or oligomer could be formed relatively easily near the target DNA or RNA sequence. [ 5 E1fecior Domain The effector component comprises an activity portion, a:n effector domain., hi some embodiments, the effector domain comprises the naturaUy-occuning activity portion of a non-nuclease protein (e.g:., deaminases). In other embodiments, the effector domain comprises a modHied amino acid sequence (e.g., substitution, deletion,. inserti.on) of a 20 naturaUy-occurring activity portion of a non.,nuclease protein. The effector domain bas an enzymatic ac1ivity, Examples of this activity include deamination activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, dismutase activity, alkylation activity, depurination activi.ty, o:xi.dation ,activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase 25 activity, polymer:ase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, DNA methylation, histone acetylation activity, or histone methylation activity.. Linker The above-mentioned two domains as wen a.~ otbets as disclosed herein can be 30 joined by means of linkers, such as, but not limited to chemical modification, peptide linkers, chemical Hnkers, covalent or non-covalent bonds. or pmtein fusion or by ,any 20 Date Re9ue / Date Received 2022-07-19 means known 1,o one skiUed in the art. The joining can be permanent or reversible, See for example US. Pat Nos. 4625014, 5057301 and 5514363, US Application Nos. 20]50182596 and 20100063258, and WO2012142515. In some embodiments, several Unkie.rs 5 can be included in order to take advan.tage of desired properties of each linker and eaich protem domain in the conjugate. For example, flexible linkers and linkers that increase the solubility of the conjugates are contemplated for use alone or with other linkers. Peptide Hnkers can be link,ed by expressing DNA encoding the liinker to one or more protein domains m th,e conjugate. Linkers can be acid cleavabl,e, photocloovable and h,eat 10 sensitive Hnkers. Methods for conjugation are well known by persons sldHed in the art and are encompassed for use in the present invention. In some embodiments. the RNA-bin.ding domain and the effector domain can be joined by a p,epdde Unker .. Peptide linkers can be linked by expressing nucl,eic acid encoding in frame th,e two domains and the linker. Optionally the linker peptide can be 15 joined at either ,or both of the amino terminus and carboxy ter.minus of the domains .. In some exaimp!es, a Hnker is a immunoglobulin hinge region linker as disclosed in U.S. Pat Nos. 6,165,476, 5,856,456, US Application Nos. 20150182596 and 2010 / 0063258 and International Application WO2012114251S. 20 Other Domains The effector fusion p.mtein. can comprise other domains. In certain embodiments. the effector fusion protein ,can comprise at least one nuclear localization signal ,(NLS). [n general, an NLS comprises a stretch of basic amino acids. Nuclear locali1,ation signals are kno,•wn in the art (see, e.g.., Lange et al., J. Biol. Chem., 2007, 282:5101-5105). The NLS 25 can be located at the N-tenninus, the C-tenninal, or in an intern.al ]ocation of the fusion protein. In some embodiments, the fusion. protein can comprise at least one ceH-penetrating domain to facilitate d,eHvery of the protein into a target cell. In one embodiment, the cellpenetrating domain can be a cell-penetrating peptide sequence. Various ceH"'.penetrating 30 peptide sequences are known in the art and examples include that of the HIV-l TAT protein, TLM oftbe human HBV, Pep-1, VP22, and a polyarg:inine p,eptide sequence. 21 Date Re9ue / Date Received 2022-07-19 In stUI oth,er embodiments, the fusion protein can comprise at least one marker domain. No11-lh1:1iting examples of marker domains includ.e fluorescent proteins, pmification tags, and epitope tags. In some embodiments, the marker domain can be a fluorescent prntein. In other embodiments, the marker domain can be a purifi,cation tag 5, and / or a:n epitope tag. See,. e.g., US 20140273233 . In one embodiment, AID was used as an example to illustrate how the system works. AID is a cytidfune deaminase that can catalyze the reaction of deamina:tion of cytosine in the cont,ext of DNA or RNA. When brought to the targeted she, AID changes a C base to U base. In dividing cells, this could lead to a C to T point mutation. 10 Alternatively, the change ,of C to U could trigger cellular DNA repair pathways~ mainly e:tclsion repair pathway, which will remove the mismatching U-0 base-'pak, and replace with a T-A, A-T, C-G, or G-C pair. As a result,. a point mutation would be generated at the target C-G site. As excision repair pathway is present in most, if not aU, somatic ceUs, recruitment of AID to th,e target site can correct a C-G base pair to others. In that ,case, if a 1:5 C-G base pair is an underlying disease causing genetic mutation in somatic tissues / ceUs, the above-described approach can be used to correct the mutation and thereby treat the disease. By the same token, if an underlying disease causing genetic mutation .i.s an A-T base pair at a sp,edfic :si.te, one can use the same approach to recruit an adenosine 20 demninase to the specific site, where adenosine deaminase can correct the A-T base pair to others. Other effec1or enzymes are expected to generate other types of changes. in basepairing. A non-exhausting list of ex.amples of DNA / RNA modifying enzymes is detailed in Ta.ble3. Table 3. Exa.mples of effector proteins th.at can be used in th.is .invention Enzyme type Cytosine, deaimfoase Genetic change C-+U / T Date Re9ue / Date Received 2022-07-19 Effector protein abbreviated AID APOBECl APOBEC3A APOBEC3B APOBBC3C APOBEC3D APOBEC3F APOBBC3G APOBEC3H 22 Adenosine A-lJG ADA deaminase ADARl DNA.Methyl. c-Met-C Dnmtl Dnmt3a trnnsferase Dnmt3b DemethyBase Met-C-C Tetl Effector protei.n. full n.ames: AID: activation induced cytidi:ne deanrlnase; a.ka AICDA APOBECI: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-Hire ] . APOBECJA: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A 5 APOBEC3B:. apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 38 APOBEC3C: apolipoprotein B mRN A editing enzyme. catalytic polypeptide-like 3C APOBEC3D: apolipoprotein B rnRN A editi1~ enzyme, catalytic polypeplide.;like 3D APOBF:CJF: .apolipoprotein B mRN A editing enzyme, catalytic polypeptide-1ike 3fl APOBECJG: apoJipoprotei:n B mRN A editi11g enzyme, catalytic polypeptide-like 3G 10 APOBEC3H: apo1ipop,rotein B mRNA editing enzyme, catalytic polypeptid.c-Jike 3H ADA: adeoosine deamirwse ADARI: adenosh:ie deam:inase acting on RNA 1 Dnmtt: DNA (cytosine-5-)-methyltnuisfemse I Dnmt:Ja: DNA (cytos.lne-5-)-methyltransferase 3 alpha 15 .Dnmt3b: DNA (cytosine-5~)-methyl.tmnsferase 3 beta Teti: methylcytosine dioxygenase The above1-described three specific components constitute the tec:hnologicail platform. Each component could be chosen from tb1e list in Table 1-3 re:specfively to 20 achieve a specific therapeutic / utility goal. In one example, a CasRcure system was constructed using (i) dCas9 from S. [)yogeneti~ as the sel1uence targeting protein, (ii) a RNA scaffold containing a guide RNA sequence, a CRISPR RNA motif, and a MS2 operator motif, and (iii) an effector fusion containing a human AID fusing to MS2 operator binding protein MCP. The s.equences for 25 the components are listed below ,')'. pyogenes dCas9 protein sequence (SEQ ID No., 1) MDKKYSIGLA,IGT'.NSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE ATRLKRT.ARRRYT'RRKNRICYLQEI FSNEMAJ<VDDSFFHRLEESFLVEEDKKHERHPIFG NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAJIMIKFRGHFLIEGDLNPDNSD 30 VDKLF"I QLVQ'.'I'YNQLFEENPlMASGVDAKAI LSARLSKSRRLENL IAQLPG.EKKNGLFGN LIALSLGLTPNFKSNFDLAEDA.KLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGAS,QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH .AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFA~TRKSEETIT'PWNF'EE 35 VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAIVDLLE'KTNRKVTVKQLKEDYF'KKIECE'DSVEISGVEDRJ<'NASLGTYHDLLKI IKDKDFLONEENEDILEDIVLTL1TLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG RLSRKLINGIRDKQ1SGKTILDFLKSDGF.ANRNEMQLIHDDSLTFKEDIQKAQVSGQGDSL 2.3 Date Re9ue / Date Received 2022-07-19 HEHIANLAGSPAIKKGILQTVKVVDELVKVM:GRHKPENIVIEMIARENQTTQKGQKNSRER MKRIE,E,G IKELGSQ,I LKEHPVENTQLQNEKL YLYYLQNGRDMYVDQELDINRLSDYDVD{1 IVPQ1S FLKDDS I DNKVLTRSDKNRG.KSDNV.E' SEEVVKKMKNYWRQLLNAKL.ITQRKFDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS S KLVSDFRKDFQFYKVRE INNYHHAHDAYLNAVVGTALI KKYPKLESEFVYGDYKVYDVRK MIAKSEQE IGKATAKYFFYSNIMNFFKTE I TLANGE fRKRPLIETNGETGE IVWDKGRDF' .A.TVRKV'LSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVA YSVLVVAKVE'KGKSKKLKSVKELLGITIMERSSFEKNPIDFLE.AKGYKEVKKDLIIKLPK YSLFELENGRK:RMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQK,QLFVE 10 QHKHYLDE I IEQI SE FSKRVI LADJ\NLDKVLSAYNKHRDKPI REQAENI IHLFT'LTNLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (Residuies und,erlined: Dl0A, H840A active site mutants) RNA scaffold expr,e:ssion ,cassette (S. J~yogenes), containing a 20-nucleotide pre>gmmmabie sequence, a CRISPR RNA motif, and an MS2 operator motif (SEQ ID No. 2): 1.5 N.2.oGTTTTAGAGC'TAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT'TGAAAAA GTGGCACCGAGTCGGTGCGCGCACATGAGGATCACCCA:TGTGCTTTTTTTG (N20: programmable sequence; UnderUned: CRISPR RNA motif; Bold: MS2 motif; 20 Italic: terminator) Effeeto,r AIU ~MCP fusi,on (SEQ ID No. 3): MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKN GCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRI 25 FTARL YFCEDRKAEPEGLRRLHRAGVOIAlMTFKDYFYCWNTFVENHERTFKAW EGLHENSVRLSROLRRILLPLYEVDDLRDAFRTLGLELKTPLGDTTHTSPPCPAPEL ~OOPMASNFTOFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSOAYKVTCSVRQ SSAQNRKYIIK,YEyPKGAWRSYLNMELTIP,IEAINSDCELIVKAMQQLLKDGNPIP SAlAAliASCUX Like the Cas protein described above, the non-nuclease effecror can also be obtained as a recombinant polypeptide. Techniques for making recombinant polypeptides are known in the art. See e.g., Creighton, !!Proteins: Structures and Molecular Principles," W.R Freeman & Co .• NY, 1983); Ausubel et al., Current Protocols in Mole,cular Biology, 35 John WHey & Sons. 2003; and Sambrook et al., Molecular Cloning, A Laboratory Manual;' Cold Spring Harbor Press, Cold Spring Harbor, NY,. 2001). 24 Date Re9ue / Date Received 2022-07-19 The above three components of the p1atfom1 / system discfosed herein can be expressed using one to three expression vectors.. The system cm1 be programmed to target virtuaUy any DNA ,or RNA sequence. Expression System S To use the platform described above, it may be desirable to express one or more of the protein and RNA components from nucleic acids that encode them. This can be p,erfonned run a v,ariety of ways. For ex.ample, the nucleic acidls encodiing the RNA scaffold or proteins can be cloned into one or more intermediate vectors for introducing into prokaryotic ,or eukaryotic cells for replication and / or transcription. Intennediate lO vector:s are typically prok:aryotic vectors, e.g., plasmids~ or shuttle vectors, or insect vectors, for storage or manipulation of the nucleic acid encoding the RNA s,caffold ,or protein for production of the RNA scaffold or protein. The nudei.c acids can also be cloned into one or more ,expression vectors, for administration to a pihmt ceH, animal c,eU, preferably a mammalian cell or a human cell, funga] cell,. bacterial ceH, or protozoan cell. [5 Accordingly, the present invention provides nucleic acids that encode any of the RNA scaffbld or proteins mentioned above.. Preferably, the nucleic acids are i.:solated and / or purified. The present invention also provides recombinant constructs or vectors having sequences encoding one or more of the RNA scaffold or proteins described above. 20 Examples of the constructs include a vector, such as a plasmid or viral vector, into whi,ch a nucleic acid sequence of the i.nvention has been inserted, in a forward or reverse orientation. In a preferred embodiment, the construct further includes regulatory sequences, including a promoter,, operably linked to the sequence. Large numbers of sui.table vectors and promoters are known to those of skill in the art, and are commerciaUy 25 available. Appropriate ,cloning and expression vectors for use with prokaryotic and eukaryo6c hosts ar,e also described in e.g., Sambrook et al. (2001~ Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press). A vector refors to a nucleic acid mol,ecule capable of transporting another nucleic acid to which it has been linked... The vector can be capable of autono:mous replication or 30 integration into a host DNA. Examples of the vector include a plasmid, cosmid, or viral vector. The, vector of this invention includes a nucleic acid in a fonn suitable for 25 Date Re9ue / Date Received 2022-07-19 expression of the nuclei.c acid in a host cell. Preferably the vector inc1udes one or more regulatory seqi:1ences ope.ratively linked to the m1cleic acid sequence to be expressed. A "regulatory s1equence" includes promoters, enhancers, and other expJ:ession control elements (e,.g,, polyadenylation signals). Regulatory sequences include those that dfoect 5, constitutive expres.sion of a nucleotide sequence, as well as inducible regulatory sequences. The design of the expression vector can depend on such factors as the choice of the host ceU to b,e trans.formed, tran.sfected, or infected, the level of expres.sion ofRNAs or proteins desired, and th,e Bike. Examples of expression vectors include chromosomal, nonchromosomal and 10 synthetic DNA sequences, bacterial plasmids, phage DNA, baculovirus,. y,east p.hismidls, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus. foWI pox virus, and pseudorabies,. However, any other vector may be used as long as it is replicable and viable in the host. The appropri.ate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, a nucleic 1 :5 acid sequence encoding one of the RNAs or proteins described above can be inserted into a.n appropriate restriction endonuclease sit:e(s) by pro,cedures known in the art. Such procedures and related sub-cloning procedures are within the scope of those sk:iUed in the a:l1. The vector may include appmpriate sequences f'or amplifying express.ion. ln 20 addition, the ,expr;ession vector preferably contains one or more selectable marker genes to provide a phenotypic traiit for selection of tran.sformed host cells such as dihydrofbfate reductase or neomycin resistance for eukaryotic cell cultures, or such as tettacycHne ,or ampiciUitn r:esistance in E. coll. The v,ectors for expressing the RNAs can include RNA Pol III promoters, to drive 25 expression of the RNAs, e.g., the HI, U6 or 7SK promoters. These human promoters allow for expression of RNAs in mammalian cells following plasmid transfection. Alternatively, a T7 promoter may be used, e .. g;, for .in vitro transcription, and the RNA can be transcribed! in vitro and purified. The v1ector 1containing the appropriate nucleic acid sequences. as described abov1e, 30 as wen as an appropriat1e promoter or control sequence, can be emp]oyed to transfonn, transfect, or infect an appropriate host to permit the host to express, the RNAs or proteins described above. Examples of suitable expression hosts indud.e bacterial. ceJls (e.g., .E. 26 Date Re9ue / Date Received 2022-07-19 coli, Streptomyces, Salmonella typhimurium), fungal ceHs (yeast), insect ceUs (e.g .. , Drosophila and Spodop.tera frugiperda (Sf9)), ani.mal cells (e.g., CHO, COS, and HEK 29:3), adenovimses, and plant cells. The selection of an appropriate host is within the scope of tbos,e skiUed in the art In. some embodiments, the present invention provides 5, methodls for producing the above mentioned RNAs or proteins. by transforming, tr.ansfocting, or infecting a host cell with an expression vector having a nucleotide sequence that encodes one of the RNAs, or polypeptides. or proteins. The host cells are then cultured under a suitable condition, which allows for the express.ion of the RN As or proteins. l O Any of the procedures known in the art for introducing foreign nuc;Reotide sequences into host ceU:s may be used. EKamples include the use of calcium phosphate transfection, polybrene. protoplast fusion, electroporation, nuc]eofection. iiposornes, microinjection. naked DNA, plasmid vectors, viral vectors. both episomal and integrati.ve, and any of the other well-known methods for introducing cloned genomic DNA, cDN~ 1:5 synthetic DNA or other foreign genetic material into a host cell. Methods Another aspect of the present invention encompasses a method for modifying a target DNA sequence (e .. g., a chromosomal sequence) or target RNA sequence in a c,en, embryo, human or non-human animab. The method comprises introducing into the cell or 20 embryo the above-described (i) a sequence-targeting protein, or a polynucleotide encoding the same, (ii) an RNA scaffold, or a DNA polynucfootide encoding the same, and (i.ii) a non-nudease effector fusion protein, or a polynucleotide encoding the same. The RNA scaffold guides the sequence-targeting protein and the fusiori protein to a target polynucleotide at a target site and the effector domain of the fusion protein modifies the 25 sequence. As disclosed herein, the sequence-targeting protein, such as a cas9 protein, is modified such that the endonuclease activity is eHminated. Ill certain embodiments, the effector protein functions as a monomer. In that case, the system of this invention can be targeted to a single site, either upstream (left) or downstream (right) of the target site as shown in Figure lB. In other embodiments;. the 30 effector protein requires dimerization for proper catalytic function. To that end, the system can be multiplexed to target sequences upstream and downstream of the target site 27 Date Re9ue / Date Received 2022-07-19 simul.taneously, therefor,e allowing the effector proteins to dimerize (Figure IC, left), Alternatively, recruitm.en:t of effector protein to a single site may be sufficient to incr,ease its affinity for neighboring effector proteins, promoting dimerization (Figure IC, right), In yet some other embodiments, a tetramer effector enzyme can be recruited and po,sitioned 5, at the target site as shown in Figure lD.. This can be achieved by dual. (Figure 1D~ Ieft) or single targeting (Figure lD, right). The system disclosed in this invention can be used to edit RNA targets too (e.g .. retrovirus inactivation). See Figure IE. In that cas,e, if the e,:ffector protein requires ,as:se.mbly of a functional oligomer, single targeting to a RNA molecule could p:mmote oligomerization as in right panels ofFigures IC and ID, 10 The target polynucleotide has no sequence limitation ,except that the sequence is immediately foUo•wed ,(:downstream or 3') by a PAM sequence. BKam.ples of PAM include, but are not limited to, NGG, NGGNG, and NNAGAA W (wherein N is defined as any nucleotide and Wis d,efined as either A or T). Other examples of PAM sequences. are given above,. and the skilled person will be able to identify further PAM sequences for use 1 :5 with a. given CRISPR protein. The target site can be in the coding region of a gene, in an intmn of a gene, in a control region between genes, etc.. The gene can be a protein coding gene or an RNA coding gene. The target p,olynucleotide can be any polynucleotide e.ndoge.nous or exogenous. to the cell. For example, the target polynucleotide can be a polynucleotide residing in the 20 nucleus of the eukaryoti,c ,cell. The target polynucleotid,e can be a sequence coding a gene product (e.g., a prntiein) or a non-coding sequence (e.g., a regul.atory pol.ynucleotidle). The protein components of this system of this invention can be introduced into the cell or embryo as an isolated protein. In one embodiment, each protein can comprise at least one ceH-penetrating domain, which facilitates cellular uptake oftbe protein. 1n other 25 embodiments, mRNA molecules or DNA molecules encoding the protein or proteins can be introduced into the cell or embryo. In. general, a DNA sequence encoding the protein is operab]y linked to a promoter sequence that will function in the cell or ,embryo of interest The DNA sequence can be linear, or the DNA sequence can be part o.f a vector. In stin other embodiments, the protein. can be introduced into the cell or embryo as an 30 RNA-protei.n complex comprising the protein and the RNA scaffold described above. In altemate embodiments, DNA encoding the protein(s) can further comprise a sequence or sequences em::oding components of the RNA scaffold. In general, the DNA 28 Date Re9ue / Date Received 2022-07-19 sequence encoding the protein and the RNA scaffold is operably Hnked to appropriate promoter control s.equenoes that all.ow the expression of the protein and the RNA scaffo]d, 1,especfrvely,. in the cell or embryo. The DNA sequence ,encoding the protein and the RNA scaffold can further comprise additional expression control, re1:,1Ulatory, and / or processing 5, sequence(s). The DNA sequence encoding the protein and the guiding RNA can be linear or can be part of a vector. In embodiments in which the RNA is introduced into the ceU via a DNA molecule encoding the RNA,. the R.NA coding sequence can be operably linked to promoter control sequence for expression of the guiding RNA in the eukaryotic cell. For example, the RNA l O coding sequence ,can be operably linked to a promoter sequence that is recognized by RNA polymerase III (Pol 1m. Examples of suitable Pol III promoters include, but are not Hmited to. mammalian U6 or Hl promoters. In exemplary embodiments. the RNA coding sequence is linked. to a mouse or human U6 promoter. ln other exempJary embodiments, the RNA ooding sequen,ce is linked to a mouse or human Hl promoter. 1 :5 The DNA molecule encoding the protein and / or RNA can be linear or circular. In some embodiments, the DNA sequence can be part of a vector. Suitable vectoi:s include plasmid vectors, phagemids, cosmids, artificial / mini-chromosomes, transposons,. and viral vectors.. Jn an ex.emplary embodiment, the DNA encoding the protein and / or RNA is present in a plasmid vector. Non-limiting examples of suitable plasmid vectors include 20 pUC, pBR322, pET, pB[uescript, and variants thereof. The vector can comprise additional expression controI se,quences (e .. g., enhancer sequences, Kozak sequenc,es, polyadenylation sequences, transcriptional termination sequences, etc.), selectable marker sequences (e.g., antibiotic resistance genes), origins of replication, and the like. The proteins components of this system of this invention (or nucleic ad.d(s) 25 encoding them) and the RNA components (or DNAs encoding them) can be introduced into a cen or embryo by a vari,ety of means. Typically., the embryo is a fertilized one-ceU stage ,embryo of the species of interest. In some embodiments. the cell or embry,o is transfected. Sui.table transfecti.on methods include calcium phos:phate-mediat,ed ttansfection, nucleofection (or electroporation), cationic polyme.r transfection (e.g., 30 DEAE-dextran or poty,ethylenimine), viraJ transduction, virosome transfecti.on,. virion transfection, liposome transfection, cationic liposome transfection,. immunoliposome b·ansfection, nonliposomal lipi.d transfection, de11drhner transfection, beat shock 29 Date Re9ue / Date Received 2022-07-19 transfection, magnetofection, lipofection, gene gun delivery, impalefection, sonoporation, optical transfec:tion, and proprietary agent-enhanced uptake of .11uclei.c acids. Tr,ansfection methodls are well known in the art (see, e.g., "Current Protocols in Mofo,cular Biology" Ausubel et at .. , John Wiley & Sons, New York, 2003 or ''Molecular Cloning: A Laboratory 5, Manual 1 ' 1 ' Sambrook & Russell., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.., 3rd edition,. 200] ). 1n other embodiments, the molecules are introduced into the cell or embryo by miicroinjection .. For example, the molecules can be injected into the pr,onuc~ei of one cell embryos. The jproteins components of this system of this invention (or nucleic acid(s) 10 encoding them) and the RNA components (or DNAs encoding them) can be introduced i.nto a ceH 0:r embryo simultaneously or sequentially. The ratio of the protei.n (or its encoding nucleic acid) to the RNA (or DNAs encoding the RNA), generally will be approximately stoichiom.etric such that they can form an RNA-protein com.pl,ex. Similady. the ratio of two different proteins (or encoding nucleic acids) will be 1 :5 approximately sto,i,chiometrk fo one embodiment, the protein components. and the RNA components (or the DNA sequence encoding them) are delivered together within the same nucleic acid or vector. The method further comprises maintaining the cell or embryo under appropdate c,onditions such that the guide RNA guides the effector protein to the targeted s:ite in the 20 target sequence, and the effector domain modifies the target sequence. In ge:1J.eral, tbe cell can be maintained under condition.s ap,propriat:e for cell gro:wth and / or maintenance. Suitable ceH culture conditions are, well known in the art and are described, for example, in Current Protocols in Molecular Biology11 Ausube1 et al., John Wiley & Sons, New York, 2003 or 11Molecular Cloning: A Laboratory Manual" Sambrook 25 & RusseU, Cold Spring Harbor Press, Cold Spring Harbor., N.Y., 3rd edition, 2001), Santiago et al .. (2008) PNAS 105:5809-5814; Moehle et al. (2007) PNAS 104:3055-3060; Umov et al. ,(2005) Nature 435:646-651; and. Lombardo et al. (2007) Nat. Biotechnology 25:.1298-1306. Those of skill. in the art appreciate that methods for culturing cells are known in the art and can and wHI vary depending on the cell type. Routine op:timizati,on 30 may be used,. in an ,cases,. t,o determine the best techniques for a particu]ar c,ell typ,e. An embryo ,can be cultured in vitro (e.g., in cell culture), Typically, the embry,o is cultured at an appropriate temperature and in appropriate media with the neces.s.ary 30 Date Re9ue / Date Received 2022-07-19 Oi / CO2 ratio to allow the expression of the proteins and RNA scaffold, if necessary .. Suitable non-limiting examples of media include M2~ Ml 6, KSOM, BMOC, and HTF media. A skiUed artisan will appreciate that culture conditions can and will va1y depending on the species of embryo. Routine optimization may be used,. in all cases, to 5, detennine the best culture condition.s for a particular species of embryo. In some cases, a cell line may be derived from an in vitro-cultured embryo (e.g., an embryonic stem ,c,elft Une). AHemative].y, an ,embryo may be cultured in vivo by transferring the embryo into a uterus of a fern ale host. Generally speaking, the female host is from the same or similar 10 species as the embryo. Preferably, the female host is p.seudo-pregnant Methods of preparing pseudo-pregnant female hosts are known i.n the art AdlditionaHy. methods of transferring an embryo into a female host are known. Culturi.ng an embryo in vi110 permits the embryo to deve.l.op and can result in a live birth of an animal derived from th,e ,embryo. Such an animal would comprise the modified chromosomal sequence in every cell of the 15 body. A variety of eukaryotlc cells are suitable for use in the method.. For example, the cell can be a human cell, a non-human marnmalian cell,. a non-mammalian vertebrate cell, an inve:rtebratie cell, an insect cell, a plant cell, a yeast cell, or a single ceJl eukaryodc organism. A vari.ety of embryos are suitable for use i.n the method. For example, the 20 embryo can be a 1-ceU, 2-cell, or 4-cell human or non-human mammalian embryo. Ex.emplary mammaHan embryos, including one cell embryos, include without limit mouse, rat,. hamster,. rodent, rabbit, feline, canine, ovine, porcine, bovine, equine, and primate embryos. In stm other embodiments, the cell can be a stem celt Suitable stem cells include without limit embryonic stem cells, ES-1ike stem cells, fetal s;tem cells, a:duh stem 25 cells, pluripotent stem ceUs, induced pluripotent stem cells,. multipotent stem cells, oligopotent st,em cells, unipotent stem cells and others. In exemplary embodiments. the cell is, a mammalian cell or the embryo is a mammalian embryo. Utilities and Applfoations The systems and methods disclosed herein have a wide vadety of utiliti.es 30 including modifying and editing (e.g;., inactivating and activating) a target polynudeotide 31 Date Re9ue / Date Received 2022-07-19 in a multiplicity of cell types. As such the systems and methods have a broad spectrum of applications in, , research and therapy. Many devaslating human diseases have one common cause: genetic alteration or mutation. The disease~,causing mutations in patients are either acquired through 5, i.nheritance from their parents or are caused by environmental, factors. These diseases include., but are not limited to, the following categories. First, some genetic disorders are caused by g:ennline mutations. One example is cystic fibrosis, which is caused by mutations at the ClFTR gene inherited from parents.. Second, some diseases, such as chronic viral infoctious diseases, are caused by exogenous environmental factors and 10 resulting genetic alt,emtions.. One example is AIDS, which is caused by insertion of the human HIV vim] genome into the genome of infected T-ceUs. Third, some neurodegenerative diseases involve genetic alterations. One example is Huntington's diseases. which is cause by expansion of GAG tri-nucleotide in the hu:n.tin.gtin gen.e of affected pati,ents. Finally, cancers are caused by various somatic mutations accumulated 1 :5 in cancer cells. Th,er,efore, correcting the disease-causing genetic mutations, or fi.1.nctiunally correcting the sequence, provides am. appea.ling therapeutic opportunity to treat these diseases. :Somatic ge11edc editing is an appealing therapeutic strategy :for many human diseases. To achieve successful therapeutic genetic editing, three critical factors are 20 considered essential!: (i) how to achieve sequence specific recognition ("sequence recognition modul,e"); (ff) how to correct the underlying mutations ("correction. m.odule"); and (Hi) how to Hnk the "correction module" to "sequence recognition module" together to achiev,e sequence specific correction. There are number of ways of achieving each individual task. Howev,er, none of the currently e~isting platfonns or technologies could 25 achieve optimal and pra.ctical somatic genetic editing. More specifically, current gene specific editing technologies are mostly based on nucleases induced DNA DSB and consequent DSB induced homologous recombination, the activity of which is Row or absent in most somatic ceUs. Thus, those technologies are of limited use for th,erapeutic c,orrections ofpathofogic.al genetic mutation.sin somatic tissues in most diseases, 30 In contrast, the system and method disclosed in this invention all.ow DNAsequence directed. ,editing of a gene or RNA transcript that does not rely on nudease activity. Th.e sy:st,ern and method do not generate DSB, or do 11.ot rely on. the DSB- 32 Date Re9ue / Date Received 2022-07-19 mediated homologous r,ecombination. Moreover, this design of the system is modular, which allows extremely fliexible and convenient way of targeting any desirable DNA or RNA sequences. In essence, this approach enables one to guide a DNA or RNA editing enzyme to virtually any DNA or RNA sequence in somatic cells, including stem cells. 5, Through precise editing th.e target DNA ,or RNA sequence, the enzyme can correct the mutated genes in genetic disorders, inactivate the viral genome in the infected ceUs, el.iminate the expression of the disease~causing protein in neurodegenera:tive diseases, or silence the oncogenic protein in cancers.. Accordingly, the system and method disclosed in this invention can be used in correcting underlying genetic alterations in diseases 10 i.ncluding the above mentioned genetic disorders:, chronic infectious disea:ses, neurodegenemtive disea:s,es, and cancer. Genetic Diseattes It is es.timat,ed that ov,er six thousands of genetic diseases are caused by known genetic mutations. Correcting the underlying disease causing mutations in the [5 pathological tissues / organs ca:n provide alleviation or cure to the diseases. For example. cystic fibrosis affects lout of ,every 1,000 people in the US. It is caused by inheritance of a mutated CFTR gene and 70%i of the patients have the same mutation, deletion of a trinucleotide leading to a deletion of phenylalanine at position 508 (ca11edl A Phe 508). A Phe 508 leads to th.e mis.location and degradation of CFTR The system and method .20 disclosed this invention ca:11 be used to co.overt a Val 509 .residue (OTT) to Phe 509 (TIT) in affe,ct:ed tissues (lung), thereby functionally correct th.e A Phe 508 mutation .. Chronic 1rifectious Diseases The :sy:sten1 and method disclosed in this invention can also be used to specifical.ly inactivate any gene in a viral genome that is incorporated into human ceUs / tissues. For 25 exampl.,e, the system and method disclosed in this invention allow one to create a stop codon for early termination of translation of the essential vira] genes, and thereby remediate or cure the chronic debilitating infectious diseases. For example, current AIDS therap,ies. ca:n reduce virid toad, but cannot totally eliminate dormant HIV from positive 'T cells. The system and m.ethod disclosed herein can be used to permanent]y inactivate one 30 or two essential H[V gene expression in the integrated HIV genome in human T-cells by i.ntrodu.ci.ng on.e or two stop codons., Another example is hepatitis B vims (HBV). The 33 Date Re9ue / Date Received 2022-07-19 system and method disclosed here can be used to specifically inactivate one or two essential HBV genes,. which are incorporated into human genome, and sil.ence HBV lifocycle. Neurodegene.rative Diseases S Some neurodegenerative diseases are caused by gain-of-function mutatfons. For exampl,e, SOD1G93A leads to development of amyotmphic laterall sclerosis (ALS). The system and method disclliosed in this invention can be used to either correct the mutati,o.n; or eliminate the mutant protein expression by introducing a stop codon or by changing a splicing site. l O Cam::.e.r:s Many genes (including tumor suppressor genes, oncogenes. and DNA repair genes) contribute to the development of cancer. Mutations in these gen.es often Read to various cancers. Using the system and method disclosed in thi.s invention, one can specificaHy target and correct these mutations.. As a result, causative onc:ogenic proteins 15 can be functionaHy .annuUed or their expression can be eliminated by introducing a point mutation at either the catalytic sites or splicing sites. Stem Cell Genetic Modification In some embodiments, stem cell or progenitor cell can be genetically modified using the system and method disclosed in this invention. Suitable cells include, ,e.g., stem 20 cells (adult stem ,eel.ls, ,embryonic stem cells, iPS cells. etc.) and progenitor ceU:s (,e.g., cardiac progenitor cells, neural progenitor cells, etc.). Suitable ceHs include mammalian stem ceUs aind progenitor ,cells, including, e.g., rodent stem c:eHs, rodent p,rogenitor c,eUs, human stem cells,. human progenitor cells, etc, Suitabl,e host ceUs include in vitro host cells, e.g., isolated host ceUS:. 25 In some embodiments, the present invention can be used for targeted and precise genetic modification of tissue ex vivo, correcting the underlying genetic defects. After the ex vivo correction, the tissues could be returned to the patients, Moreover,. the technology can be broadly used in ceU-based therapies for correcting gen,etic diseases. 34 Date Re9ue / Date Received 2022-07-19 Genetic Editing In Anim,lls and Plants The system and method described above can be used to generate a tram1genic nonhuman animal or plant having one or more genetic modific.ation of interest In some embodiments, the transgenic non-human. animal is homozygous for the genetic S modification. In some embodiments, the transgenic non-human animal is het,erozygous for the genetic modification. Jn some embodiments, the transgenic non-human animal is a verteb.rate, for exa.mple, a fish (e.g., zebra fish. gold fish, pufler fish, cav,e fish, etc.), an amphibian (frog, salamander, etc.}, a bird (e.g., chicken., turkey, etc.), a reptile (e.g., :snak:e, lizard, etc.), a mammal (e.g., an ungulate, e.g., a pig, a cow, a goat, a :sheep, etc.; a 10 l.agomorph ~e.g., a rabbil); a rodent (e.g., a rat, a mouse); a non-human primarte. The invention can be used for treating diseases in animals in a way similar to tbose for treating disease;s in humans as described above. Alternatively. it can be used to generate knock-in animal disease models bearing specific genetic muta:tion. for purposes of research, drug discovery, and target validation. The system and method described above l 5 can al.so be used for introduction of point mutations to ES ceHs or embryos of various organisms. for purpose of breeding and improving animal stocks and crop ,quality. Methods of introducing exogenous nucleic acids into plant c:eUs are wen known in the art Suitable methods .include viral infection (such as double stranded DNA viruses), transfectio.n, conjugation, protoplast fusion. electroporation, particle gun tech:noJogy, 20 calcium phosphate predpitation, direct microinjection. silicon carbide whisk,ers technology. Agrobacterium-mediated transfonnation and the like .. The choice of method is generally dependent on the type of cell being trnnsform.ed and the drrumstanc,es und,er which the transformation is taking place (i.e. in vitro, ex vivo, or in v1110), Kit 25 This invention further provides kits containing reagents for performing the above·- d'.escribed methods, including CRISPR:Cas guided target binding or conection. reaction. To that end,. one or mor,e of the reaction components, e.g., RNAs:, Cas proteins, fusion etlbctor proteins and r,elated nucleic acids, for the methods disclosed herein can be supplied in th.e form of a kit for use, 1n one embodiment., the kit comprises a CRISPR 30 protein or a nuc1eic acid encoding the Cas protein, effector prot.ein, one or more of a RNA scaffold des,cribed above, a set of RNA molecules described above. In others 35 Date Re9ue / Date Received 2022-07-19 embodiments., the kit can include one or more other reaction components. In such a kit, an appropriate am1ou11:t of one or more reaction components is provi.ded in one or more containers or head on a substrate. Examples of additional components of the kits include, but are not limited to, one 5, or more host ce.lls, one or more reagents for introducing foreign nucleotide :sequences i.nto host cellls, one or more reagents (e.g., probes or PCR primers) for detecting expression of the RNA or protein or verifying the target nucleic acid''s status, and buffers or culture media for the reactions (bu lX or concentrated fonns).. The kit may also include on,e or more of the foUowing components: supports, terminating, modifying or digestion reagents, l O osmolytes., and an apparatus for detection. The :reaction components used can be provided in a variety of forms.. For example, the components (e.g .• enzymes, RNAs, probes and / or primers) can be suspended in an aqueous solution. or as a freez.e-dried or lyophilized powder, peHet, or bead. In the latter case, th,e components., when reconstituted, form a complete mixture of components for use 1:5 in an assay. The kits of the invention can be provided at any suitable temperature. For ex.ample, for storage of kits containing protein components or complex.,es thereof in a liquid, it is prefened that they are provided and maintained below 0°C, preferably at or below -20°C,. or otherwi:s,e in a frozen state. A kit or sy:stem may c,ontain, in an amount sufficient for at least one as:say, .any 20 combination of the components described herein. In some app]ications, one or more reaction components. ma:y be provided in pre-measured single use amounts in individual, typicaUy disposable) tubes or equivalent containers. With such an arrangement, a RNAguided reaction can be performed by adding a target nucleic acid,. or a sample or ,c,eU containing the target nucleic acid, to the individual tubes directly. The amount of a 25 component supplied in the kit can be any appropriate amount and may depend on the target market to which the product is directed. The container(s) in which the components are supplied can be any conventional contai.ner that is capable of holding the :supplied fonn, for instance, microfoge tubes, microtiter plates,. ampoules, bottles, or integral testing devices,, such as fluid:ic devices, cartridges, lateral flow. or other similar devices .. 30 The kits can abo include packaging materials for holding the container or combination of containers. Typical packaging materials for such kits and systems inclllde solid matrices (e.g .• gla:s:s, plastic, paper, foil, micro-particles and the like) th.at bold the 36 Date Re9ue / Date Received 2022-07-19 reaction components or detection probes in any of a va.riety of configurations (e.g., in a vial, mi.crotiter plate well, rnicroa:rra:y, and the like). Tb.e kits may further include instructions recorded in a tangible form for use of the components. Definition S A nucleic acid or polynucleotide refers to a DNA molecule (for exampl,e, but not limited to, a d)NA or genomic DNA) or an RNA molecule (for example, but not limited to, an mRNA), and includes DNA or RNA analogs, A DNA or RNA .analog can be synthesized from nucleotide analogs. The DNA or RN A molecules may include portions that are not naturally occurring, such as modified bases, modified backbone, l O d.eoxyribonudeotides in an RNA, etc.. The nucleic acid molecule ca11 be sin.gle-stra.nded or double .. stranded. The term ''i.:solated" when referring to nucleic acid molecules or polypeptides means that the nudeic acid molecule or the polypeptide is substantially free from at least one other component with which it is associated or found together in nature. [5 As used berein, the term "guide RNA" generally refers to an RNA mol,ecufo (or a group of RNA molecules collectively) that can bind to a CRISPR protein and target the CRISPR protein to a specific location within a target DNA. A guide RNA can comprise two segments: a DNA-targeting guide segment and a protein-binding segment. The DNAtargeting segment compri:ses a nucleotide sequence that is complementary to (or at least 20 can hybridize to under ·stringent conditions) a target sequence. The protein-binding segment interacts with a CRIS]>R protein, such as a Cas.9 ,or Cas9 related polypeptide. These two segments can be located in the same RNA molecule or in two or more separate RNA molecules. When. the two segments are in sepaliate RNA molecules,. the molecule compris.ing the DNA-targeting guide segment is sometimes referred to as the CRISPR 25 RNA (crRNA), while the molecule comprising the protein-binding segment is referred to as the trans-a,ctivating RNA (tracrRNA). As used herein, the term "target nucleic add" or "target" refers 10 a nucleic acid containing a target nucleic acid sequence. A target nucleic acid may be si:ngle-s1randed or double-stranded, andl oft,en is double-stranded DNA A "target nudeic acid sequence," 30 ''target sequence" or "target region," as used herein, means a specific sequence or the complement thereof that ,one wishes to bind! to or modify using a CRISP.R system. A 37 Date Re9ue / Date Received 2022-07-19 target sequence may be within a nucleic acid in vitro or 111 vivo within the genome of a cell, which may be any form of single-stranded or double-stra11ded nrudeic acid. A "target nucleic acid strand" refers to a strand of a target nucleic acid tha:t is subject to ba:se-pairing with a guide RNA as disclosed herein.. That is, the strand of a 5, target nuclek acid that hybridizes with the crRNA and guide sequence is referred to as the "target nucleic acid strand." The other strand of the target nucleic acid, which is not complementary to the guide sequence, is referred to as the ''non-complementary strand!' In the case of double~straoded ta:rget nucleic acid (e.g., DNA), each strand can be a "ta:rget nucleic acid strand'' to design crRNA and guide RNAs and used to practice the method of 10 this invention as long as there is a suitable PAM site. As used herein, the term "derived from'1 refers to a process: whereby a first component ,(e .. g .• a first molecule), or information from that first component, i.s used to i.soiate,, derive or make a different second component (e.g., a second molecule that is different from tbe first). For example, the mammalian codon-optimi21ed Cas9 1 :5 polynuc1eotides a:r,e deriv,ed from the wild type Cas9 protein amino acid sequence. Also, the vuiant mammalian codon-optimized Cas9 polynucleotides, including the Cas9 sing]e mutant nickase (nCas9, such as nCas9D10A) and Cas9 double mutant nuU-nudease (dCas9, such as dCas9 DlOA H.840A), are derived from the polynutdeotide enc,oding the wild typ,e mammalian codon-optimized Cas9 protein. 20 As used herein th,e term '"wild type" is a term of the art understood by skilled p,ersons and means the typical form of an organism, strain, gene or characteristic ,a:s it occurs in nature a:s distinguished from mutant or variant forms, As used herein,. the term "variant" refers to a fitst composition (e.g., a first molecule), that is related to a second composition (e.g., a second molecule, also tenned a 25 1' 1'pairent11 mole,cule). The variant molecule can be derived from, is:o]ated from,, based on or homologous to the parent molecule, For example, the mutant forms of mammalian codonoptimized Cas9 (hspCas9), including the Cas9 single mutant nickase and th,e Cas9' doubJe mutant nu1l-uucleas,e, are variants of the mammalian codon-optimized wild type Cas9 (hspCa.s9), The tenn variant can be used to describe either po]ynucleotides or 30 polypeptides. As applied to polynucleotides, a variant molecule can have entire m1dootide sequence identity with tbe original parent molecule, or alternatively1 can have l,es.s tban 38 Date Re9ue / Date Received 2022-07-19 100% nucleotide s,equence identity with the parent molecule .. For example, a variant of a gene nucleotide sieque.nc,e can be a second 111,tcleotide sequence that is at least SOi0 / 4, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical in nucleotide sequence ,compare to the original nudeotide sequence.. Polynucleotide variants also include polynudeotides 5, compris.ing the entire parent polynucleoti.de, and further campris.ing additional fused nucleotide sequences. Polynucleotide variants also includes polynucleotides that are portions or subsequences of the parent polynucleotide, for exampl.e, unique subsequences (e.g., as determined by standard sequence comparison and alignment techniques) of the polynucleotides disclosed herein are also encompassed by the invention. 10 In a:rnother aspect, polynucleotide variants include nuc]eodde sequences that contain minor,. trivial or inconsequential changes to the parent nucleotide ,seque:nce. for example, minor, trivial or inconsequential changes include changes to nucleotide sequence that (i) do not change the amino acid sequence ofthe corresponding polypeptide, (i.i) occur outside the pmtein~coding open reading frame of a polynucleotide, (iii) result in deletions 1 :5 or insertions that may impact the corresponding amino acid sequence, but have Btde o,r no impact on the biologi.cal activity of the polypeptide1 (iv) the nucleotide changes result i.n the substitution of an amino acid with a chemically similar amino acid. In the case wher,e a polyn:ucleotide does not ,encode for a p.rotein (for example, a tRNA or a crRNA or a tta.tTRNA), variants of that polynucleotide can include nucleotide changes. that do not 20 result in loss of function of the polynucleotide. In another aspect, conservative variants of the discl.osed nucleotide s,equences that yield functionally identical nucleotide s.equences are encompassed by the invention. One of skill will appreciate that many variants of the disclosed nucleotide sequences are encompassed by the invention. As applied to proteins, a variant polypeptide can have entire amino acid s,equence 25 identity with the original parent polypeptide, or alternatively, can have .less than 100% amino acid identity with the parent protein. For example, a variant of an amino acid sequence can be a second amino acid sequence that is at least 50%, 600111, 70%, 80%., 90%, 95%, 98%, :99% or more identical in amino acid sequence compared to the original amino acid sequence .. 30 Polypeptide variants include polypeptides compri1sing the enti.re parent polypeptide, a:nd further comprising additional fused amino acid sequenc,es. Polypeptide vadan.ts also includes polypeptides that are portions or subseque11ces of the parent 39 Date Re9ue / Date Received 2022-07-19 polypeptide, for exampl.e, unique subsequences (e.g., as determined by standard sequence comparison and aHgnment techniques) of the polypeptides disclosed herein are also encompassed by the invention . . In another a:spect, polypeptide variants include polypeptides that contain. minor, 5, trivial or inconsequeutial changes to the parent amino acid sequeuce. For examp].,e, minor, trivial or inconsequential changes include amino acid changes (in.duding substitutions, deletions and insertions), that have little or no impact on the biological activity of the polypep,tide, and yield functionally identical polypeptides, including additions of nonfunctional peptide sequence. In other aspects, the variant polypeptides of the invention l O change the biological activity of the parent m.olecul.e; for example, muta.nt variauts of the Cas9 polypeptide tl:u1t hav,e modified or lost nuclease activity. One of skiU will appreciate that many variants of the disclosed polypeptides are encompassed by the invention. In som.e aspects, polynucleotide or polypeptide variants of the invention can include variant molecules that alter, add or delete a small percentage of the nucJeotide or 1 :5 amino acid positions, for example, typically less than about l 0%, less than about 5%, less than 4%,. less than 2% or less than 1 %. As used herein, the term "conservative substitutions" in a nucleotide or amino acid sequence refers to changes in the nucleotide seque11ce that ei.ther (i) do not result in any corresponding change in the amino acid sequence due to the redundancy of the trip,]et 20 codon code, or (ii) result in a substitution of the original parent amino acid with a:n amino acid having a chemically similar structure. Conservative substitution tables providing functiiomdly similar amino acids are well known in the art, where one amino acid residue is substituted for another amino acid residue having similar chemical properties (e.g., aroma:tiic side chains or positlve]y charged side chains),. and. therefore does not 25 substantially change the functional properties of the resulting polypeptide molecule. The following are groupings of natural amino acids that contain similar chemical properti,es, where a substitution within a group is a "conservative" amino acid substitution. This grouping indicated below is not rigid, as these natural amino adds can be placed in different gmuping when different functional properties are considered,. Amino acids 30 having nonpolar a:nd / or al.ipbatic side chains include:. glycine, alanine; valine,. leucine, iso]eucine and proline. Amino acids having polar, uncharged side chains include: serine, threonine, cysteine, methionine, asparagi.ne and glutami11e. Amino acids having aromatic 40 Date Re9ue / Date Received 2022-07-19 side chains include: phenylalanine, tyrosine and tryptophan. Amino acids having positively charged side chains include: lysiue, arginine and histidine. Amino ac:ids: having negatively charged side chains include: aspartate and glutamate. A "Cas9 mutant" or "Cas9 variant" refers to a protein or polypeptide derivative of 5, :the wild type Cas9 protein such as S. pyoge.nes Cas9 prot.eiu (i..e., SEQ ID NO: 1), e.g., a protein having one or more point mutations, insertions, deletions, truncations,. a fusion protein, or a combination tlhereof. It retains substantially the RNA targeting activity of the Cas9 protein. 'The prot.ein or polypeptide ca:n comprise, consist of, or consist ess,entially of a fragment of SEQ ID NO: 1. In general, the mutant / variant is at least 50% (e.g., any l 0 number between 50% and 100%, inclusive) identical to S.EQ 1D NO: l. The mutant / variant can bind to an RNA molecule and be targeted to a specific DNA sequence via the RNA molecule, and may additional have a nuclease activity. Examples of these d.omai.ns include RuvC Iike motifs (aa. 7-22, 759-766 and 982-989 in SEQ ID NO: 1) and HNH motif (aa 837-863). See Gasiunas et al., Proc Natl Acad Sci U S A. 2012 15 September25; 109(39): E2579-E2586 and W02013176772. "'Complementarity11 refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base-pairing or other r10n-traditio11al types. A percent complementarity indicates: the percentage of residues in a nuclek acid molecule which can form hydrogen bonds (e.g., Watson-Cri.ck 20 base pMring) with a second nucleic acid sequence (e.g., 5, 6, 7,. 8, 9, 10 out of 10 being 500 / 4, 60%, 7'0%, 80%, 90%, and 100% complementary). 11Perfecdy complementary" means that aU the contiguous residues of a nucleic acid sequence wiU hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. ''SulbstantiaUy complementary'' as used herein refers to a degree of complementarity that is at least 60%, 25 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, IO. 11, 12, l3, 14,. lS, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,. 40, 45, 50, or more nucleotides, or ref en to two nucleic acids that hybridize under stringent conditions. As used herein, ''stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly 30 hybridizes with tbe target sequence, and substantially does not hybridiz,e to non-target sequences. Stringent conditions are generally sequence-dependent, and vary depending on a number of factors.. In general, the longer the sequence, the higher the temp,erature at 41 Date Re9ue / Date Received 2022-07-19 which the sequen,ce sp,ecifically hybridizes to its target sequence . Non-Jimititrig examples of stringent ,conditions are described in detail in Tijsse11 (l.993),. Laboratory Techniques In Biochemistry And Mol,ecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter "Overvi,ew of principles of hybridization and the s.trategy of nucleic acid 5, probe assaf\ Elsevi.er,. N.Y. "Hybridization" or "hybridizing" refers to a process where compfotely or partially complementary nucleic acid strands come together under specified hybridization conditi.ons to form a double-stranded structure or region in. which the two constituent strands are joined by hydrogen bonds. Although hydrogen bonds typically fonn between 10 adenh1e and thymine or uracil (A and Tor U) or cytosine and guanine (C and G), other base pairs may form (e.g .. , Adams et al., The Biochemistry of the Nucleic Acids, l lth ,ed., 1992), As used hell'ein, 11ex:pression" refers to the proc,ess by which a polynucleotide is tra.nscribed from a DNA template (such as into and mRNA or other RNA transcript) 1:5 and / orthe process by which a transcribed mRNA is subsequently translated into peptid,e,s, polypeptides, or protei.n.s,, Transcripts and encoded polypeptides may be collectively referred to as "gene product." ff the polynucleotide is derived from genomic DNA, expres.si.on may include :splicing of the mRNA in a eukaryotic cell. The terms 1 '1'polypeptide!!, 11peptide11 and 11protei.n 11 are used interchangeably herein 20 to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modiified amino acids, and it may be inte.rrupted by non-amino ,acids. The terms also ,encompass an amino acid polymer that has been modified; for example, disulfide bond fonnation, glycosylation, lipidation, acetylation, phosphorylation, pegyla:don, or any other manipulation, such as conjugation with a labeling ,component. As 25 used herein the term "amino acid0 includes natural and / or unnatural or synthetic amino acids. including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. The term ''fusion polypeptide" or "fusion protein11 means a protein created by joining two or more polypeptide sequences together. 'The fusion polypeptides 30 encompassed in th.is inv,ention include translation products of a chimeric gene ,construct that joins th,e nucleic acid sequences encoding a first polypeptide, e.g., an RNA~binding domain,. with the nucle.i.c add sequence encoding a second pol.ypepfide, e.g., an effector 42 Date Re9ue / Date Received 2022-07-19 domain, to fonn a single open~reading frame. In other words, a "fusion polyp,eptide'" or "fusion pmteii1111 is a recon1binant protein of two or more proteins which are joined by a peptide bond or via several peptides. The fusion protein may also comprise a peptide linker between the two domains. 5, The tierm '11'li.nker1' 1 refers to any means, entity or moiety used to join two ,or more entities. A Hnker can be a covalent linker or a non-covalent linker. Examples of covalent Unkers include covalent bonds or a linker moiety covalently attached to one or more of the proteins or domains to be linked. The linker can also be a non-covalent bond, e.g;,. ,an organometaHic bond thrnugh a metal center such as platinum atom. For covalent linkages, 10 various functionaliti.es ,can be used, such as amide groups, includin.g carbo.nic aci.d derivatives, ethers, esters, including organic and inorganic esters. amino, urethane, urea and the Hke. To provide for linking, the domains can be modified by oxidation, hydroxyl.atiom., sub,stitution. reduction etc. to provide a site for cou.pling. Methods for conjugation are weU known by persons skilled in the art and are encompassed for use in 1:5 the present invention. Linker moieties include, but are not limited to, chemical linker moieties,. or for example a peptide linker moiety (a linker sequeo.ce). It will be app,reciated that modification which do not significantly decrease tl1e function of the RNA-binding domai11 and ,effector domain. are prefe11·ed. As used herein, the term 11conjugate11 or "conjugation''' or 11linked11 as used herein 20 refers to th,e attachment of two or more entities to fonn one entity. A ,conjugate encompasses both peptide-small molecule conjugates as well as p,eptide~protein / peptide conjugates. The tenns 11 subje,ct11 and "patient" are used interchangeably herein to refer to a vertebra:te, preferably a mammal, more preferably a human. Mammals include, but are not 25 limited to, murines, simians, humans, fann animals, sport animals, and pets. Tissues, ceUs and their progeny of a biological entity obtained in vivo or cultured in vitro are also e:ncompassed., In some ,embodiments, a subject may be an invertebrate animal. for exampl,e, an insect or a nematode; while in others, a subject may be a pfant or a fungus. As used herein, '1 '1treatment'1 or "treating," or 11palliating" or 11mnelior,ating" are 30 used intercha1111geably .. Th,ese terms refer to an approach for obtaining beneficial ,or desir,ed results: including but not limited to a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on. ,one 43 Date Re9ue / Date Received 2022-07-19 or more diseases, condition.s, or symptoms under treatment. For prophylactic benefit, the composition.s may be administered to a subject at risk of developing a particular disease, condition, or symptom, ,or to a subject reporting one or more of the physiological symptoms of a dis,ease, even though the disease, condition, or symptom may not have yet 5, been manifes.t,ed. As used herein, the term "contacting," when used in reference to any set of components,. includes any process whereby the components to be contacted! are mixed into same mixture (for e'Kampl,e, are added into the same compartment or solution), and does not necessarily require actual physical contact between the recited components. The l O recited components can be contacted in any order or any combination (or sub~ combination), and can include situations where one or some of the re:eited components. are subsequently removed from the mixture, optionally p.rior to addition of 0th.er recited components,.. For exampl.e, "contacting A with B and C" includes any and aU of the following situations: (i) A is mixed with C, then Bis added to the mixture; (fi) A and Bare 1:5 mixed into a mixturn; Bis removed from the mixture, and then C is added to th.e mixmre; and (iii) A is added to a mixmre of B and C., "Contacting" a. ta.rget. nucleic add or a ceU with Ollie or more reaction components, such as an Cas protein or guide RNA,. includes any or aU of the fol.lowing situation.s: (i) the target or cell is contacted with. a first co.mponent of a reaction mixture to create a mixture; then other components of the reaction mixture 20 are added in any order or combination to the mixture; and (ii) the reaction mixtur,e i:s fully formed prior to mi.xture with tb,e target or ce!L The t,enn "mixture" as used herein, refers to a combination of elements, that are interspersed and not in any particular order. A mixture is heterogeneous and not spatially separable into its different constiments. Examples of mixtures of elements include a 25 number of different elements that are dissolved in the same aqueous solution, or a number of different elements attached to a solid support at random or in no particular order in which the diffe.rent elements are not spatially distinct.. In other words. a mixture is not addressable. As disclosed her,ein, a number of ranges of values are provided. It is understood 30 that each intervening value, to the tenth of the unit of the lower limit, unl.ess the context clearly dictates otherwise, between the upper and lower limits of that range iis also speci:6.cally dis.clos,ed. Baich smaller range betwee.1.1 any stated value or interve11.ing value 44 Date Re9ue / Date Received 2022-07-19 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 small.er mn.ges may independently be included or excluded in the range, and each range where eid1er, 11ehher, or both limits are included in the smaller ranges is also encompassed within the 5, i.nventi.on, subject to any specifically excluded limit in the stated ran.ge. Where the stated range includes one or both of the limits, ranges excluding either or both of those inclucfod Umits are also included in the invention. The term "about"' gene:nd]y refers to plus or minus, 10% of the i.ndicated number. For example.) "about 100 / 4" may indicate a rang:e of 9% to 11%, and "about 20" may mean from 18-22. Other meanings of "about" may be 10 appaJ'ient from the contex:t, such as rounding off: so, for example "about 1'' may also mean from 0.S to I.4. Example 1 CRC system lied to site-specific mutation at target Cytidh1e nucl.eotldes in bacterial genome. 15 In this ex:ample, E: coli MG1655 strain was used as model. Mutati.ons in bacterial RNA polymerase subunit ~ gene (rpoB) nmder cells resistant to the antibiotic rifampidn (Jin, et al., Jmunal of Molecular Biology 202, 45-58, (1988), and Goldstein, et al., J Antibiot 67, 625-630, do:i:10.1038 / ja.2014..107 (2014)). Mutants can be isolated and analyzed individuaHy, and mutation frequency can be calculated. AID is a B-cell specific 20 protein that belongs, to APOBEC family of cytidine deaminases: and is involved .in somatic ll,ypermuta.d.on and class :switch recombination during 1:mtibody diversification and afi:ii.nity matumtion (Odegard, et al., Nat Rev fmmunol 6, 573-583 (2006), and Noia, et al. Annual Re111ew t?f Biachemistry 16, l-22, doi:doi:10.11416 / annurev . biochem.76.061705.090740 (2007)). Thus., for these set of el!:perimen.ts, rpoB gene from coli MG1655 is target,ed 25 using AID as a notMtuclease effector protein .. Constructs and system co11figurations Inducible promoters AU protein-coding co.nstructs were designed under the control of a Tet inducible pmmoter. Anhydrotetracycline (ATc; Sigma) was used as inducer at a co11centra.tion of 30 30 nM. 45 Date Re9ue / Date Received 2022-07-19 Ca.v9 contttructs A central Feature of the present system is the introduction of preeis.e nucleotide modifications without generating DSBs. To this end, nuclease deficient versions of Cas9 were used as DNA targeting modules, namely catalytically deficient Cas9 (Cas90uwH840A, S dCa.s9) a.ind Cas9 ni,ckas,es. (nCas9ornA or nCas9u114oA) (Jinek, M. et al,, .Science 137, 8],6- 82], doi:]0.1126 / sdence. 1225829 (2012)). Cas9 nickases have been used to reduce offtarget DSB, by offset double DNA nicking (Ran, F.. A. et al., Cell 154, BS0.-.1389, doi:10.1016 / j.cell.2013.08.021 (2013)i and Shen., B. et al., Nat .Meth 11, 399-402, doi:10.1038 / nmeth.2857 (2014)) and dCas9 has been engineered to perform a variety of 10 activities independent of nuclease activity. See Fujit~ T. et al., Biochemictd ,1nd biophysical re~etirch commurric,,ticms 439, 132~136, (2013), Perez.-Pin~ P. et 1:11. Not Meth 10, 973-976, doi:10.1038 / nmeth.2600 (2013), Mali,. P. et al. Nat Biotechnol 31, 833- 838. doi:IOJ038 et alJnbt.2•675 (2013), Zalatan, J. G. et al., Cell 160, 339-350, doi:10.1016 / j.cell.2014.1 L052 (2015). Qi, L. S. et al,., Cell 152,. H73-U83, 15 dloi:I0..1016 / j,ce1L2013.02.022 (2013), Larson, M. H. ,et al., Nature protocols 8, 2180- 2196. doi:10..]038 / nprot.2013.132 (2013), Hilton, I. B. et al'., Nat Biotec:h 33, 510-517, dloi:10J038 / nbt.3199 (2015), Thakore, P. I. et al., Nat Meth 12, l 143-J 149, doi:10.1038 / nmethJ.63 (2015), Chen, .B. et tll., Cell 155, 1479-14'91, doi:lOJ0l6 / j,,cell.2013.12.001 (2013), and Fu, Y. et al., Nature communiccttions 1, 20 doi:IOJ038 / ncomms11707 (2016). Therefore, these variants are large]y considered safe and represented perfect candidates to develop the system presented in this :study. Targeted / '.ecruit,mmt system The system was engineered as an RNA scaffold-mediated recruitment platform. A schematfo re,pi:esentaitiou including schematic of constructs used in tllis study illustrated 25 in FIG. ]A Cas9 variants were designed as stand-alone constructs,. while gRNAs were engineered as chimeric RNA species in which phage RNA scaff ol.ds are synthetically fused to the 3' end of CRISPR RNA scaffold. Phage RNA scaffolds recruit :spedfic RNA binding proteins that are in tum tethered to non-nuclease effector proteins (FIG. lB). The RNA scaffo.ld recruiting system is derived from phage MS.2 and its interacting partner 30 MS2 coat protein (MCP). 46 Date Re9ue / Date Received 2022-07-19 Targeting gRNA The target is the bacterial rpoB gen.e. Mutations in three clusters, together call,ed rifampicin resistance-determining region (RRDR), confer the ceUs resistance to the antibiotic rifampicin (Ri~) (Goldstein, et al., J Antibiot 67, 625-,630, S doi:l0.1038 / ja.2014.107 (2014)). A set of four gRNAs was desi.gned to target criticaW amino acids along RRDR Cluster I sequence (i.e. S512, D5 l6, H526 and S531; FIG. 2A). Jin, et al., . / mm"Jal qf molecular biology 202, 45-58, (1988) and Jin, D, J. et al., MetJuxls in Enzymo,k,gy Vol. Volume 273 300-319 (Academiic Press, 1996) Experimental approach 10 Chemi,cally competent .E. cc)li MG1655 cells were transfonned with 10-20 ng of total DNA comprised of a combination of plasmids encoding for the constructs descrihed i.n Secti.on 1. After transformation, cells were selected and induced i.n Luria-Berlani broth containing the appropriate antibiotics, After selection / induction,. OD was measur,ed, ,cells were seriaUy diluted and 108 to 104 cells were plated in LB agar plates containing 15 rifampicin (120 μM). Two hundred cells were plated in selective agar plates without rifaimpidn for plating efficiency . After overnight incubation, colonies were counted ,and mutation frequency was scored. In addition, rpoB gene from i.solated colonies was amplified by PCR and sequenced in order to map muta:ti,ons. Results 20 1'argeted.recniitment of AJD led to site-spec{fi.c c:orwers'ioJ'I of C to 1'. A set of four gRNAs targeting .,poB's RRDR (cluster I) region was used to recruit AID to target sites (Fig .. 2A). CRC targeting with rpoB TS-4, and to a lesser ext,end with rpaB_TS-3, i.n.creased the survival fraction ofMG1655 cells in rifampicin media (Fig. 2B, 2C). Sequence analysis of clones derived from treatment rpoB TS-4 revealed high 25 specificity, mutating C 1592 to T, with the concomitant amino acid change from serine 531 to phenylalanine, a mutati.on known to render Rif cells (Peterse:n-Mabrt, et al., Natitre 418~ 99-104 (2002), Xu, M., et al., Jaurnal of Bacteriology 187, 2783-2792, doi: l0.1128 / JB.187.8.278.3-2792,2005 (2005). and Zenkin, N., et al., Antimicrobial Agents and Chemothen1py 49, 1587-1590, doi:10.1128 / AAC.49.4.1587-1590.2005 30 (2005)) (Fig. 20). The mutation distributions of rpoB_TS-3, rpoB_TS-4 and scr:amble are summarized in Fig. 2E. The highly increased mutation frequency observed and the 47 Date Re9ue / Date Received 2022-07-19 iocatio11 of modified nudeotide on treatment 1710B_TS.4, and with reduced efficiency on treatment. rpoB TS-3, suggest that the target cytosine must be positioned on the unpaired strand left by C:RISPR R-loop, preferentially closer to the 5' end of the pmtospacer (i.e. mutation fret1uency TS4>TS3, both targeting and modifying the same nucleotide, Figs. 5, 2A, 2C and 2E). Thi.s. is consistent with the notion that AID actiive]y deaminates cytosine residues on single strand DNA (Odegard, et aL, Nat Rev Jmrmmol 6, 573-583 (2006), Noia, et al., Annual Review of Biachemistry 76, l-22, doi:doi:10.1146 / annurev.biocbem.76.061705.090740 (2007), Smith, H C., et al., Seminars in Cell & Developmental Biology 23, 258-268, 10 doi:10.1016 / Jsemcdb.2011.l0.004 (20I2), and Ranganathan, V., et at, Nature communication. ., 5, doi:l0 .. l038 / ncomms5516 (2014)). A schematic representation of the targeting mod,el is shown .Fig. 2F. CRC .modularity Changing the targeting module from dCas9 to nCas9 nw8 increases efficacx of C 15 .to TIA conversion Changing the targ:eting module from dCas9 to nCas9moA increased the effi.ciency of the system in terms of survival fraction on rifampicin plates from 18 to 43 fo]d over the control (Fig. 3A). Mutadan analysis revealed the same specificity as in AIDCRC treatment for target nucleotide. ln this case C1592 was modified in 100% of the clones, 75% 20 mutated C to T and 25% mutated C to A (Fig. 3B). TiJ.!JM;lte4 reQDJitment Qf Qther DQn~ny.cleruie eff~g]:Qf$, APQBEC3G a:nd AP,QBECl. is able to introduce site-specific conversion of C to T / A 111 add.ition to AID as effector protein, we tested other cytid.ine deaminases from the APOBEC family, namely APOBEC3G and APOBEC:1 (Fig. 4A), APOBECl 25 i.ncreased the targeted mutation frequency compared to the prototype system, AI°CRCornAAPOBEC3G is less active than the prototype system. Mutation analysis: of AP1) 1CRCmoA treated cells with rpaB TS~4 as targeting construct showed 100% Cl 592>T conversion. In additi.on, 25% of amal.y.z.ed clones were double mutants, converting Cl S90>T, without amino acid cbange (Fig. 4B). 30 lncf\easing the number of RNA recruitment scaffolds enhances mu1a:tion fr,eguen,cy without alteri.ng_ s12,,ecificity of C to Tl A conversion. 48 Date Re9ue / Date Received 2022-07-19 Addfog tandem multimeric recruiting scaffolds could potentially increase effector presence on the target regi.on and therefore e11han.ce the system's efficiency. To this end, we engineeredl rpo:B TS-4 to include two MS2 loops (2xMS2). We compared the targeting efficiency between .rpoB _ TS-4 with one MS2 loop ( lxMS2) and rpoB_ TS-4 2xMS2 (Fig. 5, SA). The results indicate that increasing the number of recruiting loops in fact ,enhances the mutation frequency in terms of Rif', suggesting increased presence of effector prntein. Mutation analysis of AmCRCou1A treated cell with 1poB~TS-4_2xMS2 as targeting construct, showed that Cl592 nucleotide was modified in 1000 / o of the clones1. 62 .. 5% mutated C to T and 37.5% mutated C to A (Fig. 58). These results suggest that 10 engineering the recruiting modules does not affect the system's targeting sp,ecificity Taken together, tl::u,se r,esults indicate that the modular design of the C:RC system facilitates the engineering process and opens the possibility to further improving the system. Example 2 CRC system led to site-specific nucleotide conversion in mammalian systems 15 Experimental des1g11.: Engineering the system for mammalian expression We next sought to engineer the system for mmnmaliat1 expression. To this end, we recapituiated the prok:aryotic AIDCRCDlOA system as a multicistronic construct, using a mammalian codon. optimized 11Cas9.ornA followed by AID MCP fusion separated by a se1f-cleavable P2A jpeptide. The constructs were cloned under the control ofUbiquitin C 2.0 promoter. gRNA __2xMS2 ,cassettes were cloned under the control of U6 or HJ promoter, for targets with 5'-G or S'~A, respectively (Ranganathan, V., et al., Nature communications 5,. doi:I0J038 / ncomms5516 (2014)). A schematic repr:esentati.on of the constructs used an these set of experiments is illustrated in Fig. 6A. Targeting extmchromosoma / DNA: EGFP reverse mutation as~s·ay 25 EGFP was ,eugineered t,o harbor a loss of function point mutation (197 A>G,. Y66C) thait destroys its fluurophore, therefore rendering the protein non-fluorescent (m:EGFPY66c). The expression ve,ctor of the mutant GFP is then transfected into mamm.a]ian cells and serves as a substrate of th,e system. The aim of this experiment was to "correct" this lossof::. function mutation. When the "corrected" gene is transcribed and translated, the 49 Date Re9ue / Date Received 2022-07-19 correction wm restore protein function, which can be visualized as fluorescent ,celBs under the fluorescence microscope. Experimental appmach Approximately 7x.105 293T cells wer::e transfected with rn μg. of a combination of S DNA ,comprising the target plasmid encoding nfEGFPY66c, AIDCRCowA and gRNA constructs. For comparison, base editor 3rd generation system (BE3,. Komor,. A. C., et al., Nature advance online publication. doi: 10 .103 8 / natu:re 17946) was used in the:se set of experiments. BE3 is a slightly similar system with a different recruitment mechani:sm, di.rect fusion of Cas9 with APOBECl, and includes a peptide that inhibits uracil DNA lO glycosybtse, an e.nzyme i.nvolved in DNA .repair. After overn.ight htcubation, ,ceUs were anailyzed under fluorescence microscope to observe GFP signal. Res11lts It was found that the above CRC system was able to modify target nudeotide in extrachromosomal. DNA,. restoring protein function. Since the target cytosine is located 15 on the template strand (TS, -). two gRNAs were designed to bind t.he non-template strand (NT,+) around the target nucleotide (Fig. 6B). The target cytosine is located on positions 5 and 12 within 11JEG1P1 ' 66c_NT-I and ,ijEGl1'PY66c_NT-2 protospacers, respectively. 293T cells were transfocted with DNA encoding for nCas9oioA. AJD_MCP, gRNAs ( 1I,EGJ?P166c_NT-1 or 111£GFPY66c NT-2 or scramble), and the target constmct, 2.0 11rEGFPY6 6C. EGF'P signal. was detected on cells treated with ,!fE'GFP1•66c_NT-1 and 11f1j:GFPr'6r,c __ NT.:2, but not with scramble (Fig. 6C). EOFP signal was greater in , / £Gf'1)n,r,c _NT -1 treated cells compared to q,EGF'Pr66c_NT-2 due ro the position of the target cytos:ine. n.,tEGFP:r:66c_;NT~l lik!ely makes the targeted C more accessible to AID (Fig. 6C, central and right panels). 1n addition, the CRC platform was compared with a 25 different gene editing system (BE3), which utilized a di.rect fusion of the cytidine deaminase pmtein to Cas9 protein for recruitment and required a co-expression of an inhibitor of uracH DNA glycosylase (UGI) to improve efficiency. It was unexpectedly tbund that the CRC syst,em, where the effector and the sequence-targeting module were Hnked via the RNA scatlbld. was much more efficient than the .BE3 system, even without 30 local UNG inhibition (without the expression of the uracil DNA g)ycosylase inhibitor UGI) (Fig.6C, 6D, and 7B). 50 Date Re9ue / Date Received 2022-07-19 Thes,e results confirm the findings from the bacterial system and .indicate that the system effi.ci.ently deaminates specific cytosine residues in ,extmchromosomal DNA in human cells in a programmable fashion. Quantitation of GFP positive cells from treatments with AIDCRC010A and BE3 using nJE.GFP166C -'NT-1 as a targeting gRNA 5, suggests that CRC :system. has a better conversion efficiency than BF3 (Fig. 60). Example 3 CRC system ~ed to site-specific nucleotide conversion in endogenous gene .in mammalian ceUs Targeting an endogenous locus: Chinese hamster .HPRT gene Encouraged by the positi.ve results observed from the bacteri.al negative :selection lO system, we decided to use a similar approach in mammalian.. Hypoxanthine--guanine phosphoribosy[ transferase (HPRT) is an enzyme involved in purine metabolism, and mutations along its ,coding sequence are known to cause resistance to the antimetabolite 6- thiog11uinine ,(,6-TGR) (01Neill, J. P. et al., Nature 269, 815-816 (1977)). For these experiments we aimed to mutate HPRT gene with CRC system in order to disrupt its 15 function foUowed by selection of mutant cells with 6-TG for further analysis. Experimental approach Approximately 7x105 Chinese ha:m.ster V79-4 c,ell:s were transfec.ted with 10 μg of a combination of DNA comprising A 111CRCnwA construct and gRNA HPRT_T:S-1 expression vector. For comparison, cells were also treated with BE3 and gRNA .20 HPR.'(.TS-].. Treated and untreated cells were grown following a mammalian mutagenesis protocol previously described (Klein, C. B., et al.,. in Current Protocol:\' in Toxicology (John Wiley & Sons, Inc., 2001)). Bri,efly, after transfecti.on cells were maintained for seven days before 6-TG selection for mutation fixati.on and turnover of preexisting HPRT mRNA and protein. Cells were selected with 60 μiM 6-TG for 14 days 2.5 to allow 6-TGR colonies to form. Colonies were counted to estimate mutation frequency, a:nd individual colonies were isolated and propagated individually for sequencing analysis. One gRNA was designed to target exon 3 from the Chinese hamster HPRT gene (Fig. 7A). The gRNA targets codon 74 encoding for phenylalanine and mutations in this 30 residue have been implicated in reduced HPRT protein stability (Davidson, B. L., et al., 51 Date Re9ue / Date Received 2022-07-19 Gene 63, 331-336, doi:http: / / dx.doi.org / l0J016 / 0378-lll9 (88)90536-7 (1988)). V79-4 ceHs were transfected with DNA encoding for ,•,mcRCmOA or BE3 constructs together with the targeting gRNA expression vector. AIDCRCmoA system led to mutation n:mdering the cell resistant to 6-TG treatment with a higher efficiency than BE3 system (t.e; 140- 5 versus 40-fold higher than untreated cells, respectively; Fig. 7B), The resul.ts show that the CRC system is abl,e to target and modify specific DNA sequences in an endogenous mammaHan focus. The foregoing examples and description of the preferred embodiments should be taken as Hlustrating, .rather than as limiting the present invention as defined by the claims. 10 As will be readily appreciated, numerous variations and combinations of the features set forth above can be utili.zed without departing from the present invention as set forth in the claims. Su.ch variatfons are .not regarded as a departure from the scope of the inventi,on. and all such variations are int,ended to be included within the scope of the invention. 52 Date Re9ue / Date Received 2022-07-19

Claims

Application No. 3168241 Our Ref: 28020-71 (096738.00730) 53 149803266.1 CLAIMS 1. A host cell comprising: (i) a CRISPR protein, (ii) an RNA scaffold, said RNA scaffold comprising (a) a nucleic acid-targeting motif comprising a guide RNA sequence that is complementary to a target nucleic acid sequence, (b) a CRISPR motif capable of binding to the CRISPR protein, and (c) a recruiting RNA motif, and (iii) a non-nuclease effector fusion protein, said non-nuclease effector fusion protein comprising (a) an RNA binding domain capable of binding to the recruiting RNA motif, (b) a linker, and (c) an effector domain that has an enzymatic activity for DNA / RNA modification.

2. A method for producing the cell of claim 1, comprising introducing into an isolated host cell (a) the CRISPR protein, (b) the RNA scaffold, and (c) the non-nuclease effector fusion protein.

3. The method of claim 2, wherein the CRISPR protein is introduced into the host cell by a nucleic acid encoding the CRISPR protein.

4. The method of claim 2 or 3, wherein the non-nuclease effector fusion protein is introduced into the host cell by a nucleic acid encoding the non-nuclease effector fusion protein.

5. The method of any one of claims 2-4, wherein the RNA scaffold is introduced into the host cell by a nucleic acid encoding the RNA scaffold.

6. The method of any one of claims 2-5 comprising culturing the host cell under a suitable condition, which allows for the expression of the RNA scaffold, the CRISPR protein or the nonnuclease effector fusion protein.

7. A cell modified using a system, wherein the system comprises: CA 3168241 Date reçue / Received date 2024-12-18 Application No. 3168241 Our Ref: 28020-71 (096738.00730) 54 149803266.1 (i) a CRISPR protein or a polynucleotide encoding the same, (ii) an RNA scaffold or a polynucleotide encoding the same, said RNA scaffold comprising (a) a nucleic acid-targeting motif comprising a guide RNA sequence that is complementary to a target nucleic acid sequence, (b) a CRISPR motif capable of binding to the CRISPR protein, and (c) a recruiting RNA motif, and (iii) a non-nuclease effector fusion protein or a polynucleotide encoding the same, said non-nuclease effector fusion protein comprising (a) an RNA binding domain capable of binding to the recruiting RNA motif, (b) an effector domain that has an enzymatic activity for DNA / RNA modification, and (c) a linker joining the RNA binding domain and the effector domain.

8. The cell of claim 1 or 7 or the method of any one of claims 2-6, wherein the CRISPR protein comprises the sequence of dCas9 or nCas9 of a species selected from the group consisting of Streptococcus pyogenes, Streptococcus agalactiae, Staphylococcus aureus, Streptococcus thermophilus, Neisseria meningitidis, and Treponema denticola.

9. The cell of any one of claims 1, 7 and 8 or the method of any one of claims 2-6 and 8, wherein the recruiting RNA motif and the RNA binding domain are a pair selected from the group consisting of: a telomerase Ku binding motif and Ku protein or an RNA-binding section thereof, a telomerase Sm7 binding motif and Sm7 protein or an RNA-binding section thereof, a MS2 phage operator stem-loop and MS2 coat protein (MCP) or an RNA-binding section thereof, a PP7 phage operator stem-loop and PP7 coat protein (PCP) or an RNA-binding section thereof, a SfMu phage Com stem-loop and Com RNA binding protein or an RNA-binding section thereof, and a non-natural RNA aptamer and corresponding aptamer ligand or an RNA-binding section thereof.

10. The cell of any one of claims 1 and 7-9 or the method of any one of claims 2-6 and 8-9, wherein the enzymatic activity is deamination activity, dismutase activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, CA 3168241 Date reçue / Received date 2024-12-18 Application No. 3168241 Our Ref: 28020-71 (096738.00730) 55 149803266.1 recombinase activity, polymerase activity, ligase activity, photolyase activity or glycosylase activity.

11. The cell of any one of claims 1 and 7-10 or the method of any one of claims 2-6 and 8-9, wherein the cell is selected from the group consisting of: an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, an adult stem cell, a pluripotent stem cell, an induced pluripotent stem cell, a multipotent cell, an oligopotent stem cell, a unipotent cell, a plant cell, an algal cell, an animal cell, in invertebrate cell, a vertebrate cell, a fish cell, a frog cell, a bird cell, a mammalian cell, a pig cell, a cow cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non-human primate cell, and a human cell.

12. The cell or the method of claim 11, wherein the cell is derived from a human or non-human subject.

13. The cell or the method of claim 12, wherein the human or non-human subject has a genetic mutation of a gene or has a pathogen or is at risk of exposing to the pathogen.

14. The cell or the method of claim 13, wherein the subject has a disorder caused by the genetic mutation or is at risk of having the disorder.

15. The cell of any one of claims 1 and 7-14 or the method of any one of claims 2-6 and 8-14, wherein the CRISPR protein does not have a nuclease activity. CA 3168241 Date reçue / Received date 2024-12-18