RNA scaffold

Abstract

Provides an optimized RNA scaffold for precise genome targeting effector systems. [Solution] An RNA scaffold is provided comprising (a) tracrRNA and (b) an RNA motif with an extended sequence.

Description

[Technical Field]

[0001] This invention relates to RNA scaffolds for CRISPR systems. [Background technology]

[0002] CRISPR-Cas technology is developing rapidly, and the range of CRISPR applications is expanding rapidly (Lau, The CRISPR Journal, Vol 1, No 6). The main component of the CRISPR system is the guide RNA (gRNA), which forms part of the RNA scaffold. This RNA scaffold first directs the CRISPR system to a desired target in the genome, and then delivers a biologically active effector to that target to perform the desired function. The RNA scaffold must deliver the effector in the correct orientation and conformation so that it can function effectively in a specific manner that yields the desired output without causing off-target effects. Therefore, an optimized RNA scaffold is necessary for a precise genome-targeting effector system.

[0003] The inventors have designed RNA scaffolds optimized for enhanced target performance. The RNA scaffolds, systems, and methods provided herein enable precise modification of the genome while simultaneously minimizing the potential for off-target effects, thereby making the methods and systems particularly suitable for therapeutic applications. [Overview of the project] [Means for solving the problem]

[0004] In the first embodiment, the present invention is (a) tracrRNA and; (b) RNA motif having an extended sequence, It provides an RNA scaffold containing [the specified element].

[0005] In one embodiment, the RNA scaffold according to the first embodiment further comprises a crRNA containing a guide RNA sequence. The RNA scaffold according to the first embodiment includes one or more modifications. The RNA motif is ligated to the 3' end of the tracrRNA via a linker. In a preferred embodiment, the linker is single-stranded RNA or a chemical bond. The single-stranded RNA linker contains 0 to 10 nucleotides, preferably 2 to 6 nucleotides.

[0006] In one embodiment, the RNA scaffold according to the first embodiment includes tracrRNA fused to crRNA containing a guide RNA sequence that forms a single RNA molecule. In another embodiment, the RNA scaffold according to the first embodiment includes tracrRNA and crRNA containing a guide RNA sequence synthesized as separate RNA molecules. In any embodiment, tracrRNA hybridizes to crRNA via a repeat:anti-repeat region. When tracrRNA is synthesized as a single RNA molecule as shown in Figure 10B, it includes an anti-repeat region, a tetraloop, and the 3' constant region of gRNA. When tracrRNA is synthesized as separate RNA molecules, it includes an anti-repeat region and the 3' constant region of sgRNA, and the tetraloop is absent as shown in Figure 10D. The anti-repeat region of tracrRNA hybridizes to the repeat region of crRNA. In a preferred embodiment, the repeat:anti-repeat region is extended.

[0007] The RNA scaffold of the present invention comprises one or more RNA motifs, in this case one Alternatively, multiple RNA motifs may include one or more modifications. These one or more modifications may be located at the 5' and / or 3' ends of the one or more RNA motifs. The RNA scaffold of the present invention may include one or more modifications, including a substitution of a 10-position A base to 2-aminopurine (2AP). The RNA scaffold may use 2'-deoxy-2-aminopurine or 2'-ribose-2-aminopurine. The RNA scaffold of the present invention may have one or more modifications to the backbone and / or sugar moiety of the RNA scaffold. The extended sequence of the RNA motif is a double-strand extension, in which case the extended sequence of the RNA motif contains 2 to 24 nucleotides. In one embodiment, a 4-nucleotide extension results in a stem having 23 nucleotides in total length. In another embodiment, a 10-nucleotide extension results in a stem having 29 nucleotides in total length. In another embodiment, a 16-nucleotide extension results in a stem having 35 nucleotides in total length. In another embodiment, a 26-nucleotide extension results in a stem having 45 nucleotides in total length.

[0008] The RNA scaffold of the present invention comprises one or more RNA motifs that bind to an aptamer-binding molecule. The one or more RNA motifs are selected from the following aptamers: MS2, Ku, PP7, SfMu, and Sm7. For example, the MS2 aptamer binds to an MCP protein. In a preferred embodiment, the RNA scaffold comprises one recruiting MS2 RNA motif. In another embodiment, the RNA scaffold comprises two recruiting MS2 RNA motifs. In a preferred embodiment, the MS2 aptamer is wild-type MS2, mutant MS2, or a variant thereof. The mutant MS2 used herein is C-5, F-5 hybrid, and / or F-5 mutant. The RNA motif of the RNA scaffold according to the present invention recruits an effector module. The effector module disclosed herein comprises an RNA-binding domain that can bind to the RNA motif, and an effector domain. Preferred effector domains are selected from the following: reporter, tag, molecule, protein, microparticle, and nanoparticle. In a preferred embodiment, the effector domain is a DNA modifying enzyme. Preferred DNA modifying enzymes are selected from: AID, CDA, APOBEC1, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, or other APOBEC family enzymes, ADA, ADAR family enzymes, or tRNA adenosine deaminases.

[0009] In a second embodiment, the present invention is as follows: (a) CRISPR protein; (b) The crRNA of the present invention as defined above; (c) The RNA scaffold of the present invention as defined above; (d) aptamer binding molecule; (e) Effects module We provide a system for gene modification, including the modification of genes.

[0010] The system according to the second embodiment comprises components (a) to (e), which are delivered in the form of nucleic acids, protein complexes, and / or expressed by any suitable expression vector. The system provided herein may include a CRISPR protein fused to one or more uracil DNA glycosylase (UNG) inhibitor peptides (UGIs). In a preferred embodiment, the CRISPR used in the system according to the second embodiment is a class 2 type II CRISPR protein, such as Cas9. The CRISPR protein and / or effector module used in the system according to the second embodiment may include one or more nuclear localization signals (NLS). The CRISPR protein may be a class 2 Cas protein that is either nuclease null or has nickase activity.

[0011] The effector module used in the system according to the second embodiment may be an effector fusion protein comprising an RNA-binding domain capable of binding to an RNA motif and an effector domain. The system according to the second embodiment comprises an RNA motif and the following: Telomerase Ku-binding motif and Ku protein or its RNA-binding section, Telomerase Sm7 binding motif and Sm7 protein or its RNA binding section, MS2 phage operator stem-loop and MS2 coat protein (MCP) or its RNA-binding section, PP7 phage operator stem-loop and PP7 coat protein (PCP) or its RNA-binding section, SfMu phage Com stem-loop and Com RNA-binding protein or its RNA-binding section, An effector module containing a pair of RNA-binding domains selected from the group consisting of the following may be used.

[0012] In a third embodiment, the present invention provides a method for genetically modifying cells, comprising the steps of introducing into cells and / or expressing a system according to the second embodiment in the cells. The method according to the third embodiment may be used to genetically modify cells to correct gene mutations, inactivate gene expression, change gene expression levels, or alter intron-exon splicing, but is not limited thereto. Genetic modifications by the method provided in the third embodiment are point mutations, which may optionally introduce immature stop codons, disrupt start codons, disrupt splicing sites, or correct gene mutations. [Brief explanation of the drawing]

[0013] [Figure 1]Figure 1A shows a system comprising the following three structural and functional components: (1) a sequence targeting component (e.g., a Cas protein); (2) an RNA scaffold for sequence recognition and effector recruitment, including crRNA, tracrRNA, and RNA motifs; and (3) an effector module (e.g., a non-nuclease DNA modifying enzyme, such as AID fused to a small protein that binds to an RNA motif). More specifically, as shown in Figure 1A, the components of the RNA scaffold-mediated recruitment platform include: a sequence targeting component 1 (e.g., dCas9 or nCas9D10A); an RNA scaffold 2 containing a cRNA including a guide RNA (and repeat: repeat of anti-repeat stem) 2.1 for sequence targeting, a tracrRNA 2.2 for Cas protein binding, and an RNA motif 2.3 for recruiting the effector module; and an effector module 3 containing an effector domain 3.1 (e.g., cytidine deaminase) fused to an RNA aptamer ligand 3.2. Figure 1B shows a schematic diagram of the RNA scaffold-mediated recruitment complex at the target sequence: Cas9 (or dCas9 or nCas9) binds to tracrRNA, and the RNA motif (e.g., aptamer) recruits effector modules, which form an active RNA scaffold-mediated recruitment system capable of editing target residues on unpaired DNA within the CRISPR R-loop. [Figure 2] The figure shows (A) an MS2 hairpin sequence with a C-5 substitution, and (B) an MS2 hairpin sequence containing an F-5 variant sequence with an additional substitution from A to d2AP at the indicated position A-10. [Figure 3] This figure shows RNA motifs containing MS2 stem extensions of (A) 4nt, (B) 10nt, (C) 16nt, and (D) 26nt compared to wild-type MS2. [Figure 4] This figure shows a module of an RNA scaffold containing tracrRNA, an RNA motif with an extension sequence, and crRNA containing a guide RNA sequence. [Figure 5]Figure showing variations in the phenotypic interference of the TRAC Ex3 SA splicing site caused by a synthetic aptamer due to the change from cytosine base to thymine base. Synthetic crRNA:tracrRNA (with or without aptamer) with electroporated nCas9-UGI-UGI and rApobec1 and hAID deaminases. [Figure 6] Figure showing variations in the base changes of the TRAC Ex3 SA splicing site caused by a synthetic aptamer due to the change from cytosine base to thymine base. Synthetic crRNA:tracrRNA (with or without aptamer) with electroporated nCas9-UGI-UGI and rApobec1 and hAID deaminases. Data are shown as the percentage of T sequenced at the indicated target C residue when measured by Sanger sequencing. [Figure 7] Figure showing HEK Site2 editing by tracrRNA containing a 4nt or 16nt extension of the MS2 hairpin sequence with nCas9-UGI-UGI and rApobec1 deaminase. Data are shown as the percentage of T sequenced at the indicated target C residue when measured by Sanger sequencing. [Figure 8] Figure showing HEK Site2 and HEK Site3 editing by tracrRNA containing one or two MS2 hairpins at the 3' end of an RNA motif with nCas9-UGI-UGI and hAID deaminase. Data are shown as the percentage of T sequenced at the indicated target C residue when measured by Sanger sequencing. [Figure 9-1]A figure showing base editing efficiency at different target gene loci using various RNA scaffold designs. Figures 9A - C: Figures showing the effects of the position and number of MS2 aptamers and the extension of the repeat:anti-repeat upper stem on APOBEC-1-mediated base editing. Base editing was measured at 3x target gene loci, and the sequences and C residues within the base editing target window are shown in Table 5 in Example 1. The RNA scaffolds incorporated either a single copy (1xMS2) or two copies (2xMS2) of the MS2 aptamer and were positioned at either the tetra-loop (TL), stem-loop 2 (SL2), or 3' of the RNA scaffold (3'). Additionally, some designs incorporated a 14-base extension of the repeat:anti-repeat upper stem (7bp extension US). Data are shown as the percentage of Ts sequenced at the indicated target C residues when measured by Sanger sequencing. Error bars represent the standard deviation of the mean from 3 replicate experiments. Figures 9D - H: Editing by APOBEC-1 was measured at 5 additional gene loci by the best 1xMS2_3' 7bp extension US prior to testing with 2xMS2_3' 7bp-extension US. The sequences and C residues within the base editing target window are shown in Table 5 in Example 1. Data are shown as the percentage of Ts sequenced at the indicated target C residues when measured by Sanger sequencing. Error bars represent the standard deviation of the mean from 3 replicate experiments. Figure 9I: A figure showing a comparison of the effects of different lengths of extension of the repeat:anti-repeat upper stem on aptamer-dependent APOBEC-1-mediated base editing. 2bp, 5bp, 7bp, and 10bp sgRNA processing upper stem extensions as well as the non-extended upper stem (1xMS2_3') sgRNA were included in the analysis. Data are shown as the percentage of Ts sequenced at the indicated target C residues when measured by Sanger sequencing. Error bars represent the standard deviation of the mean from 3 replicate experiments. [Figure 9-2] The same as above. [Figure 9-3] The same as above. [Figure 10-1]This figure shows an annotated diagram representing different parts of an RNA scaffold when synthesized as a single molecule or as separate molecules. Figure 10A: RNA scaffold synthesized as a single molecule with two MS2s as disclosed in Prior Art International Publication No. 2017011721. Figure 10B: RNA scaffold synthesized as a single molecule with one MS2 as described herein. Figure 10C: Anti-repeat: RNA scaffold synthesized as a single molecule with one MS2 having a 7 bp extension on either side of the repeat region. Figure 10D: RNA scaffold synthesized as separate molecules without a tetraloop. Figure 10E: RNA scaffold synthesized as separate molecules with a 2AP modification at position 10 of the MS2 stem-loop. Figure 10F: RNA scaffold synthesized as separate molecules with a 2AP modification at position 10 of the F-5 variant of the MS2 stem-loop. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 11-1] This figure shows base editing using chemically synthesized C-5 or F-5 1xMS2_3' tracrRNAs with rApobec1 deaminase crRNA and mRNA in nCas9-UGI-UGI U2OS stable cells. The gene sites targeted by each cRNA are (A)CR0118_PDCD1, (B)CR0107_PDCD1, (C)CR0057-TRAC_EX3, (D)CR0151_CD2, (E)HEK Site2, (F)CR0121_PDCD1, and (G)CR0165_CIITA. The data are shown as the percentage of T sequences at the indicated target C residues, measured by Sanger sequencing. [Figure 11-2] Same as above. [Figure 12]This figure shows base editing in nCas9-UGI-UGI U2OS stable cells using chemically synthesized C-5 or F-5 1xMS2_3' tracrRNA accompanied by the crRNA and mRNA of hAID deaminase. The gene sites targeted by each cRNA are (A) CR0151_CD2, (B) CR0121_PDCD1, and (C) CR0165_CIITA. The data are shown as the percentage of T sequences at the indicated target C residues, measured by Sanger sequencing. [Figure 13] This figure shows base editing by chemically synthesized 1xMS2_3'sgRNAs(C-5), 1xMS2_3'_7bp-extended_US sgRNAs(C-5) (including a 7-base pair extension of the upper stem of the repeat:anti-repeat), or 1xMS2_3' tracrRNA (C-5) with the crRNA and mRNA of hAID deaminase in nCas9-UGI-UGI U2OS stable cells. The gene sites targeted by each crRNA are (A)TRAC_22550571, (B)PDCD1_241852953, and (C)CTNNB1. The data are shown as the percentage of T sequences at the indicated target C residues, measured by Sanger sequencing. [Figure 14] This figure shows base editing in nCas9-UGI-UGI U2OS stable cells using chemically synthesized C-5 or F-5 1xMS2_3' tracrRNA with rApobec1 deaminase crRNA and various levels of mRNA. The target sites by each cRNA are (A) HEK Site2 and (B) CR0107_PDCD1. [Modes for carrying out the invention]

[0014] This invention relates to novel RNA scaffolds for targeting genomes and delivering functional effectors. Such functional effectors include enzymes, reporters, tags, molecules, proteins, microparticles, and nanoparticles.

[0015] One application of the present invention relates to CRISPR gene editing and screening. The present invention can be used in any CRISPR gene editing system. The application of the present invention involves recruiting an effector module to a target DNA sequence in the genome. The present invention involves the use of an RNA scaffold. The present invention has particular applications in CRISPR base editing systems, such as RNA scaffold-mediated recruitment systems.

[0016] An example of an RNA scaffold-mediated recruitment system includes the following functional components: (1) a CRISPR / Cas-based module manipulated for sequence targeting; (2) an RNA scaffold-based module for guiding the platform to a target sequence and for recruiting effector modules; and (3) an effector module, such as cytidine deaminase (e.g., activation-inducible cytidine deaminase, AID).

[0017] In a first embodiment, an RNA scaffold comprising: (a) tracrRNA; and (b) an RNA motif with an extension sequence is provided herein. As disclosed herein, the RNA scaffold is optimized for enhanced gene editing. The RNA scaffold-mediated recruitment system is a complex of several components, including an RNA scaffold, which needs to be assembled in a specific manner to perform a precise function. The complex must reach the precisely correct orientation and conformation so that it can effectively edit the genome in a specific manner such as locating a specific portion of the genome and resulting in a desired output. Furthermore, the complex must effectively recruit and deliver a biologically active effector module, such as an enzyme, in the correct orientation / conformation to edit the genome while maintaining enzymatic activity without causing significant off-target effects. Previous base editing systems have resulted in poor or limited editing in many regions.

[0018] To overcome these problems, the inventors introduced one or more modifications to the RNA scaffold-mediated recruitment system, particularly the RNA scaffold, which was identified through trial and error.

[0019] While we do not wish to be bound by any theory, some of these modifications are thought to induce conformational changes in the components of the RNA scaffold-mediated recruitment system. Significant improvements have been observed with the use of the RNA scaffolds disclosed herein. Advantageously, the optimized system, including the RNA scaffold, which itself contains an RNA motif with an extended sequence, possesses superior flexibility, stability, positioning, and affinity, thereby efficiently editing previously resistant regions, including therapy-related loci, while maintaining performance. The novel RNA scaffold expands the repertoire of editable targets and enhances the efficiency of gene editing.

[0020] RNA scaffold-mediated recruitment system Conventional nuclease-dependent precise genome editing for mutation correction typically requires the introduction of DNA double-strand breaks (DSBs) and activation of homologous recombination-dependent repair (HDR) pathways.

[0021] In recent years, RNA-mediated base editing systems have also been developed. This system recruits a base editing enzyme to a target DNA sequence via the RNA component of the CRISPR complex. The system contains a modified gRNA with a reprogrammable RNA aptamer at its 3' end, which recruits a congeneral aptamer ligand fused to an effector (e.g., a deaminase effector). Using this system, targeted nucleotide modification has been achieved with high precision in prokaryotic cells and eukaryotic cells such as mammalian cells; see International Publications 2018129129 and 2017011721. A novel second generation of RNA-mediated base editing systems with enhanced specificity and efficiency was tested in prokaryotic cells and further improved in mammalian cells. This second-generation system / platform exhibits high specificity, high efficiency, and low off-target tendencies. Modular design completely separates the nucleic acid modification module from the nucleic acid recognition module. Therefore, RNA-mediated base editing systems offer an alternative to the recruitment of effectors by fusion to or direct interaction with sequence-targeting proteins, which can not effectively separate the sequence-targeting function from the nucleic acid modification function. The present invention disclosed herein is an RNA scaffold-mediated recruitment system, which is a modified modular design of an RNA-mediated base editing system. Various modifications are incorporated into the components of the system, thereby improving the flexibility, specificity, and efficiency of the system. The novel RNA scaffold-mediated recruitment system is not limited to base editing and has several potential applications, such as genome editing, genome screening, and genome tagging, providing a powerful tool for genetic engineering and therapeutic development.

[0022] Schematic diagrams of exemplary RNA scaffold-mediated recruitment systems for use in the manner provided herein are illustrated in Figures 1A and 1B. The system comprises the following three structural and functional components: (1) a sequence targeting component (e.g., a Cas protein); (2) an RNA scaffold for sequence recognition and effector recruitment, comprising crRNA, tracrRNA, and RNA motifs; and (3) an effector module (e.g., a non-nuclease DNA modifying enzyme, e.g., an AID fused to a small protein that binds to an RNA motif). More specifically, as shown in Figure 1A, the components of the RNA scaffold-mediated recruitment platform are as follows: Sequence targeting component 1 (e.g., dCas9 or nCas9) D10A The RNA scaffold 2 contains a cRNA including a guide RNA (and repeat:anti-repeat stem) 2.1 for sequence targeting, a tracrRNA 2.2 for Cas protein binding, and an RNA motif 2.3 for recruiting the effector module, as well as an effector module 3 containing an effector domain 3.1 (e.g., cytidine deaminase) fused to an RNA aptamer ligand 3.2. Figure 1B shows a schematic diagram of the RNA scaffold-mediated recruitment complex at the target sequence: Cas9 (or dCas9 or nCas9) binds to the tracrRNA, the RNA motif (e.g., aptamer) recruits the effector module, and forms an active RNA scaffold-mediated recruitment system that can edit target residues on unpaired DNA within the CRISPR R-loop. These three components can be constructed in a single expression vector or multiple separate expression vectors, or they can be introduced in DNA-free form (mRNA or protein and chemically synthesized RNA molecules). The three specific components as a whole, and in combination thereof, constitute the activation of the technical platform. Figure 1B shows the three components of the RNA scaffold in a specific 5' to 3' order, but the components can also be arranged in a different order if necessary, for example, for optimization for different Cas protein variants.

[0023] As disclosed herein, there are several clear distinctions between recruitment mechanisms: RNA scaffold-mediated recruitment systems and direct fusion of Cas9 to effector protein systems (BE systems). The modular design of the RNA scaffold-mediated recruitment system allows for flexible system engineering. Modules are interchangeable, and many combinations of different modules can be achieved simply by swapping the nucleotide sequences of the recruiting RNA aptamer and its homologous ligand. On the other hand, recruitment of effectors by direct fusion or direct interaction of the sequence-targeting unit with the protein component always requires reengineering of a new fusion protein, which is technically more difficult and unpredictable. Furthermore, the RNA scaffold-mediated recruitment system likely promotes oligomerization of the effector protein, while direct fusion, due to steric hindrance, would likely hinder oligomer formation.

[0024] Due to its relative ease of use and scalability, CRISPR / Cas-based The gene system is poised to dominate the therapeutic landscape, making it an attractive gene editing technology for developing novel applications with therapeutic value. As disclosed herein, the RNA scaffold-mediated recruitment system utilizes a particular embodiment of the CRISPR / Cas system. To overcome the limitations associated with the DSB and HDR requirements for conventional CRISPR / Cas gene editing systems, a remarkable gene editing method called base editing (BE) has been developed, utilizing the DNA targeting ability of Cas9 lacking double-strand break activity, such as dCas9 or nCas9, combined with the DNA editing function of APOBEC-1, an enzyme member of the APOBEC family of DNA / RNA cytidine deaminases. By directly fusing a deaminase effector to a nuclease-deficient Cas9 protein called dCas9, these tools, called base editors, can introduce targeted point mutations into genomic DNA or RNA without producing DSBs or requiring HDR activity. Essentially, the BE system utilizes a nuclease-deficient CRISPR / Cas9 complex as a DNA targeting mechanism, where the mutant Cas9 acts as an anchor to directly recruit cytidine or adenine deaminase via protein-protein fusion.

[0025] On the other hand, RNA scaffold-mediated recruitment systems take a different approach. More specifically, in these RNA scaffold-mediated recruitment systems, the RNA component of the CRISPR / Cas9 complex functions as an anchor for effector recruitment by incorporating RNA motifs such as aptamers into the RNA molecule. The RNA aptamer then recruits an effector molecule, such as an effector fused to an RNA aptamer ligand. Compared to recruitment by direct protein fusion or other recruitment approaches using protein components, the mechanism of these RNA scaffold-mediated recruitment systems has several distinct features that are potentially advantageous for both system engineering and achieving better functionality. For example, it has a modular design in which nucleic acid sequence targeting function and effector function belong to different molecules, thereby allowing for the independent reprogramming of functional modules and the multiplexing of other systems. Reprogramming of the RNA scaffold-mediated recruitment system requires only changes to the RNA aptamer sequence in the gRNA and the replacement of the congeneral RNA aptamer ligand-fused effector. It does not require the re-engineering of individual functional Cas9 fusion proteins. In addition, the effector module is smaller in size, which can potentially allow for more efficient oligomerization of the functional effector. Moreover, since RNA scaffold-mediated recruitment does not require the generation of Cas9 fusion proteins, which further increases the gene / transcription size of Cas9, the system can potentially be constructed in a more efficient manner for packaging and delivery by viral vectors, non-viral vectors, mRNA molecules, mechanical means, or protein components.

[0026] As disclosed herein, the present invention provides further engineering of RNA scaffold-mediated recruitment systems for precise gene editing. As demonstrated herein, the optimized RNA scaffold-mediated recruitment system exhibits several important differences compared to previous RNA scaffold-mediated base editing systems such as those described in International Publication No. 2018129129 and International Publication No. 2017011721 (which are incorporated herein by reference in their entirety). First, the optimized RNA scaffold-mediated recruitment system exhibits substantially increased on-target effects compared to first- and second-generation RNA scaffold-mediated base editing systems, while still maintaining low or no detectable off-target effects. Second, the optimized RNA scaffold-mediated recruitment system has greater flexibility due to modifications incorporated into various components of the system, such as extension sequences at the 3' end of the RNA motif. Third, the optimized RNA scaffold has improved positioning of the RNA motif relative to tracrRNA. It has a 3D obstruction.

[0027] a. Array targeting module The sequence targeting components of the methods and systems provided herein typically utilize Cas proteins from the CRISPR / Cas system derived from bacterial species as sequence targeting proteins.

[0028] In embodiments, the Cas protein may be a mutant Cas protein, a dCas protein having a mutation in its nuclease catalytic domain and therefore lacking nuclease activity, or an nCas protein having a partial mutation in one of its catalytic domains and therefore lacking nuclease activity for generating DSBs. The Cas protein is specifically recognized by a tracrRNA component of an RNA scaffold that guides the Cas protein to its target DNA or RNA sequence. The latter is flanked by a 3'PAM.

[0029] Cas protein Various Cas proteins can be used in the present invention. Cas proteins, CRISPR-related proteins, or CRISPR proteins are mutually interchangeable and refer to proteins or proteins derived from the CRISPR-Cas class 1 or class 2 system having RNA-induced DNA binding. Non-limiting examples of suitable CRISPR / Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3( Examples include Cse4 (or CasE), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966. For example, Koonin and Makarova, 2019, origins and evolution of crispr cas systems, Review Philos Trans R Soc Lond B Biol Sci. May 13, 2019; 374(1772).

[0030] In one embodiment, the Cas protein is derived from a Class 2 CRISPR-Cas system. In a preferred embodiment, the Cas protein is a Class 2 Type 2 Cas system. In an exemplary embodiment, the Cas protein is or is derived from a Cas9 protein. The Cas9 protein in question is found in *Streptococcus pyogenes*, *Streptococcus thermophilus*, *Streptococcus sp.*, *Nocardiopsis dassonvillei*, *Streptomyces pristinaespiralis*, *Streptomyces viridochromogenes*, *Streptomyces viridochromogenes*, *Streptosporangium roseum*, and *Alicyclobacillus acidocardarius*. Bacillus pseudomycoides (Bacillus pseudomycoides) ides), Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis erginosa aeruginosa), Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile difficile), Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanchtis Haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp.), Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Legionella pneumophila, Francisella novicida, gamma proteobacterium HTCC5015, Parasutterella excrementihominis, Sutterella wadsworthensis, Sulfurospirillum sp. SC ADC, Ruminobacter sp. RM87, Burkholderiales bacterium 1 1 47. Bacteroidetes, Oral Taxon 274 str. F0058, Wolinella succinogenes. Burkholderiales bacteria YL45, Ruminobacter amylophilus, Campylobacter sp. P0111, Campylobacter sp. RM9261, Campylobacter lanienae strain RM8001, Campylobacter lanienae strain P0121, Turicimonas muris, Legionella londiniensis, Salinivibrio sharmensis, Leptospira sp. isolate FW.030, Moritella sp.) isolate NORP46, Endozoicomonas genus S-B4-1U, Tamilnaduibacter salinas salinus), Vibrio natriegens, Arcobacter skirrowii, Francisella philomiragia, Francisella hispaniensis, or Parendozoicomonas haliclonae It may be derived from haliclonae.

[0031] Generally, Cas proteins contain at least one RNA-binding domain, which interacts with a guide RNA. The Cas protein may be a wild-type Cas protein or a modified version lacking nuclease activity or possessing only single-strand nicking activity. The Cas protein can be modified to increase nucleic acid binding affinity and / or specificity, alter enzymatic activity, and / or change other properties of the protein. For example, the nuclease (i.e., DNase, RNase) domain of the protein can be modified, deleted, or inactivated. Alternatively, the protein can be cleaved to remove domains not essential to its function. The protein's activity can also be optimized by cleaving or modification.

[0032] In some embodiments, the Cas protein may be a variant of the wild-type Cas protein (e.g., Cas9) or a fragment thereof. In other embodiments, the Cas protein may be obtained from a mutant Cas protein. For example, the amino acid sequence of the Cas9 protein may be modified to alter one or more properties of the protein (e.g., nuclease activity, affinity, stability, etc.). Alternatively, domains of the Cas9 protein that are not involved in RNA targeting may be removed from the protein so that the modified Cas9 protein is smaller than the wild-type Cas9 protein. In some embodiments, the system of the present invention utilizes a Cas9 protein derived from Streptococcus pyogenes, such as one encoded in bacteria or one that is codon-optimized for expression in mammalian cells.

[0033] A variant 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, fusion proteins, or combinations thereof. Such variants possess at least one or both of RNA-induced DNA-binding activity or RNA-induced nuclease activity. Generally, the modified version is at least 50% (e.g., any number between 50% and 100%, e.g., 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, and 99%) identical to the wild-type protein, e.g., SEQ ID NO: 1.

[0034] Cas protein (and other protein components described in this invention) are recombinant The recombinant polypeptide can be obtained as a polypeptide. To prepare the recombinant polypeptide, the nucleic acid encoding it can be ligated to another nucleic acid encoding a fusion partner, such as glutathione-s-transferase (GST), a 6x-His epitope tag, or the M13 gene 3 protein. The resulting fusion nucleic acid expresses a fusion protein in a suitable host cell, which can be isolated by methods known in the art. The isolated fusion protein can be further processed, for example by enzymatic degradation, to remove the fusion partner and obtain the recombinant polypeptide of the present invention. Alternatively, the protein can be chemically synthesized using conventional methods known in the art, or produced by recombinant DNA techniques described herein using methods known in the art.

[0035] The Cas protein described in the present invention may be provided in a purified or isolated form, or may be part of a composition. Preferably, in the case of a composition, the protein is first purified to a certain degree of purity, more preferably to a higher level of purity (e.g., about 80%, 90%, 95%, or 99%, or higher). The composition according to the present invention may be any type of desired composition, but is typically an aqueous composition suitable for use as a composition for RNA induction targeting or for encapsulation within a composition. Those skilled in the art are well aware of the various substances that can be included in such nuclease reaction compositions.

[0036] To carry out the methods disclosed herein for modifying target nucleic acids, the target protein can be produced in target cells by mRNA, protein-RNA complexes (RNPs), or any suitable expression vector. Examples of expression vectors include chromosomal, non-chromosomal, and synthetic DNA sequences, bacterial plasmids, minicircles, phage DNA, baculoviruses, yeast plasmids, vectors obtained from combinations of plasmids and phage DNA, and viral DNA, such as vaccinia, adenovirus, fowlpox virus, and pseudorabies virus. Further details are described in the Expression Systems and Methods section below.

[0037] As disclosed herein, nuclease-dead Cas9 (dCas9, e.g., from Streptococcus pyogenes D10A, H840A mutant protein) or nuclease-defective nickase Cas9 (nCas9, e.g., from Streptococcus pyogenes D10A mutant protein) can be used. dCas9 or nCas9 can also be obtained from various bacterial species. Table 1 lists a non-inclusive list of Cas9 examples and their corresponding PAM requirements. Synthetic Cas substitutes, such as those described in Rauch et al., Programmable RNA-Guided RNA Effector Proteins Built from Human Parts. Cell, Vol. 1, No. 178, June 27, 2019, pp. 122-134 e12, can also be used.

[0038] [Table 1]

[0039] UGI In some embodiments of the present disclosure, the sequence targeting components described above include a fusion between (a) a CRISPR protein and (b) a first uracil DNA glycosylase (UNG) inhibitor peptide (UGI). For example, the fusion protein may include a Cas protein fused to the UGI, such as a Cas9 protein. Such a fusion protein may exhibit increased nucleic acid editing efficiency compared to a fusion protein that does not contain a UGI domain. In some embodiments, the UGI includes a wild-type UGI sequence or one having the following amino acid sequence:sp|P14739|UNGI_BPPB2:Uracil-DNA glycosylase inhibitor (UGI)MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML Sequence ID No. 2.

[0040] In some embodiments, the UGI proteins provided herein include UGI fragments and proteins homologous to UGI or UGI fragments. For example, in some embodiments, UGI includes a fragment of the amino acid sequence described above. In some embodiments, UGI includes an amino acid sequence homologous to the amino acid sequence described above or an amino acid sequence homologous to a fragment of the amino acid sequence described in the above UGI sequence. In some embodiments, a protein containing UGI or a fragment of UGI or a homolog of UGI or a UGI fragment is referred to as a "UGI variant." UGI variants share homology to UGI or its fragments. For example, a UGI variant is at least about 70% (e.g., at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%) of wild-type UGI or a UGI sequence as described above.

[0041] Suitable UGI proteins and nucleotide sequences are provided herein, and further suitable UGI sequences are known to those skilled in the art, such as, for example, Wang et al., Uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 encodes a binding protein specific for uracil-DNA glycosylase. J Biol. Chem. 264:1163~1171 (1989); Lundquist et al., Site-directed mutagenesis and characterization of uracil-DNA glycosylase inhibitor protein. Role of specific ca rboxylic amino acids in complex formation with Escherichia coli uracil-DNA glycosylase. J Biol.Chem.272:21408~21419(1997);Ravishankar et al., X-ray analysis of a complex of Escherichia coli uracil DNA glycosylase (EcUDG) with a proteinaceous inhibitor.The structural elucidation of a prokaryotic UDG.Nucleic Acids Res. 26:4880~4887 (1998); and Putnam et al., Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and This includes the one published in its complex with Escherichia coli uracil-DNA glycosylase. J Mol. Biol. 287:331~346 (1999), the entire contents of which are incorporated herein by reference.

[0042] b. RNA scaffolds and effector mobilization for sequence recognition: The second component of the platform disclosed herein is an RNA scaffold having the following three subcomponents: a crRNA containing a guide RNA sequence, a trans-activated CRISPR RNA (tracrRNA), and an RNA motif with an extension sequence. This scaffold may be either a single RNA molecule or a complex of multiple RNA molecules. The crRNA containing the guide RNA of the RNA scaffold is linked to the tracrRNA by a repeat:anti-repeat region consisting of a 7bp lower stem and a 4bp upper stem with a 4nucleotide bulge structure inserted between them. When the RNA scaffold is expressed as a single molecule, the repeat:anti-repeat region is linked by a tetraloop containing four nucleotides, as shown in Figure 10B. When the RNA scaffold is expressed as multiple RNA molecules, the tetraloop is absent, and the repeat:anti-repeat region links the RNA molecules of crRNA and tracrRNA, as shown in Figure 10D.

[0043] As disclosed herein, a programmable guide RNA, a tracrRNA, and a crRNA comprising a Cas protein together form a CRISPR / Cas-based module for sequence targeting and recognition, while the RNA motif recruits effector molecules, such as base editing enzymes, via RNA-protein binding pairs, which then carry out gene modifications. Thus, the RNA scaffold connects the effector module (e.g., base editing enzyme) to the sequence recognition module (e.g., a type II Cas protein). The RNA scaffolds disclosed herein include one or more modifications.

[0044] Programmable Guide RNA (crRNA) One of the key sub-components is the programmable guide RNA. Due to its simplicity and efficiency, the CRISPR-Cas system has been used to perform genome editing in cells of various organisms. The specificity of this system is determined by base pairing between the target DNA and the custom-designed guide RNA. By manipulating and regulating the base pairing properties of the guide RNA, any desired sequence can be targeted, as long as a PAM sequence adjacent to the target sequence exists.

[0045] Among the subcomponents of the RNA scaffold disclosed herein, the guide sequence provides targeting specificity. It includes a region that is complementary to a pre-selected target site of interest and to which hybridization is possible. In various embodiments, the target specifying component of the guide sequence is The g component may contain approximately 10 to more than 25 nucleotides. For example, the base-pairing region between the guide sequence and the corresponding target site sequence may be approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25 nucleotides long. In exemplary embodiments, the guide sequence is approximately 17 to 20 nucleotides long, for example, 20 nucleotides.

[0046] Furthermore, crRNA has a constant 3' region of a targeting sequence. This sequence forms a repeat:anti-repeat stem that ligates crRNA to the tracrRNA component of the RNA scaffold. The constant 3' sequence of crRNA is complementary to the 5' sequence of tracrRNA, thus forming a double-stranded stem. The repeat:anti-repeat region of the RNA scaffold can be divided into three parts: the lower stem, the bulge, and the upper stem. The lower stem is a 7bp form with both Watson-Crick and non-Watson-Crick base pairing; it is accompanied by a bulge structure of 4 nucleotides that follows. The upper stem consists of a 4bp structure. When synthesized as a single RNA molecule, tracrRNA contains the anti-repeat region, tetraloop, and 3' constant region of sgRNA. When synthesized as separate RNA molecules, tracrRNA contains the anti-repeat region and 3' constant region of sgRNA, but the tetraloop is absent.

[0047] One requirement for selecting a suitable target nucleic acid is that it has a 3' PAM site / sequence. Each target sequence and its corresponding PAM site / sequence are referred to herein as Cas targeting sites. One of the best-characterized systems, a class 2 CRISPR system, such as type II enzymes, requires only the Cas9 protein and a guide RNA complementary to the target sequence that affects the target cleavage. For example, a class 2 type II CRISPR system of Streptococcus pyogenes, such as Cas9, uses a target site having N12-20NGG, where NGG represents the PAM site derived from Streptococcus pyogenes, and N12-20 directly represents the 12-20 nucleotides from 5' to the PAM site. Additional PAM site sequences derived from other bacterial species include NGGNG, NNNNGATT, NNAGAA, NNAGAAW, and NAAAAC. For example, U.S. Patent Application Publication No. 20140273233, International Publication No. 2013176772, Cong et al., (2012), Science 339 (6121):819~823, Jinek et al., (2012), Science 337 (6096):816~821, Mali et al., (2013), Science 339 (6121):823~826, Gasiunas et al., (2012), Proc Natl Acad Sci US A. 109 (39):E2579~E2586, Cho et al., (2013) Nature Biotechnology 31, 230~232, Hou et al., Proc Natl Acad Sci US A. September 24, 2013; 110(39):15644~9, Mojica et al., Microbiology. See March 2009;155(Pt 3):733~40 and www.addgene.org / CRISPR / . The contents of these documents are incorporated in their entirety hereby by reference.

[0048] The target nucleic acid strand can be either of two strands on the genomic DNA in the host cell. Examples of such genomic dsDNA include, but are not limited to, host cell chromosomes, mitochondrial DNA, and stably maintained plasmids. However, it should be understood that the method of the present invention can be carried out with other dsDNA present in the host cell, such as unstable plasmid DNA, viral DNA, and phagemid DNA, regardless of the nature of the host cell's dsDNA, as long as a Cas targeting site is present. The method of the present invention can also be carried out with RNA.

[0049] tracrRNA In addition to the guide sequences described above, the RNA scaffold of the present invention includes additional active or inactive subcomponents. For example, the scaffold has tracrRNA. For instance, the scaffold can be a hybrid RNA molecule in which the crRNA described above, including the programmable guide RNA, is fused to the tracrRNA to mimic a natural crRNA:tracrRNA double strand. Shown below is an exemplary hybrid crRNA:tracrRNA gRNA sequence, SEQ ID NO: 3:5'-(20nt guide)GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU-3'. Various tracrRNA sequences are known in the art, examples of which include the following tracrRNAs and their active moieties. When used herein, the active moiety of tracrRNA retains the ability to form complexes with Cas proteins such as Cas9, dCas9, or nCas9. Methods for producing crRNA-tracrRNA hybrid RNAs (also known as single guide RNAs or sgRNAs) are known in the art. In one embodiment in which the crRNA and tracrRNA are provided as a single gRNA (sgRNA), the two components are linked together by a tetrastem loop. In some embodiments, the repeat:antirepeat region is extended. On either side of the repeat:antirepeat region, there is an extension of 2, 3, 4, 5, 6, 7 bases or more than 7 bases. In a preferred embodiment, the repeat:antirepeat region has a 7-nucleotide extension on either side of the upper stem, as shown in Figures 10C and 10D. The 7-base extension on either side of the upper stem results in a region that is 14 base pairs longer. If the RNA scaffold is synthesized as a single RNA molecule, as shown in Figure 10C, a 7-base extension on either side of the upper stem results in an upper stem having a total of 11 bases on either side and a total length of 22 nucleotides.When the RNA scaffold is synthesized as two separate RNA molecules, as shown in Figure 10D, a 7-base extension on either side of the upper stem results in an upper stem having a total of 11 bases on either side and a total length of 25 nucleotides. In one embodiment, when the RNA scaffold is synthesized as a single RNA molecule, the total length of the upper stem of the repeat:anti-repeat region is 22 nucleotides. In another embodiment, when the RNA scaffold is synthesized as two separate RNA molecules, the total length of the upper stem of the repeat:anti-repeat region is 25 nucleotides. In yet another embodiment, the extension can be more than 7 bases.

[0050] For example, see International Publication No. 2014099750, U.S. Patent Application Publication No. 20140179006, and U.S. Patent Application Publication No. 20140273226. The contents of these documents are incorporated herein by reference in their entirety. TracrRNA sequences for Streptococcus pyogenes Cas9 with various truncations and extensions are shown below.

[0051] [ka]

[0052] In some embodiments, the tracrRNA is derived from Strep pyogenes.

[0053] In some embodiments, tracrRNA and crRNA containing a guide sequence are two separate RNA molecules that together form a functional guide RNA and part of an RNA scaffold. In this case, tracrRNA must be able to interact (usually by base pairing) with crRNA containing a guide sequence in order to form the two parts of guide crRNA:tracrRNA.

[0054] RNA motif A third subcomponent of the RNA scaffold is the RNA motif, which substantially recruits the effector module (base editing enzyme) to the target DNA. The RNA motif is also called the recruiting RNA motif. This binding is important for the gene editing systems and methods disclosed herein. The RNA scaffold disclosed herein may have one or more RNA motifs.

[0055] Prior art methods for recruiting effector / DNA editing enzymes to target sequences involve the direct fusion of the effector protein to dCas9. While direct fusion of the effector enzyme (e.g., dCas9) to the protein required for sequence recognition has been successful in activating or repressing sequence-specific transcription, protein-protein fusion designs can introduce spatial constraints, which are not ideal for enzymes that need to form multi-complexes for their activity. Indeed, most nucleotide editing enzymes (e.g., AID or APOBEC3G) require the formation of dimers, tetramers, or higher-position oligomers for their DNA editing catalytic activity. Direct fusion to dCas9, which anchors to DNA in a fixed conformation, would hinder the formation of a functional oligomeric enzyme complex at the correct location.

[0056] In contrast, the RNA scaffold-mediated recruitment systems and methods provided herein are based on the recruitment of RNA scaffold-mediated effector proteins. More specifically, the platform utilizes various RNA motif / RNA-binding protein binding pairs. For this purpose, aptamer-binding molecules, such as RNA-binding proteins (e.g., MS2 envelope protein, MCP), specifically bind to RNA motifs (e.g., MS2 operandi). The RNA scaffold is designed such that the RNA motif is linked to the RNA scaffold via a linker sequence at the 3' end of the tracrRNA. The linker may be single-stranded RNA or a chemical bond. In one embodiment, the single-stranded linker contains 0 to 10 nucleotides, preferably 2 to 6 nucleotides. The single-stranded sequence may contain GC nucleotides. Advantageously, the linker, e.g., the single-stranded linker, separates the loop of the RNA motif from the loop of the bulky stem of the tracrRNA. One or more RNA motifs disclosed herein have an extension sequence. In a preferred embodiment, the extension sequence is a double-stranded extension. The extension sequence varies in length, containing 2 to 24 nucleotides. In some embodiments, the one or more RNA motifs include one or more modifications. The one or more modifications may be at the 5' and / or 3' ends of the one or more RNA motifs.

[0057] As a result, the RNA scaffold components of the platform disclosed herein are engineered RNA molecules, which include not only crRNA for specific DNA / RNA sequence recognition and tracrRNA for Cas protein binding, but also RNA motifs for effector recruitment (Figure 1B). Thus, recruited effector modules can be recruited to target sites by their ability to bind to RNA motifs. Due to the flexibility of RNA scaffold-mediated recruitment, functional monomers, and dimers, tetramers, or oligomers, can be formed relatively easily near target DNA or RNA sequences. These RNA motif / binding protein pairs can be obtained from naturally occurring sources (e.g., RNA phages, or yeast telomerase) or can be artificially designed (e.g., RNA aptamers and their corresponding binding protein ligands). A non-comprehensive list of examples of recruiting RNA motif / RNA binding protein pairs that can be used in the methods and systems provided herein is summarized in Table 2.

[0058] [Table 2]

[0059] The sequences for the above join pairs are listed below.

[0060] 1. Telomerase Ku-binding motif / Ku heterodimer a. Ku-bonded hairpin

[0061] [ka]

[0062] [ka]

[0063] The ">" symbol separates the two dimers. 2. Telomerase Sm7 binding motif / Sm7 homoheptamer a. Sm consensus site (single strand)

[0064] [ka]

[0065] [ka]

[0066] 3. MS2 phage operator stem-loop / MS2 outer layer protein a. MS2 phage operator stem loop

[0067] [ka]

[0068] [ka]

[0069] 4. PP7 phage operator stem-loop / PP7 coat protein a. PP7 phage operator stem loop

[0070] [ka]

[0071] [ka]

[0072] 5. SfMu Com stem-loop / SfMu Com binding protein a. SfMu Com stem loop

[0073] [ka]

[0074] a. SfMu Com-binding protein SEQ ID NO: 20

[0075] [ka]

[0076] The RNA scaffold can be either a single RNA molecule or a complex of multiple RNA molecules. For example, the guide RNA, tracrRNA, and RNA motif may be three segments of a single long RNA molecule. Alternatively, one, two, or three of them may be on another molecule. In the latter case, the three components can be linked together to form the scaffold via covalent or non-covalent bonding, such as Watson-Crick base pairing.

[0077] In one embodiment, the RNA scaffold may include two separate RNA molecules. The first RNA molecule may include a crRNA comprising a programmable guide RNA and a region that can form a stem double structure together with a complementary region. The second RNA molecule may include a tracrRNA and an RNA motif, in addition to a complementary region. Through this stem double structure, the first and second RNA molecules form the RNA scaffold of the present invention. In one embodiment Each of the first and second RNA molecules contains a sequence (approximately 6 to 20 nucleotides) in which bases pair with the sequence of the other. Similarly, tracrRNA and RNA motifs can reside on different RNA molecules and be carried along with other stem double structures.

[0078] The RNA and associated scaffolds of the present invention can be prepared by various methods known in the art, such as cell-based expression, in vitro transcription, and chemical synthesis, or a combination thereof. The ability to chemically synthesize relatively long RNAs (200-mers or longer) makes it possible to produce RNAs with specialized functions superior to those possible with the four basic ribonucleotides (A, C, G, and U).

[0079] The Cas protein-guide RNA scaffold complex can be prepared by recombinant techniques or in vitro translation-transcription systems using host cell systems known in the art. Details of such systems and techniques can be found, for example, in International Publication Nos. 2014144761, 2014144592, 2013176772, U.S. Patent Application Publication No. 20140273226, and U.S. Patent Application Publication No. 20140273233, the contents of which are incorporated herein by reference in their entirety. The complex can be isolated or purified, at least to some extent, from the cellular material of a cell or from the in vitro translation-transcription system in which it was produced.

[0080] qualification The RNA scaffolds disclosed herein may include one or more modifications.

[0081] Such modifications may include the inclusion and / or removal of at least one non-naturally occurring nucleotide, a modified nucleotide, or an analog thereof. Examples of such modifications, but not limited to, include the addition of nucleotides to extend sequences, nucleotide substitutions, the addition of linker sequences, the removal of nucleotides, and alteration of the positioning of various components of the RNA scaffold. One or more modifications may be to the backbone and / or sugar moieties of the RNA scaffold.

[0082] Nucleotides can be modified in the ribose, phosphate, and / or base moieties. Modified nucleotides may include 2'-O-methyl analogs, 2'-fluoro analogs, or 2'-deoxy analogs, or 2'-ribose analogs. The nucleic acid backbone can be modified; for example, a phosphorothioate backbone may be used. The use of locked nucleic acids (LNAs) or bridged nucleic acids (BNAs) may also be possible. Further examples of modified bases, but not limited to these, include 2-aminopurines, 5-bromouridine, 5-methylcytidine, 5-methoxyuridine, pseudouridine, inosine, and 7-methylguanosine. These modifications can be applied to any component of the RNA scaffold. These modifications can be applied to any component of the CRISPR system. In preferred embodiments, these modifications are made to RNA components, such as guide RNA sequences.

[0083] In some embodiments, the RNA scaffold or its subsections described above may include one or more modifications, such as base modifications and skeletal modifications, to provide nucleic acids having novel or enhanced features (e.g., improved stability).

[0084] Modified skeletons and modified nucleoside bonds Suitable nucleic acids containing modifications include nucleic acids containing modified skeletons, bases, sugars, or non-natural nucleoside bonds. Nucleic acids (having a modified skeleton) have phosphorus in the skeleton. This includes materials that retain atoms and materials that do not have phosphorus atoms in their skeleton.

[0085] Suitable modified oligonucleotide skeletons containing a phosphorus atom include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, as well as methyl and other alkyl phosphonates such as 3'-alkylene phosphonates, 5'-alkylene phosphonates, and chiral phosphonates, phosphineates, phosphoramidates such as 3'-aminophosphoamides and aminoalkylphosphoamides, phosphorodiamidates, thionophosphoamides, thionoalkyl phosphonates, thionoalkyl phosphotriesters, selenophosphates and boranophosphates having a normal 3'-5' bond, their 2'-5' bond analogues, and those having inverted polarity where one or more internucleotide bonds are 3'-3', 5'-5', or 2'-2' bonds. Suitable oligonucleotides with reverse polarity include a single 3'-3' bond in most internucleotide bonds, i.e., a single reverse nucleoside remains, which can be basic (the nucleic acid base either lacks a hydroxyl group or has a hydroxyl group in place). Various salts (e.g., potassium or sodium), mixed salts, and free acid forms are also included.

[0086] In some embodiments, the nucleic acid of the subject comprises one or more phosphorothioate and / or heteroatom nucleoside bonds, particularly -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2- (known as the methylene (methylimino) or MMI skeleton), -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -ON(CH3)-CH2-CH2- (in this case, the natural phosphate diester nucleotide bond is represented as -OP(=O)(OH)-O-CH2-). MMI-type nucleoside bonds are disclosed in U.S. Patent No. 5,489,677, referenced above. Preferred amide nucleoside bonds are disclosed in U.S. Patent No. 5,602,240.

[0087] For example, nucleic acids having a morpholino backbone structure, such as that disclosed in U.S. Patent No. 5,034,506, are also preferred. For example, in some embodiments, the nucleic acid of the subject includes a six-membered morpholino ring instead of a ribulose ring. In some of these embodiments, phosphorodiamidate or other non-phosphodiester nucleoside bonds are replaced with phosphodiester bonds.

[0088] Suitable modified polynucleotide skeletons that do not contain phosphorus atoms have skeletons formed by short-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short-chain heteroatoms or heterocyclic nucleoside bonds. These include those having morpholino bonds (partially formed from the sugar moiety of the nucleoside); siloxane skeletons; sulfide, sulfoxide, and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; riboacetyl skeletons; alkene-containing skeletons; sulfamate skeletons; methyleneimino and methylenehydrazino skeletons; sulfonate and sulfamide skeletons; amide skeletons; and others having mixed N, O, S, and CH2 constituent parts.

[0089] Imitations The nucleic acids of the subject may be nucleic acid mimes. The term “mime,” when applied to polynucleotides, is intended to encompass polynucleotides in which only the furanose ring or both the furanose ring and the internucleotide bond are replaced with non-furanose groups; substitution of only the furanose ring is also called a sugar substitute in the art. Heterocyclic base moiety Alternatively, the modified heterocyclic base moiety is maintained for hybridization with a suitable target nucleic acid. One such nucleic acid, a polynucleotide mimeograph known to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PAN, the sugar backbone of the polynucleotide is replaced by an amide atom-containing backbone, particularly an aminoethylglycine backbone. The nucleotide is maintained and directly or indirectly binds to the aza nitrogen atom of the amide portion of the backbone.

[0090] One polynucleotide mimeograph reported to possess excellent hybridization properties is peptide nucleic acid (PNA). The backbone of a PNA compound is two or more linked aminoethylglycine units, which give PNA to an amide-containing backbone. The heterocyclic base moiety directly or indirectly binds to the aza nitrogen atom of the amide moiety of the backbone. Representative U.S. patents describing the preparation of PNA compounds include, but are not limited to, U.S. Patents 5,539,082; 5,714,331; and 5,719,262.

[0091] Another class of polynucleotide mimetics being studied is based on linked morpholino units (morpholino nucleic acids) having heterocyclic bases bonded to a morpholino ring. Several linking groups have been reported for linking morpholino monomer units in morpholino nucleic acids. One class of linking groups has been selected to give nonionic oligomeric compounds. Nonionic morpholino-based oligomeric compounds are less likely to have undesirable interactions with cellular proteins. Morpholino-based polynucleotides are nonionic mimetics of oligonucleotides that are less likely to form undesirable interactions with cellular proteins (Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503~4510). Morpholino-based polynucleotides are disclosed in U.S. Patent No. 5,034,506. Various compounds belonging to the morpholino class of polynucleotides, having various different linking groups that connect monomeric subunits, have been prepared.

[0092] A further class of polynucleotide mimetics is called cyclohexenyl nucleic acid (CeNA). The furanose ring normally present in DNA / RNA molecules is replaced by a cyclohexenyl ring. CeNA DMT-protected phosphoramidite monomers have been prepared and used in the synthesis of oligomeric compounds based on classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides with specific CeNA-modified positions have been prepared and studied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). Generally, the incorporation of CeNA monomers into DNA strands increases the stability of DNA / RNA hybrids. CeNA oligoadenylates formed complexes with RNA and DNA complement that exhibited stability similar to that of the native complex. Studies of incorporating CeNA structures into native nucleic acid structures have been demonstrated by NMR and circular dichroism to facilitate conformational adaptation.

[0093] Further modifications include locked nucleic acids (LNAs) in which a 2'-hydroxyl group is bonded to the 4' carbon atom of the sugar ring, thereby forming a 2'-C,4'-C-oxymethylene bond and thus a bicyclic sugar moiety. This bond can be methylene (-CH2-), where the group bridges the 2' oxygen atom and the 4' carbon atom, and n is either 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455~456). LNAs and LNA analogs exhibit very high bithermal stability with complementary DNA and RNA (Tm = +3 to +10°C), stability against 3'-exonuclease degradation, and good solubility. LANs containing potent non-toxic antisense oligonucleotides have been described (Wahlestedt et al., Proc. Na tl. Acad. Sci. USA, 2000, 97, 5633~5638).

[0094] The synthesis and preparation of LNA monomers adenine, cytosine, guanine, 5-methylcytosine, thymine, and uracil, as well as their oligomerization and nucleic acid recognition properties, are described (Koshkin et al., Tetrahedron, 1998). (54, 3607-3630). LNA and its preparation are also described in International Publication Nos. 98 / 39352 and International Publication Nos. 99 / 14226.

[0095] Modified sugar portion The nucleic acid of the subject may also contain one or more substituted sugar moieties. Preferred polynucleotides include sugar substituents selected from: OH; H; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-Co-alkyl, in which case alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-C 10 Alkyl or C2-C 10 They can be alkenyls and alkynyls. Particularly preferred are O((CH2) n O)m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON((CH2) n CH3)2, where n and m are from 1 to about 10. Other suitable polynucleotides are as follows: C1 - C 10 Lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O - alkaryl or O - aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, a group that improves the pharmacokinetic properties of the oligonucleotide, or a group that improves the pharmacodynamic properties of the oligonucleotide, and sugar substituents selected from other substituents having similar properties. Suitable modifications include 2'-methoxyethoxy (also known as 2'-O - CH2CH2OCH3, 2'-O-(2 - methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486 - 504), i.e., an alkoxyalkoxy group. Further suitable modifications include 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group (also known as 2'-DMAOE as described in the examples below), and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O - dimethyl - amino - ethoxy - ethyl or 2'-DMAEOE), i.e., 2'-O - CH2 - O - CH2 - N(CH3)2. <00004S8>

[0096] Other suitable sugar substituents include methoxy(-O-CH3), aminopropoxy(-OCH2CH2CH2NH2), allyl(-CH2-CH=CH2, -O-allylCH2-CH=CH2), and fluoro(F). The 2'-sugar substituent can be at the arabino(upper) or ribo(lower) position. A preferred 2'-arabino modification is 2'-F. Similar modifications can be made at other positions of the oligomeric compound, particularly on the 3'-terminal nucleotide or at the 3' position of the sugar and the 5' position of the 5'-terminal nucleotide in 2'-5' bonded oligonucleotides. The oligomeric compound may also have sugar mimetic compounds, such as a cyclobutyl moiety instead of pentofuranosyl sugar.

[0097] Base modification and substitution The nucleic acids of the subject matter may also include modifications or substitutions of nucleic acid bases (often simply referred to in the art as “bases”). As used herein, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases are, Other synthetic and natural nucleic acid bases, e.g., 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3)uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thiouracil This includes mine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, in particular 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, as well as 3-deazaguanine and 3-deazaadenine, etc. Further modified nucleic acid bases include tricyclic pyrimidines, such as phenoxazinecytidine (1H-pyrimido(5,4-b)(1,4)benzoxazine-2(3H)-one), phenothiazinecytidine (1H-pyrimido(5,4-b)(1,4)benzothiadin-2(3H)-one), G-clamps, such as substituted phenoxazinecytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazine-2(3H)-one), carbazolecytidine (2H-pyrimido(4,5-b)indole-2-one), pyridoindolecytidine (H-pyrimido(3',2':4,5)pyrrolo(2,3-d)pyrimidine-2-one), and others.

[0098] The heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced by other heterocyclic bases, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Further nucleic acid bases are disclosed in U.S. Patent No. 3,687,808, The Concise Encyclopedia of Polymer Science and Engineering, pp. 858-859, edited by Kroschwitz, JI, and published by John Wiley & Sons. This includes what is disclosed in 1990, what is disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and what is disclosed by Sanghvi, YS, Chapter 15, Antisense Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B., eds., CRC Press, 1993. Some of these nucleic acid bases are useful for increasing the binding affinity of oligomeric compounds. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase the stability of nucleic acid double helix by 0.6-1.2°C (Sanghvi et al., ed., Antisense). (Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), for example, it is a suitable base substitution when combined with 2'-O-methoxyethyl sugar modification.

[0099] The modifications disclosed herein can be incorporated into various locations on the RNA scaffold, such as the tetraloop of sgRNA, the repeat:antirepeat region of the crRNA:tracrRNA component, any location on the tracrRNA, such as the 5' end, 3' end, stemloop 1, 2, or 3, and the RNA motif. The modifications disclosed herein, but are not limited to, the extension of the repeat:antirepeat of the two-part sgRNA or crRNA:tracrRNA component, the positioning of the RNA motif at the 3' end of the tracrRNA motif, the linking of the RNA motif to the CRISPR motif by a linker, the modification of nucleotides of the RNA motif, and R One possible explanation is that it's an extension of the NA motif.

[0100] Positioning of RNA motifs RNA motifs can be located at various positions on the RNA scaffold, as described in Example 1. The RNA scaffold of the present invention may have one MS2 RNA motif or two MS2 RNA motifs. The RNA motif, e.g., an MS2 aptamer, can be located at the 3' end of tracrRNA, the tetraloop of sgRNA, the stemloop 2 of tracrRNA, and the stemloop 3 of tracrRNA. The positioning of the aptamer, e.g., an MS2 aptamer, is important due to steric hindrance caused by bulky loops. In a preferred embodiment, the MS2 aptamer is located at the 3' end of the CRISPR motif. Advantageously, the positioning of the MS2 aptamer at the 3' end of the CRISPR motif is via a gap, thus reducing steric hindrance from other bulky loops on the RNA scaffold.

[0101] Linker An RNA motif can be bound to a tracrRNA motif via a linker. This linker may be single-stranded RNA or a chemical bond. The single-stranded RNA linker may have 2, 3, 4, 5, 6, 7, or more than 7 nucleotides. Advantageously, the linker sequence provides flexibility to the RNA scaffold. The linker sequence may contain GC nucleotides.

[0102] Modification of RNA motif nucleotides Modifications can be made to RNA motifs, such as aptamer sequences. In preferred embodiments, the RNA motif includes one or more modifications. For example, preferred modifications are for C-5 and F-5 aptamer variants. In preferred embodiments, the modification to the aptamer is the substitution of adenine at position 10 with 2-aminopurine (2-AP). Advantageously, the substitution induces a conformational change, resulting in higher affinity compared to wild-type MS2. While we do not wish to be bound by any theory, the conformational change induced by 2-AP is thought to result in the formation of a hydrogen bond between the extracyclic amino group of the 2-AP nucleotide at position 10 and carbonyl B59 in the backbone. The replacement of the MS2 hairpin sequence with a higher affinity MS2 sequence is thought to result in increased gene editing efficiency because the amino acid substitution helps align the RNA stem-loop to a conformation that is more recognized by the coat protein.

[0103] Suitable modifications to RNA motifs include, for example, 2'-deoxy-2-aminopurine, 2'-ribose-2-aminopurine, phosphorothioate modifications, 2'-O-methyl modifications, 2'-fluoro modifications, and LNA modifications, as listed above. Advantageously, the modifications help increase stability and promote stronger binding / folding of the desired hairpin structure.

[0104] Other suitable modifications may be at the 5' and / or 3' ends of the one or more RNA motifs.

[0105] RNA motif extension The length of RNA motif extensions can be variable. Extensions to RNA motifs can range from 2 to 24 nucleotides. Extensions to RNA motifs can range from more than 24 nucleotides. Figures 3A-D show several extensions to recruiting RNA motifs compared to wild-type MS2, and the sequences for the extensions are shown below. Figure 3A shows a 4-nucleotide (2 bp) extension, which results in a stem with 23 nucleotides in total length (SEQ ID NO: 21). Figure 3B shows a 10-nucleo extension. Figure 3C shows a 16-nucleotide (8 bp) extension, which results in a stem with 29 nucleotides in total length (SEQ ID NO: 22). Figure 3D shows a 26-nucleotide (13 bp) extension, which results in a stem with 45 nucleotides in total length (SEQ ID NO: 24). Advantageously, the extension of the RNA motif increases the flexibility of the motif. The extension of the RNA motif can be double-stranded or single-stranded. Double-stranded extension provides greater stability of the RNA scaffold. In a preferred embodiment, the extension of the RNA motif is double-stranded.

[0106] Sequences for RNA motif extension:

[0107] [ka]

[0108] key: GC linkers are underlined, nucleotide extensions are shown in bold, and aptamers are in italics.

[0109] Repeat: Anti-repeat area crRNA and tracrRNA may be provided as sgRNA or as two separate components. crRNA hybridizes to tracrRNA via a repeat:antirepeat region. The repeat region of crRNA hybridizes to the antirepeat region of tracrRNA. The repeat:antirepeat region may be extended to increase the flexibility, correct folding, and stability of the components. The repeat:antirepeat region may be extended by 2, 3, 4, 5, 6, 7 bases or more than 7 bases on either side of the region. The repeat:antirepeat region may be extended by a total of 14 nucleotides. The repeat:antirepeat may also include other modifications disclosed above.

[0110] combination of modifiers An RNA scaffold may have one or more of the modifications mentioned above. One or more modifications to an RNA scaffold may be one or more of the modifications mentioned above, for example, an extension of a repeat:anti-repeat region, an extension of a recruiting RNA motif, or a nucleotide substitution to 2AP. The one or more modifications may be on different components of the RNA scaffold, for example, an extension of the repeat:anti-repeat region or two-part crRNA:tracrRNA of sgRNA, and an extension of an RNA motif. The one or more modifications may be on the same component of the RNA scaffold, for example, an extension of an RNA motif and a nucleotide substitution of an RNA motif. There may be two or more, three or more, four or more, or five or more modifications. In one embodiment, the modifications may be an extension of an RNA motif and / or a nucleotide substitution of an RNA motif. For example, the modifications may be an extension of an RNA motif or a nucleotide substitution of an RNA motif. In other cases, the RNA motif may have an extended length and nucleotide substitutions.

[0111] Aptamer In some embodiments, the aptamer-binding protein may be the wild-type protein, a variant of the wild-type protein, or a variant thereof. An example of the RNA motif used herein is the MS2 aptamer. The RNA motif binds to an aptamer-binding molecule. The MS2 motif specifically binds to the MS2 bacteriophage coat protein (MCP). An in vitro selection process was repeated to obtain a family of aptamers. Two of the aptamer family members include the MS2 C-5 variant and the MS2 F-5 variant. One notable difference between wild-type MS2 and the C-5 and F-5 variants is the substitution of a uracil nucleotide to cytosine at position 5 of the aptamer loop. The F-5 variant has been reported to have a higher affinity for the coat protein compared to the wild-type and other members of the aptamer family. Preferably, both the C-5 and F-5 variants are used as aptamers in the present invention. In one embodiment, the MS2 aptamer is wild-type MS2, the variant MS2, or a variant thereof. In another embodiment, the MS2 aptamer comprises C-5 and / or F-5 variants. The MS2 protein bound to the CRISPR motif may be a single copy (i.e., one MS2 loop) or a double copy (i.e., two MS2 loops). In a preferred embodiment, the RNA scaffold has one RNA motif. In other embodiments, the RNA scaffold has two or more, three or more, or four or more RNA motifs. In yet another embodiment, the RNA scaffold has two RNA motifs.

[0112] C. Effector Module A third component of the platform disclosed in the present invention is a non-nuclease effector. The effector module disclosed herein includes an RNA motif and an RNA-binding domain that can bind to the effector domain. Effector domains used herein include, but are not limited to, enzymes, reporters, tags, molecules, proteins, microparticles, and nanoparticles. In one embodiment, the effector domain is a DNA-modifying enzyme.

[0113] The effector is not a nuclease and does not possess any nuclease activity, but may possess other types of DNA modifying enzyme activity, such as base editing. Examples of enzymatic activity, but not limited to these, include deamine activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, dismutase activity, nickase activity, alkylation activity, depurine activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, fotriase activity, or glycosylase activity. In some embodiments, the effector has cytidine deaminase (e.g., AID, APOBEC3G), adenosine deaminase (e.g., ADA), DNA methyltransferase, and DNA demethylase activity. In some embodiments, the effector is derived from different vertebrate species having different activity properties.

[0114] In a preferred embodiment, this third component is a conjugate or fusion protein having an RNA-binding domain and an effector domain. These two domains may be joined via a linker.

[0115] In some embodiments, effectors are not required for certain cell types (e.g., cancer cell overexpression deaminase). In such cases, endogenous effectors (e.g., APOBEC, AID, etc.) can be gene-edited to include the recruitment module, and therefore, exogenous editors are also not required. This means that the desired editor can be expressed. Applicable to cell types—e.g., lymphocyte-like (B+T) cells and certain cancer cells. In addition, nickase activity does not necessarily have to originate from the Cas module but can be recruited from effectors—for example, dCas9 may have aptamers to recruit both nickase and editor by the same gRNA recruitment. The effector proteins used herein may be wild-type enzymes, genetically modified enzymes, or chimeric enzymes.

[0116] RNA-binding domain While various RNA-binding domains can be used in the present invention, the RNA-binding domains of Cas proteins (e.g., Cas9) or their variants (e.g., dCas9) should not be used. As mentioned above, direct fusion to dCas9, which anchors to DNA in a fixed conformation, would prevent the formation of a functional oligomeric enzyme complex at the correct location. Instead, the present invention utilizes various other RNA motif / RNA-binding protein binding pairs. Examples of these are listed in Table 2.

[0117] In this method, effector proteins can be recruited to the target site by the ability of their RNA-binding domain to bind to a recruiting RNA motif. Due to the flexibility of RNA scaffold-mediated recruitment, functional monomers, and dimers, tetramers, or oligomers can be formed relatively easily near the target DNA or RNA sequence.

[0118] Effector Domain An effector component includes an active site, i.e., an effector domain. In one embodiment, effector domains used herein include, but are not limited to, enzymes, reporters, tags, molecules, proteins, microparticles, and nanoparticles. In some embodiments, the effector domain includes a naturally occurring active site in a non-nuclease protein (e.g., deaminase). In other embodiments, the effector domain includes a modified amino acid sequence (e.g., substitution, deletion, insertion) of a naturally occurring active site in a non-nuclease protein. The effector domain has enzymatic activity. Examples of this activity include deamine activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photoryase activity, glycosylase activity, DNA methylation activity, histone acetylation activity, or histone methylation activity. Some modifications in non-nuclease proteins (e.g., deaminases) can help reduce off-target effects. For example, as described below, mutating Ser38 in AID to Ala can reduce the recruitment of AID to off-target sites.

[0119] Linker The two domains mentioned above and others disclosed herein can be joined by linkers, for example, but not limited to, chemical modifications, peptide linkers, chemical linkers, covalent or non-covalent bonds, or protein fusion, or any means known to those skilled in the art. Such joining may be permanent or reversible. See, for example, U.S. Patent Nos. 4,625014, 5057301, and 5514363, U.S. Patent Publication Nos. 20150182596 and 20100063258, and International Publication No. 2012142515, the contents of which are incorporated herein by reference in their entirety. In some embodiments... Several linkers can be included to utilize the desired properties of each linker and protein domain in the conjugate. For example, flexible linkers and linkers that increase the solubility of the conjugate are intended for use alone or in combination with other linkers. Peptide linkers can be conjugated to one or more protein domains in the conjugate by expressing DNA that encodes the linker. The linkers can be acid-cleavable, photocleavable, and heat-sensitive linkers. Methods of conjugation are well known to those skilled in the art and are incorporated for use in the present invention.

[0120] In some embodiments, the RNA-binding domain and the effector domain can be joined by a peptide linker. The peptide linker can be joined by expressing a nucleic acid encoding the two domains and the linker in frame. If necessary, the linker peptide can be joined at either the amino-terminus, carboxyl-terminus, or both of the domains. In some examples, the linker is an immunoglobulin hinge region linker disclosed in U.S. Patent No. 6,165,476, No. 5,856,456, U.S. Patent Application Publication No. 20150182596, No. 2010 / 0063258, and International Application No. 2012 / 142515, each of which is incorporated herein by reference in its entirety.

[0121] Other domains Effector fusion proteins may contain other domains. In certain embodiments, effector fusion proteins may contain at least one nuclear localization signal (NLS). Generally, NLSs consist of a stretch of basic amino acids. Nuclear localization signals are known in the art (see, for example, Lange et al., J. Biol. Chem., 2007, 282:5101-5105). NLSs may be located at the N-terminus, C-terminus, or internal position of the fusion protein.

[0122] In some embodiments, the fusion protein may include at least one cell-permeable domain to facilitate protein delivery to target cells. In one embodiment, the cell-permeable domain may be a cell-permeable peptide sequence. Various cell-permeable peptide sequences are known in the art, examples of which include those of the HIV-1 TAT protein, human HBV TLM, Pep-1, VP22, and polyarginine peptide sequences.

[0123] In further other embodiments, the fusion protein may include at least one marker domain. Non-limiting examples of marker domains include fluorescent proteins, purified tags, and epitope tags. In some embodiments, the marker domain may be a fluorescent protein. In other embodiments, the marker domain may be a purified tag and / or an epitope tag. See, for example, U.S. Patent Application Publication No. 20140273233.

[0124] In one embodiment, AID was used as an example to demonstrate how the system works. AID is a cytidine deaminase that can catalyze the deaminement reaction of cytidine in relation to DNA or RNA. When delivered to a targeted site, AID changes C bases to U bases. In dividing cells, this can cause a point mutation from C to T. Alternatively, the change from C to U can trigger the cellular DNA repair pathway, primarily the excision repair pathway, which will remove the mismatched U-G base pair and replace it with a TA, AT, CG, or GC pair. As a result, a point mutation will occur at the target CG site. The excision repair pathway is used in most, if not all, cell cells. Because it is present in cells, the recruitment of AID to the target site can modify the CG base pair to another. In that case, if the CG base pair is a fundamental disease-causing gene mutation in somatic tissue / somatic cells, the approach described above can be used to modify the mutation, thereby treating the disease.

[0125] Similarly, if the underlying disease-causing gene mutation is an AT base pair at a specific site, the same approach can be used to recruit adenosine deaminase to that specific site, in which case adenosine deaminase can modify the AT base pair to another. Other effector enzymes are expected to produce other types of modifications at the base pair. A non-comprehensive list of examples of DNA / RNA modifying enzymes is detailed in Table 3.

[0126] [Table 3]

[0127] The three specific components described above constitute the technical platform. Each component can be selected from the list in Tables 1-3 to achieve a specific therapeutic / utility objective.

[0128] The RNA scaffold-mediated recruitment system can be constructed using (i) dCas9 / nCas9 derived from Streptococcus pyogenes as a sequence-targeting protein, (ii) an RNA scaffold containing a guide RNA sequence, tracrRNA, and crRNA containing an RNA motif, such as an MS2 operator motif, and (iii) an effector module containing human AID fused to the MS2 operator-binding protein MCP. The sequences for these components are listed below:

[0129] [ka]

[0130] [ka]

[0131] [ka]

[0132] [ka]

[0133] RNA scaffold expression cassette (Streptococcus pyogenes) containing a 20-nucleoside programmable sequence, a CRISPR RNA motif (tracrRNA), and an MS2 operator motif:

[0134] [ka]

[0135] (N 20 : Programmable sequence; underlined: CRISPR RNA motif (tracrRNA); Bold: MS2 motif; Italic: Terminator; Bold italic: GC Linker; Bold underline: Extension to MS2) The above RNA scaffold contains one MS2 loop (1xMS2). Below is an RNA scaffold containing two MS2 loops (2xMS2), in which case the MS2 scaffold is underlined:

[0136] [ka]

[0137] Effector AID-MCP Fusion:

[0138] [ka]

[0139] key:

[0140] [ka]

[0141] [ka]

[0142] As with the Cas protein described above, non-nuclease effectors can also be obtained as recombinant polypeptides. Techniques for producing recombinant polypeptides are well known in the art.

[0143] As described herein, mutating Ser38 to Ala in AID can reduce the recruitment of AID to off-target sites. The DNA and protein sequences of both wild-type AID and AID_S38A (phosphorylation-deficient, pnAID) are listed below: wtAID cDNA (Underlined bold text indicates Ser38 codon)

[0144] [ka]

[0145] wtAID protein (underlined in bold is Ser38):

[0146] [ka]

[0147] AID_S38A cDNA (Underlined bold indicates S38A variant)

[0148] [ka]

[0149] AID_S38A protein (underlined in bold indicates S38A variant)

[0150] [ka]

[0151] Exemplary arrangement Some exemplary sequences developed in this study are shown below. A Protein sequence of the RNA scaffold-mediated recruitment system nu construct (SEQ ID NO: 35):

[0152] [ka]

[0153] key:

[0154] [ka]

[0155] A Protein sequence of the RNA scaffold-mediated recruitment system nu.2 construct (SEQ ID NO: 36)

[0156] [ka]

[0157] key:

[0158] [ka]

[0159] Protein sequence of the RNA scaffold-mediated recruitment system (SEQ ID NO: 37):

[0160] [ka]

[0161] key:

[0162] [ka]

[0163] The 2xUGI base editor sequence is represented by sequence number 186.

[0164] Several exemplary RNA sequences of the gRNA constructs used in this study are shown below. Each contains one or two copies of the 5' end to the 3' end, a customizable target, a gRNA scaffold, and an MS2 aptamer. 1. Sequence of the gRNA_MS2 construct (SEQ ID NO: 38):

[0165] [ka]

[0166] key:

[0167] [ka]

[0168] 2. Sequence of the gRNA_2xMS2 construct (SEQ ID NO: 39):

[0169] [ka]

[0170] key:

[0171] [ka]

[0172] The three components of the platform / system disclosed herein can be expressed using one, two, or three expression vectors. The system can be programmed to target substantially any DNA or RNA sequence. Similar RNA scaffold recruitment systems could be constructed by changing the modular components of the system, for example, any suitable Cas orthologue, deaminase orthologue, and other DNA modifying enzymes.

[0173] Cell type / therapeutic use The RNA scaffold recruitment system of the present invention can be used to genetically modify cells, for example, but not limited to, animal cells, fungal cells, and plant cells. In a preferred embodiment, the RNA scaffold of the present invention can be used to genetically modify human cells. The present invention can be applied to primary cell lines, immortalized cell lines, and primary cells isolated from humans. Examples of human cells include, but not limited to, differentiated cells or stem cells. Preferred human cells include those obtained from any of the three germinal layers, namely the endoderm, mesoderm, and ectoderm. For example, human cells are those found in the following organs: skeletal muscle, skeleton, dermis of the skin, connective tissue, urogenital system, heart, blood (lymphocytes), and spleen (mesoderm); stomach, colon, liver, pancreas, bladder; urethral mucosa, epithelial portion of the trachea, lungs, pharynx, thyroid gland, parathyroid gland, intestinal tract (endoderm); or cells found in the central nervous system, retina and lens, head and sensory organs, ganglia and nerves, pigment cells, head connective tissue, epidermis, hair, and mammary glands (ectoderm). In a preferred embodiment, the RNA scaffold is used to genetically modify primary immune cells or immune cell lines. Immune cells include T cells, NK cells, B cells, CD34+ hematopoietic stem progenitor cells (HSPCs), and other cells involved in lymphocyte production, as well as cells of the blood, bone marrow, spleen, lymph nodes, and thymus. Immune cells, particularly primary immune cells, that are naturally present in host animals or patients, or obtained from induced pluripotent stem cells [iPSCs], can be genetically modified. Immune cells include T cells, NK cells, B cells, pluripotent cells, and other cells involved in the production of immune cells and lymphocytes, as well as blood, bone marrow, and spleen. Examples include hematopoietic stem cells (HSCs), which are pluripotent cells that can differentiate into cells of organs, lymph nodes, and the thymus.

[0174] Methods for genomic manipulation in cells in vitro, in vivo, or ex vivo (e.g., methods for altering or manipulating the expression of one or more genes or one or more gene products) are also provided herein. In particular, the methods provided herein are useful for target base editing disruption in mammalian cells.

[0175] In another embodiment, a method for targeting a disease for base editing modification is provided herein. As is established in the art, the target sequence may be any disease-associated polynucleotide or gene. Examples of useful applications of mutation or “modification” of endogenous gene sequences include modification of disease-associated gene mutations, modification of sequences encoding splicing sites, modification of regulatory sequences, modification of sequences causing gain-of-function mutations, and / or loss-of-function mutations, as well as targeted modification of sequences encoding structural features of proteins.

[0176] In some cases, it would be advantageous to genetically modify cells using the methods described herein so that the cells express chimeric antigen receptors (CARs) and / or T cell receptors (TCRs). “Chimeric antigen receptors (CARs)” are sometimes referred to as “chimeric receptors,” “T-body,” or “chimeric immune receptors (CIRs).” As used herein, the term “chimeric antigen receptors (CARs)” means an artificially constructed hybrid protein or polypeptide comprising an extracellular antigen-binding domain (e.g., a single-chain variable fragment (scFv)) of an antibody operably linked to a transmembrane domain, and at least one intracellular domain. Generally, the antigen-binding domain of a CAR has specificity for a particular antigen expressed on the surface of a target cell of interest. For example, T cells can be engineered to specifically express a CAR against CD19 on a B-cell lymphoma. In the case of allogeneic antitumor cell therapy not limited by donor matching (TCRs and HLA markers), cells can be engineered to knock in the nucleic acid encoding the CAR, but they can also be knocked out of the genes that are factors in donor matching.

[0177] Where used herein, the terms “genetically modified” and “genetically engineered” are mutually interchangeable and mean prokaryotic or eukaryotic cells containing exogenous polynucleotides, regardless of the method used for insertion. In some cases, effector cells are made or modified by human hands (e.g., using recombinant DNA technology) or modified to contain nucleic acid molecules that do not exist in nature, obtained from such molecules (e.g., by transcription, translation, etc.). Effector cells containing exogenous, recombinant, synthetic, and / or otherwise modified polynucleotides are considered engineered cells.

[0178] Cell therapy and ex vivo therapy Various embodiments of the present invention also provide cells that are produced or used according to any other embodiment of the present invention for use in therapy. In one embodiment, the present invention relates to a method for producing therapeutic cells, such as T cells engineered to express a chimeric antigen receptor (CAR-T) or T cells engineered to express a T cell receptor (TCR-T). CAR-T / TCR-T cells may be obtained from primary T cells or differentiated from stem cells. Suitable stem cells include, but are not limited to, mammalian stem cells, such as human stem cells, such as, but are not limited to, hematopoietic stem cells, neural stem cells, embryonic stem cells, induced pluripotent stem cells (iPSCs), mesenchymal stem cells, mesodermal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, and retinal stem cells. Stem cells are not limited to these, but examples include mammalian stem cells, such as mouse stem cells, and mouse embryonic stem cells.

[0179] In various embodiments, the present invention may be used to modify the expression of a single gene or multiple genes in various types of cells or cell lines, such as, but not limited to, cells derived from eukaryotic cells, such as human cells, in order to knock out base changes. The present invention may be used for multiple modifications, i.e., one or more base edits, which can be introduced simultaneously or sequentially. The technique may be used for many applications, such as, but not limited to, gene knockout to prevent graft-versus-host disease by making non-host cells non-immunogenic to the host, or to prevent host-versus-graft disease by making non-host cells resistant to host attack. These approaches are also related to the development of allogeneic (commercial) or autologous (patient-specific) cell-based therapies.Such genes, though not limited to these, include T cell receptors (TRAC), major histocompatibility complex (MHC class I and class II tissue) genes, e.g., B2M, co-receptors (HLA-F, HLA-G), genes involved in innate immune responses (MICA, MICB, HCP5, STING, DDX41, and Toll-like receptors (TLR)), inflammation (NKBBiL, LTA, TNF, LTB, LST1, NCR3, AIF1), heat shock proteins (HSPA1L, HSPA1A, HSPA1B), complement cascades, regulatory receptors (NOTCH family members), antigen processing (TAP, HLA-DM, HLA-DO), titer or sustained increase (e.g., PD-1, CTLA-4, and other members of the B7 family of checkpoint proteins), and genes involved in immunosuppressive immune cells (e.g., FOXP3 and interleukin(I)). Examples include genes involved in T-cell interactions with the tumor microenvironment (but not limited to these, such as cytokine receptors, e.g., TGFB, IL-4, IL-7, IL-2, IL-15, IL-12, IL-18, IFN-gamma), genes involved in the causes of cytokine release syndrome (but not limited to these, such as IL-6, IFN-gamma, IL-8 (CXCL8), IL-10, GM-CSF, MIP-1α / β, MCP-1 (CCL2), CXCL9, and CXCL10 (IP-10)), genes encoding antigens targeted by CAR / TCR (e.g., endogenous CS1 for which the CAR is designed), or other genes found to be beneficial for CAR-T / TCR-T (e.g., TET2) or other cell-based therapies, e.g., CAR-NK, CAR-B). For example, see DeRenzo et al., Genetic Modification Strategies to Enhance CAR T Cell Persistence for Patients With Solid Tumors. Front. Immunol., February 15, 2019.

[0180] The technology may also be used to knock down or modify genes that are involved in the fracturing of immune cells, such as T cells and NK cells, or genes that alert the immune system of a patient or animal when foreign cells, particles, or molecules have entered the patient or animal, or genes that encode proteins that are currently therapeutic targets used to impair or boost the immune response, such as DC52 and PD1, respectively.

[0181] One application is to manipulate HLA alleles in bone marrow cells to increase haplotype matching. These manipulated cells can then be used in bone marrow transplants to treat leukemia. Another application is to manipulate the negative regulatory element of the fetal hemoglobin gene in hematopoietic stem cells to treat sickle cell anemia and β-thalassemia. The negative regulatory element will mutate, and the expression of the fetal hemoglobin gene will affect hematopoietic stem cells. It is reactivated in cells and compensates for loss of function caused by mutations in adult α or β hemoglobin genes. Further applications include manipulating iPS cells to produce allogeneic therapeutic cells for various degenerative diseases, such as Parkinson's disease (loss of neuronal cells) and type 1 diabetes (loss of pancreatic beta cells). Another exemplary application is manipulating HIV-resistant T cells by inactivating the CCR5 gene and other genes encoding receptors necessary for HIV to enter cells.

[0182] Types of gene modification Accordingly, methods for targeted disruption of the transcription or translation of a target gene are provided herein. In particular, such methods include targeted disruption of the transcription or translation of a target gene by disruption of the start codon, introduction of an immature stop codon, and / or targeted disruption of an intron / exon splicing site.

[0183] By using the methods described herein, one or more genes of interest can be knocked in and / or knocked out in primary cells, thereby improving efficiency and reducing the rate of off-target indel formation. In preferred embodiments, the methods are used for multiplexed base editing, including gene knock-in, gene knockout, and missense mutations.

[0184] As described in the following paragraphs and examples, our streamlined approach to genome engineering employs base editors (e.g., third and fourth generation base editors, adenine base editors) for target gene disruption by knockout and missense mutations, as well as target gene knock-in in the presence of a DNA donor template. The methods described herein are well suited to studies of hematopoietic cell biology and gene function, modeling of diseases such as primary immunodeficiency disorders, modification of disease-causing point mutations, and the creation of novel cell products (e.g., T cell products) for therapeutic applications.

[0185] Delivery of components to cells A preferred method for delivering base editing components to cells is provided in the following examples of this specification.

[0186] In embodiments provided herein, the RNA scaffold is chemically synthesized RNA, which is introduced into cells by any preferred technique, such as electroporation. Base editing enzyme components and class 2Cas enzyme components can be introduced into cells as mRNA or proteins.

[0187] In embodiments, components such as base editors and guide molecules can be delivered to cells in vitro, ex vivo, or in vivo. Optionally, a viral or plasmid vector system is employed for the delivery of the base editing components described herein. Preferably, the vector is a viral vector, e.g., a lentiform or baculovirus vector, or preferably an adenovirus / adeno-associated virus (AAV) vector, but other means of delivery are also known (e.g., yeast systems, microvesicles, means of attaching gene guns / vectors to gold nanoparticles, etc.), and these are assumed. In certain embodiments, a nucleic acid encoding gRNA and a base editing fusion protein are packaged in one or more viral delivery vectors for delivery to cells. Suitable viral delivery vectors include, but are not limited to, adenovirus / adeno-associated virus (AAV) vectors and lentiform virus vectors. Optionally, non-viral delivery methods known in the art can be used to introduce nucleic acids or proteins into mammalian cells. The protein can be delivered by a pharmaceutically acceptable vehicle or, for example, encapsulated in liposomes. Other means of delivery are also known (e.g., yeast systems, microvesicles, means of attaching gene guns / vectors to gold nanoparticles, etc.) and are assumed. In some cases, cells are electroporated for the uptake of gRNA and base editors (e.g., BE3, BE4, ABE). In some cases, after the introduction of gRNA, base editors, and vectors by electroporation, the DNA donor template is delivered as an adeno-associated virus type 6 (AAV6) vector by adding the viral supernatant to the culture medium.

[0188] The rate of insertion or deletion (indel) formation can be measured by appropriate methods. For example, Sanger sequencing or next-generation sequencing (NGS) can be used to detect the rate of indel formation. Preferably, contact results in less than 20% off-target indel formation during base editing. Contact results in at least a 2:1 ratio of intended and unintended products during base editing.

[0189] Expression system To use the platform described above, it may be desirable to express one or more of the protein and RNA components from the encoding nucleic acid. This can be carried out in various ways. For example, an RNA scaffold or a nucleic acid encoding a protein can be cloned into one or more intermediate vectors for introduction into prokaryotic or eukaryotic cells for replication and / or transcription. For the production of RNA scaffolds or proteins, the intermediate vector for storage or manipulation of the RNA scaffold or the nucleic acid encoding the RNA scaffold or protein is typically a prokaryotic vector, such as a plasmid or shuttle vector, or an insect vector. The nucleic acid can also be cloned into one or more expression vectors for administration to plant cells, animal cells, preferably mammalian or human cells, fungal cells, bacterial cells, or protist cells. Thus, the present invention provides nucleic acids encoding any of the RNA scaffolds or proteins mentioned above. Preferably, the nucleic acid is isolated and / or purified.

[0190] The present invention also provides recombinant constructs or vectors having sequences encoding one or more RNA scaffolds or proteins as described above. Examples of constructs include vectors in which the nucleic acid sequence of the present invention is inserted in the forward or reverse direction, such as plasmids or viral vectors. In preferred embodiments, the construct further includes regulatory sequences operably linked to the sequence, such as promoters. Many suitable vectors and promoters are known to those skilled in the art and are commercially available. Cloning and expression vectors suitable for use with prokaryotic and eukaryotic hosts are known in the art.

[0191] A vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. Vectors can autonomously replicate or integrate into host DNA. Examples of vectors include plasmids, cosmids, or viral vectors. The vectors of the present invention contain nucleic acids in a form suitable for nucleic acid expression in host cells. Preferably, the vector includes one or more regulatory sequences operably ligated to the nucleic acid sequence to be expressed. Examples of "regulatory sequences" include promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). Regulatory sequences include those that direct the constitutive expression of nucleotide sequences and inducible regulatory sequences. The design of an expression vector may depend on factors such as the selection of host cells to be transformed, transfected, or transduced, the desired level of RNA or protein expression, and so on.

[0192] Examples of expression vectors include chromosomal, non-chromosomal, and synthetic DNA sequences, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral DNA, such as vaccinia, adenovirus, fowlpox virus, and pseudorabies virus. However, any other vector may be used as long as it is replicable and viable in a host. Suitable nucleic acid sequences can be inserted into vectors by various procedures. Generally, nucleic acid sequences encoding one of the RNAs or proteins described above can be inserted into suitable regulatory endonuclease sites by procedures known in the art. Such procedures and related subcloning procedures are within the scope of the art.

[0193] The vector may contain a suitable sequence for amplifying expression. In addition, the expression vector may preferably contain one or more selectable marker genes, such as dihydrofolate reductase, to provide a phenotypic trait for the selection of transformed host cells. This includes, for example, neomycin resistance for eukaryotic cell culture, or tetracycline resistance, or ampicillin resistance in E. coli.

[0194] A vector for expressing RNA may include an RNA Pol III promoter to drive the expression of RNA, such as the HI, U6, or 7SK promoter. These human promoters enable RNA expression in mammalian cells after plasmid transfection. Alternatively, for example, the T7 promoter may be used for in vitro transcription, and the RNA can be transcribed and purified in vitro.

[0195] Using a vector containing the appropriate nucleic acid sequence and the appropriate promoter or control sequence described above, an appropriate host can be transformed, transfected, or infected so that the host can express the RNA or protein described above. Examples of suitable expression hosts include bacterial cells (e.g., Escherichia coli, Streptomyces, Salmonella typhimurium), fungal cells (yeast), insect cells (e.g., Drosophila and Spodoptera frugiperda (Sf9)), various animal cells (e.g., CHO, COS, and HEK293), adenoviruses, and plant cells. The selection of an appropriate host is within the scope of those skilled in the art. In some embodiments, the present invention provides a method for producing the RNA or protein mentioned above by transforming, transfecting, or infecting a host cell with an expression vector having a nucleotide sequence encoding one of the RNAs or a polypeptide or protein. The host cell is then cultured under suitable conditions that allow for the expression of the RNA or protein.

[0196] Any procedure known in the art can be used to introduce a foreign nucleotide sequence into a host cell. Examples thereof include calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and the use of any other well-known method for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into a host cell.

[0197] Cell culture The method further comprises maintaining the cells under appropriate conditions such that the guide RNA directs the effector protein to a target site in the target sequence and the effector domain modifies the target sequence.

[0198] Generally, cells can be maintained under conditions appropriate for cell growth and / or maintenance. Suitable cell culture conditions are well known in the art, and those skilled in the art understand that methods for culturing cells are known in the art and can vary depending on the cell type. In all cases, routine optimization can be used to determine the optimal technique for a particular cell type.

[0199] Cells useful for the methods provided herein can be newly isolated primary cells or can be obtained from frozen aliquots of primary cell cultures. In some cases, the cells are electroporated for uptake of the gRNA and base editing fusion protein. As described in the examples below, electroporation conditions for some assays (e.g., for T cells) can include 1400 volts, a pulse width of 10 milliseconds, and 3 pulses. After electroporation, the electroporated T cells can be allowed to recover in cell culture medium and then cultured in T cell growth medium. In some cases, the electroporated cells can be allowed to recover in cell culture medium for about 5 to about 30 minutes (e.g., about 5, 10, 15, 20, 25, 30 minutes). Preferably, the recovery cell culture medium does not contain antibiotics or other selected agents. In some cases, the T cell growth medium is Complete CTS OpTmizer T cell growth medium.

[0200] Use ​The RNA scaffold of the present invention can be used for the following applications: genome editing, genome screening, therapeutic cell generation, genome tagging, epigenome editing, karyotype engineering, cromotin imaging, transcriptome and metabolic pathway engineering, genetic circuit engineering, cell signaling sensing, cellular event recording, phylogenetic information reconstruction, gene drives, DNA genotyping, miRNA quantification, in vivo cloning, site-directed mutagenesis, genome diversification, and in situ proteomic analysis.

[0201] Applications include research into human diseases, such as cancer immunotherapy, antiviral therapy, bacteriophage therapy, cancer diagnosis, pathogen screening, microbiome remodeling, stem cell reprogramming, immunogenomic engineering, vaccine development, and antibody production.

[0202] definition Nucleic acids or polynucleotides mean DNA molecules (e.g., cDNA or genomic DNA, but not limited to these) or RNA molecules (e.g., mRNA, but not limited to these), and include DNA or RNA analogs. DNA or RNA analogs can be synthesized from nucleotide analogs. DNA or RNA molecules may contain parts that do not exist in nature, such as modified bases, modified backbones, or deoxyribonucleotides in RNA. Nucleic acid molecules may be single-stranded or double-stranded. Those skilled in the art will understand that uracil is a nucleotide that replaces thymine in RNA form. The DNA sequences disclosed herein have thymine nucleotides, and the corresponding RNA sequences have uracil nucleotides at the same positions.

[0203] The term “isolated,” when referring to nucleic acid molecules or polypeptides, means that the nucleic acid molecules or polypeptides are substantially free from at least one other component to which they are associated or found together in nature.

[0204] As used herein, the term “guide RNA” generally refers to CRISPR-type RNA. Guide RNA refers to an RNA molecule (or collectively, a group of RNA molecules) that can bind to a protein and direct the CRISPR protein to target a specific location within the target DNA. Guide RNA can include two segments: a DNA targeting guide segment and a protein-binding segment. The DNA targeting segment contains a nucleotide sequence that is complementary to (or at least capable of hybridizing under stringent conditions with) the target sequence. The protein-binding segment interacts with the CRISPR protein, such as Cas9 or a Cas9-related polypeptide. These two segments can be located on the same RNA molecule or on two or more separate RNA molecules. When these two segments are on separate RNA molecules, the molecule containing the DNA targeting guide segment is sometimes called CRISPRRNA (crRNA), while the molecule containing the protein-binding segment is sometimes called trans-activating RNA (tracrRNA).

[0205] As used herein, the terms “target nucleic acid” or “target” mean a nucleic acid containing a target nucleic acid sequence. A target nucleic acid can be single-stranded or double-stranded, and is often double-stranded DNA. “Target nucleic acid sequence,” “target sequence,” or “target region” means, as used herein, a specific sequence or its complement that you wish to bind or modify using the CRISPR system. A target sequence may be present in nucleic acids in vitro or in the genome of a cell in vivo, and it may be any form of single-stranded or double-stranded nucleic acid.

[0206] "Target nucleic acid strand" means the strand of target nucleic acid used for base pairing with the guide RNA disclosed herein. That is, the strand of target nucleic acid that hybridizes with the crRNA and guide sequence is called the "target nucleic acid strand." Other strands of target nucleic acid that are not complementary to the guide sequence are called "non-complementary strands." In the case of double-stranded target nucleic acids (e.g., DNA), each strand can be a "target nucleic acid strand" for designing the crRNA and guide RNA, and they can be used to carry out the method of the present invention, as long as suitable PAM sites are present.

[0207] As used herein, the term “derived from” means a process used to isolate, derive, or construct a different second component (e.g., a second molecule different from the first) from a first component (e.g., a first molecule) or information derived from that first component. For example, mammalian codon-optimized Cas9 polynucleotides are derived from the amino acid sequence of the wild-type Cas9 protein. Furthermore, variant mammalian codon-optimized Cas9 polynucleotides, such as Cas9 single-stranded mutant nickase (nCas9, e.g., nCas9D10A) and Cas9 double-stranded mutant nuclease-deficient (dCas9, e.g., dCas9 D10A H840A), are also derived from polynucleotides encoding the wild-type mammalian codon-optimized Cas9 protein.

[0208] As used herein, the term “wild type” is a term of the art as understood by those skilled in the art, and means a typical form of an organism, lineage, gene, or trait that occurs in nature, distinct from a variant or mutated form.

[0209] As used herein, the term “variant” means a first composition (e.g., the first molecule) relating to a second composition (e.g., the second molecule, also called the “parent” molecule). A variant molecule may originate from, be isolated from, be obtained based on, or be homologous to the parent molecule. For example, variant forms of mammalian codon-optimized Cas9 (hspCas9), including Cas9 single-stranded mutant nickase and Cas9 double-stranded mutant nickase deletion, are variants of mammalian codon-optimized wild-type Cas9 (hspCas9). The term “variant” describes either a polynucleotide or a polypeptide. It can be used for that purpose.

[0210] When applied to polynucleotides, a variant molecule may have total nucleotide sequence identity to the original parent molecule, or alternatively, less than 100% nucleotide sequence identity to the parent molecule. For example, a variant of a gene nucleotide sequence may be a second nucleotide sequence that has at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more identity to the original nucleotide sequence in a nucleotide sequence comparison. A polynucleotide variant also includes a polynucleotide that includes the entire parent polynucleotide and further includes an additional fusion nucleotide sequence. A polynucleotide variant also includes a polynucleotide that is a part or subsequence of the parent polynucleotide, and unique subsequences of polynucleotides disclosed herein (such as those determined by standard sequence comparison and alignment techniques) are also covered by the present invention.

[0211] In another embodiment, a polynucleotide variant includes a nucleotide sequence that involves minor, trivial, or insignificant changes to the parent nucleotide sequence. For example, minor, trivial, or insignificant changes include changes to a nucleotide sequence such that (i) they do not alter the amino acid sequence of the corresponding polypeptide, (ii) they occur outside the protein-coding open reading frame of the polynucleotide, (iii) they result in a deletion or insertion that may affect the corresponding amino acid sequence but has little or no effect on the biological activity of the polypeptide, and (iv) the nucleotide change results in an amino acid substitution by a chemically similar amino acid. If the polynucleotide does not encode a protein (e.g., tRNA, or crRNA, or tracrRNA), variants of that polynucleotide may include nucleotide changes that do not result in a loss of function of the polynucleotide. In another embodiment, conserved variants of the disclosed nucleotide sequence that result in a functionally identical nucleotide sequence are also included in the invention. Those skilled in the art will understand that many variants of the disclosed nucleotide sequence are included in the invention.

[0212] When applied to proteins, a variant polypeptide may have the same entire amino acid sequence as the original parent polypeptide, or alternatively, it may have less than 100% amino acid identity with respect to the parent protein. For example, an amino acid sequence variant may be a second amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical in amino acid sequence to the original amino acid sequence.

[0213] Polypeptide variants include polypeptides comprising the entire parent polypeptide and further comprising additional fusion amino acid sequences. Polypeptide variants also include polypeptides that are a part or partial sequence of the parent polypeptide, and unique partial sequences of polypeptides disclosed herein (such as those determined by standard sequence comparison and alignment techniques) are also covered by the present invention.

[0214] In another embodiment, polypeptide variants include polypeptides comprising small, trivial, or insignificant changes to the parent amino acid sequence. For example, small, trivial, or insignificant changes include amino acid changes (including substitutions, deletions, and insertions) that result in a functionally identical polypeptide, including the addition of non-functional peptide sequences, which have little or no effect on the biological activity of the polypeptide. In another embodiment, the variant polypeptides of the present invention modify the biological activity of a parent molecule, for example, a variant variant of a Cas9 polypeptide having altered nuclease activity or losing nuclease activity. Those skilled in the art will know that many of the disclosed polypeptides It is understood that variants of this invention are included in the present invention.

[0215] In some embodiments, the polynucleotides or polypeptides of the present invention may include variant molecules that modify, add, or delete a small percentage of nucleotide or amino acid positions, for example, typically less than about 10%, less than about 5%, less than 4%, less than 2%, or less than 1%.

[0216] As used herein, the term "conservative substitution" in a nucleotide or amino acid sequence means a change in the nucleotide sequence that is either (i) one that results in no corresponding change in the amino acid sequence due to the redundancy of the triplet codon code, or (ii) one that results in substitution of the original parent amino acid with an amino acid having a chemically similar structure. Tables of conservative substitutions that provide functionally similar amino acids are well known in the art and, in this context, one amino acid residue is substituted with another amino acid residue having similar chemical properties (e.g., an aromatic side chain or a positively charged side chain), and thus does not substantially alter the functional properties of the resulting polypeptide molecule.

[0217] The following are groupings of natural amino acids having similar chemical properties, where substitutions within a group are "conservative" amino acid substitutions. This grouping shown below is not strict as these natural amino acids can be placed in different groupings when considering different functional properties. Amino acids having nonpolar side chains and / or aliphatic side chains include the following: glycine, alanine, valine, leucine, isoleucine, and proline. Amino acids having polar uncharged side chains include the following: serine, threonine, cysteine, methionine, asparagine, and glutamine. Amino acids having aromatic side chains include the following: phenylalanine, tyrosine, and tryptophan. Amino acids having positively charged side chains include the following: lysine, arginine, and histidine. Amino acids having negatively charged side chains include the following: aspartate and glutamate.

[0218] "Cas9 variant" or "Cas9 variant" means a protein or polypeptide derivative of the wild-type Cas9 protein, such as the Streptococcus pyogenes Cas9 protein, e.g., a protein having one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof. They substantially retain the RNA targeting activity of the Cas9 protein. The protein or polypeptide may contain, consist of, or substantially consist of fragments of the Streptococcus pyogenes Cas9 protein. Generally, the variant / variant is at least 50% (e.g., any number between 50% and 100%) identical to the Streptococcus pyogenes Cas9 protein. The variant / variant may be able to bind to RNA molecules, target specific DNA sequences via RNA molecules, and additionally possess nuclease activity. Examples of these domains include the RuvC-like motif (amino acids 7-22, 759-766, and 982-989 of the S. pyogenes Cas9 protein) and the HNH motif (amino acids 837-863). See Gasiunas et al., Proc Natl Acad Sci US A. September 25, 2012;109(39):E2579-E2586 and International Publication No. 2013176772.

[0219] "Complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence by either traditional Watson-Crick base pairing or other non-traditional types of base pairing. The percentage of complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, and 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively). "Complete complementarity" means that all adjacent residues of a nucleic acid sequence are compatible. This means forming hydrogen bonds with the same number of adjacent residues in the second nucleic acid sequence. "Substantially complementary," as used herein, means a degree of complementarity such as at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% for a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or two nucleic acids that hybridize under stringent conditions.

[0220] As used herein, “stringent conditions” for hybridization mean conditions under which nucleic acids complementary to the target sequence predominantly hybridize with the target sequence and substantially do not hybridize with non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on several factors. Generally, the longer the sequence, the higher the temperature at which it specifically hybridizes with its target sequence. A non-limiting example of stringent conditions is found in Tijssen (1993), Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes Part I, Second Chapter “Overview of principles of hybridization and the strategy of nucleic acid This is described in detail in “Probe Assay”, Elsevier, NY.

[0221] "Hybridization" and "hybridizing" refer to the process by which fully or partially complementary nucleic acid strands assemble under specific hybridization conditions to form a double-stranded structure or region in which two constitutive strands are joined by hydrogen bonds. Hydrogen bonds are typically formed between adenine and thymine or uracil (A and T or U), or between cytidine and guanine (C and G), but other base pairs can also be formed (e.g., Adams et al., The Biochemistry of the Nucleic Acids, 11th edition, 1992)).

[0222] As used herein, “expression” means the process by which polynucleotides are transcribed from a DNA template (e.g., to mRNA or other RNA transcripts), and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may collectively be called “gene products.” When polynucleotides are obtained from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells.

[0223] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to mean polymers of amino acids of any length. Such polymers may be linear or branched, and may contain modified amino acids, which may be interspersed with non-amino acids. The terms also encompass modified amino acid polymers, such as those modified by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or any other operation, such as conjugation with labeling components. Where used herein, the term “amino acid” encompasses natural and / or unnatural or synthetic amino acids, such as glycine and both D- and L-isomers, as well as amino acid analogs and peptidomimetics.

[0224] The terms "fusion polypeptide" or "fusion protein" refer to a protein created by joining two or more polypeptide sequences together. The fusion polypeptides included in this invention are nucleic acid sequences encoding a first polypeptide, for example, RNA-bound domains. The translation product of a chimeric gene construct includes a nucleic acid sequence encoding a second polypeptide, such as an effector domain, which is joined to one of the domains to form a single open reading frame. In other words, a “fusion polypeptide” or “fusion protein” is a recombinant protein of two or more proteins joined by a peptide bond or several peptides. A fusion protein may also include a peptide linker between the two domains.

[0225] The term "linker" means any means, entity, or part used to join two or more entities. A linker can be a covalent linker or a non-supply linker. Examples of covalent linkers include covalent bonds or linker moieties covalently attached to one or more proteins or domains to be linked. Linkers can also be non-covalent bonds, such as organometallic bonds with a metal center, such as a platinum atom. In the case of covalent bonds, various functionalities can be used, such as amide groups, e.g., carbonate derivatives, ethers, esters, e.g., organic and inorganic esters, aminos, urethanes, ureas, and the like. To provide linkage, a site for coupling can be provided by modifying the domain by oxidation, hydroxylation, substitution, reduction, etc. Methods of conjugation are well known to those skilled in the art and are included for use in the present invention. Linker moieties include, but are not limited to, chemical linker moieties, or, for example, peptide linker moieties (linker sequences). It is understood that modifications that do not significantly impair the function of the RNA-binding domain and effector domain are preferred.

[0226] As used herein, the terms “conjugate,” “conjugation,” or “linked” mean the combination of two or more entities to form a single entity. Conjugates encompass both peptide-small molecule conjugates and peptide-protein / peptide conjugates.

[0227] The terms “subject” and “patient” are used interchangeably herein to mean vertebrates, preferably mammals, more preferably humans. Mammals include, but are not limited to, mice, monkeys, humans, livestock, sports animals, and pets. Tissues, cells, and their offspring of biological entities obtained in vivo or cultured in vitro are also included. In some embodiments, the subject may be an invertebrate, such as an insect or nematode, while in other embodiments, the subject may be a plant or fungus.

[0228] As used herein, “treatment,” “to treat,” “to alleviate,” and “to improve” are interchangeable. These terms mean an approach to obtain a beneficial or desired outcome, such as, but not limited to, therapeutic and / or preventive effects. Therapeutic effect means any therapeutically related improvement in or to one or more diseases, conditions, or symptoms under treatment. In the case of a preventive effect, the composition may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, in which case the disease, condition, or symptom may not yet be apparent.

[0229] As used herein, the term “to bring into contact” encompasses any process in which the components to be brought into contact are mixed in the same mixture (e.g., added to the same compartment or solution) and do not necessarily require actual physical contact of the enumerated components. The components may be brought into contact in any order or in any combination (or partial combination), and may include situations in which one or more of the enumerated components are subsequently removed from the mixture before the other enumerated components are added. For example, “bringing A into contact with B and C” includes all of the following situations: (i) A is mixed with C, and then B is added to the mixture; (ii) A and B are mixed into the mixture; B is removed from the mixture, and then C is added to the mixture; and (iii) A is added to the mixture of B and C. “Bringing a target nucleic acid or cell into contact with one or more reaction components, such as a Cas protein or guide RNA” includes all of the following situations: (i) the target or cell is brought into contact with a first component of the reaction mixture to form a mixture; then the other components of the reaction mixture are added to the mixture in any order or combination; and (ii) the reaction mixture is fully formed before mixing with the target or cell.

[0230] As used herein, the term "mixture" refers to a combination of elements scattered in no particular order. A mixture is heterogeneous and its different components are not spatially separable. Examples of elemental mixtures include several different elements dissolved in the same aqueous solution, or several different elements mounted on a solid support in a disordered or random order such that the different elements are not spatially distinguishable. In other words, a mixture is not addressable.

[0231] As disclosed herein, several ranges of values ​​are provided. Unless otherwise specified, it should be understood that each intermediate value between the upper and lower limits of that range is also specifically disclosed, down to a unit of one-tenth of the lower limit. Each smaller range between any mentioned value or intermediate value in a mentioned range and any other mentioned value or intermediate value in a mentioned range is included within the present invention. The upper and lower limits of these smaller ranges are independently included within the present invention, depending on any explicitly excluded limits in the mentioned range, and whether either limit value is included in the smaller range, neither is included, or both are included. If a mentioned range includes one or both of the upper and lower limits, the range excluding one or both of these included upper and lower limits is also included within the present invention. The term “about” generally means plus or minus 10% of the indicated value. For example, “about 10%” may refer to a range from 9% to 11%, and “about 20” may refer to 18 to 22. Other meanings of "approximately" can be obvious from the context, such as rounding, and therefore, for example, "approximately 1" could mean between 0.5 and 1.4.

[0232] Various exemplary embodiments of the compositions and methods according to the present invention are described in the following examples. [Examples]

[0233] Example 1 Modification of RNA scaffolds sgRNA sequence design A complete list of the sgRNA designs used and their sequences is shown in Table 4. All sgRNA designs are based on *Streptococcus pyogenes* sgRNA consisting of a target-specific 20nt spacer sequence, a 76nt b-constant region sgRNA sequence, and a 7nt poly-T U6 termination signal. All modifications are made to the constant components of the sgRNA and consist of inclusion of RNA aptamer hairpins and / or extension of stem repeats:antirepeats. A single copy (1xMS2) or two copies (2xMS2) of the MS2 hairpin sequence (C5 variant) were incorporated into either the tetraloop, stemloop 2, or 3' of the sgRNA. For 2xMS2 tracrRNA, there are two designs: one integrating two copies of the C5 MS2 variant into the 3' of the sgRNA, and another positioned in stemloop 2. A second design was pursued, consisting of a modified C5 variant and an engineered MCP protein-binding f6 aptamer assimilated at the 3' of the sgRNA. The f6 aptamer is a different variant used for the 2xMS2 plasmid design. Repeats: Various extensions of the upper stem of the anti-repeat were incorporated into either side of the stem, each time incorporating a native Streptococcus pyogenes sequence.

[0234] [Table 4-1]

[0235] [Table 4-2]

[0236] Plasmid design With the exception of sgRNA, all components of the base editing system were encoded in a single vector and expressed as a single polycistronic unit derived from the CMV promoter. This vector encoded the expression of APOBEC-1-MCP fusion protein, and nCas9 (D10A), fused to UGI by a C-terminal nCas9-UGI fusion protein, had two copies of SV40 NLS adjacent to the C-terminus of nCas9 and the N-terminus of UGI. Furthermore, the vector's encoding of turboRFP expression allows for monitoring of transfection efficiency.

[0237] The sgRNA components of the base editing system are expressed in separate vectors, and in this case, expression is driven by the RNA polymerase III U6 promoter. NA was expressed as a single unit containing the crRNA and tracrRNA components of Streptococcus pyogenes Cas9, linked by the artificial tetraloop described earlier. A list of sgRNA target sgRNA sequences is shown in Table 5. If a target did not have a 5'G, G was added as needed to the expression from the U6 promoter.

[0238] The expression design for the BE4max base editor was as previously described.

[0239] [Table 5]

[0240] Cell culture and transfection HEK293 cells were cultured in DMEM (Dulbecc's modified Eagle medium) supplemented with 10% FBS. 24 hours prior to transfection, 50,000 cells were seeded into a single well of a 24-well plate to achieve a culture density of approximately 70% for transfection. After 24 hours, the cells were lipid-transfected using Lipofectamine 3000 reagent (ThermoFisher Scientific) with 200 ng of plasmid DNA (150 ng of pinpoint / BE4max vector and 50 ng of sgRNA expression vector).

[0241] Cell lysis and flow cytometry 72 hours after transfection, the culture medium is removed, the cells are washed once with PBS, and then transfected with 100 μl of TrypLE-expressing enzyme (ThermoFisher Scientific). Therefore, the cells were removed from the well. Next, the dissociated cells were centrifuged at 300x rpm for 5 minutes at room temperature, and the supernatant was decanted. The pelleted cells were washed once with PBS, centrifuged again at 300x rpm for 5 minutes, the supernatant was decanted, and then the pelleted cells were resuspended in 100 μl of PBS. 20 μl of the resuspended cells were transferred to a 96-well plate and incubated with 36 μl of Direct PCR lysis reagent (Viagen Biotech) under the following conditions: 55°C for 30 minutes, followed by 95°C for 30 minutes, and then the cell lysate was stored at -20°C. The remaining 80 μl of resuspended cells were transferred to a 96-well plate and pelletized by centrifuging at 300x rpm for 5 minutes at room temperature. The supernatant was decanted, and the cells were resuspended in 50 μl of MACS buffer (Miltenyi Biotec) supplemented with 0.5% BSA for flow cytometry analysis. All flow cytometry was performed using iQue3 (Sartorius). PCR amplification and target region One μl of cell lysate obtained using DirectPCR lysis reagent was used for each PCR reaction. Q5 Hi-Fi 2x Master Mix (NEB) was used for amplification of the sgRNA target site, and the reaction mixture was prepared as follows.

[0242] [Table 6]

[0243] The PCR reaction was performed under the following thermal cycling conditions.

[0244] [Table 7]

[0245] The primers used and their annealing temperatures are detailed in Table 6 below.

[0246] [Table 8]

[0247] Example 2 Base editing efficiency of modified RNA scaffolds RNA synthesis All crRNAs and tracrRNAs were synthesized by Horizon Discovery using either 2'-acetoxyethyl orthoester (2'-ACE) or 2'-tert-butyldimethylsilyl (2'-TBDMS) protection chemistry. The aforementioned chemical modifications were included, such as two 2'-O-methylnucleotides and two phosphorothioate bonds (2xMS modification) at the 5' end of crRNAs and the 3' end of tracrRNAs. RNA oligos were 2'-deprotected / desalted and purified by either high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE). Prior to electroporation, the oligos were resuspended in 10 mM Tris buffer at pH 7.5.

[0248] Electroporation HEK 293T cells (ATCC, #CRL-11268) were electroporated using the Invitrogen® Neon® transfection system, 10 μL kit. 50,000 cells, 1 μg of mRNA, and a mixture of 6 μM synthetic crRNA:tracrRNA were electroporated at 1150 V for 20 ms and 2 pulses. mRNA (obtained from TriLink or transcribed in vitro in-house by standard methods) was mixed with nCas9-UGI and MCP-AID or MCP-APOBEC in a 3:1 molar ratio. Cells were seeded in 96-well plates using complete serum growth medium and harvested after 72 hours for further processing.

[0249] Cell processing The cells were dissolved in 100 μl of buffer containing proteinase K (Thermo Scientific, #FEREO0492), RNase A (Thermo Scientific, #FEREN0531), and Phusion HF buffer (Thermo Scientific, #F-518L) at 56°C for 30 minutes. Following this, the cells were thermally inactivated at 95°C for 5 minutes. This cell lysate was used to prepare 200-400 nucleotide PCR amplicons extending to the region containing the base editing site. The unpurified PCR amplicons were subjected to Sanger sequencing using Genewiz.

[0250] Editorial Analysis Base editing efficiency was calculated from AB1 files using the Chimera analysis tool fitted with the open-source tool BEAT. Chimera identifies editing efficiency by first subtracting background noise to define the expected variability in the sample. This allows for evaluation of editing efficiency without the need to normalize to control for the sample. After this, Chimera removes all outliers from the noise using the Central Absolute Deviation (MAD) method and then evaluates the editing efficiency of the base editor over the length of a 20 bp input guide sequence.

[0251] Example 3 A base editing system applied to human primary immune cells using lentiviral integrated sgRNA. In this example, primary human PanT lymphocytes were used to demonstrate the usefulness of base-editing mRNA components in primary immune cells in the presence of constitutively expressed sgRNA using RNA aptamers under the control of the PolIII promoter. Pan T cells were activated using anti-CD3 and anti-CD28 and then transduced using enriched and concentrated lentiviral particles. Successfully transduced cells were selected using promycin selection to ensure that >95% of the population had at least one copy of the lentiviral insertion. During selection, the T cells were reactivated with anti-CD3 and anti-CD28 and then electroporated with mRNA components for both deaminase-MCP and nCas9-UGI-UGI components. The cells were then incubated for a further 72–96 hours, and the cells were inspected for surface knockout by flow cytometry, and base editing was inspected by targeted PCR amplification and Sanger sequencing.

[0252] Example 4 A base editing system applied to primary human immune cells using synthetic crRNA and tracrRNA-aptamer guides. In this example, primary human Pan T lymphocytes were used to demonstrate the usefulness of a base editing system with crRNA and aptamer-modified tracrRNA components in primary immune cells. Pan T cells were activated using anti-CD3 and anti-CD28, and then electroporated with deaminase-MCP, nCas9-UGI-UGI components, tracrRNA-aptamers, and mRNA components for both crRNA. The cells were then incubated for a further 72–96 hours, and the cells were examined for surface knockout by flow cytometry, and base editing was examined by targeted PCR amplification and Sanger sequencing.

[0253] The data demonstrate that base editing systems can edit primary immune cells using modified crRNA and tracrRNA-aptamers with mRNA components, without requiring DNA integration into the genome (by lentiviral cassettes). The results show that deaminase specificity by different RNA aptamers and Apobec1 has preference for single RNA motifs, while AID deaminase prefers dual RNA motifs in this context. The results demonstrate the high utility of base editing systems for modifying specific bases for functional protein knockout, as demonstrated by surface staining, flow cytometry, and DNA-level modifications.

[0254] material and method guide Internally generated data was used to specify the base editing window calculated at a set distance from the PAM motif ((NGG)). This data was used to develop algorithms to predict phenotypic or gene knockout applicable guide sequences for the following genes: TRAC, TRBC1, TRBC2, PDCD-1, B2M, and CD52 (Table 7). crRNAs and tracrRNAs were synthesized using Horizon Discovery (formerly Dharmacon) and Agilent. Synthetic crRNA sequence (SEQ ID NO: 86)

[0255] [ka]

[0256] 2'OMe(m) and phosphorothioate(*) modified residues Synthetic 1xMS2 tracrRNA-aptamer sequence (SEQ ID NO: 87):

[0257] [ka]

[0258] 2'OMe(m) and phosphorothioate (*) modifications Synthetic 2xMS2 tracrRNA-aptamer sequence (SEQ ID NO: 88):

[0259] [ka]

[0260] 2'OMe(m) and phosphorothioate(*) modified residues Lentivirus sgRNA sequence (SEQ ID NO: 89)

[0261] [ka]

[0262] mRNA component creation Messenger RNA molecules were custom-constructed using Trilink with modified nucleotides: pseudouridine and 5-methylcytosine. mRNA composition The raw material was translated into the following proteins: deaminase AID = NLS-hAID-linker-MCP, deaminase Apobec1 = NLS-rApobec1-linker-MCP, and Cas9-UGI-UGI = NLS-nCas9-UGI-UGI-NLS.

[0263] Plasmid construction By incorporating additional selectable markers (e.g., antibiotics, fluorescent proteins) into the lentiviral construct, we ensured that a single integrated copy was present within the genome of the target cell population. Sequences for specific guide sequences were cloned into overhangs generated by type IIS restriction enzyme sites (by T4 DNA ligase technique). The target construct extended to the Cas9 scaffold and aptamer sequences prior to the stop sequence, ensuring that the guide sequence was fully within the frame (G nucleotide inclusion if not at 5' of the sequence) for efficient transcription from the human U6 PolIII promoter. Plasmid clones were checked by Sanger sequencing and restriction digestion QC before scaling up to large-scale plasmid preparation (e.g., maxiprep).

[0264] Creation of lentiviral particles Using a third-generation plasmid system (Horizon Discovery), sgRNA-aptamer lentiviral constructs were constructed in functional lentiviral particles. The viral particles were then concentrated by diafiltration and dispensed for transduction.

[0265] lentivirus trait introduction T cells were activated for >48 hours, transduced at a MOI of 0.1 using plates treated with retronectin (T100B, Takara-bio), and incubated overnight at 37°C and 5% CO2.

[0266] Frozen T cell culture Frozen CD3+ T cell sources (Hemacare) were thawed and then cultured in Immunocult XT medium (STEMCELL Technologies) with 1x penicillin / streptomycin (Thermofisher) at 37°C and 5% CO2.

[0267] T cell electroporation 48–72 hours after activation, T cells were electroporated using a Neon Electroporator (Thermofisher) or a 4D Nucleofector (Lonza). For the Neon Electroporator, the conditions for both deaminase-MCP and nCas9-UGI-UGI were 1600v / 10ms / 3 pulses in a 10μl cuvette with 250k cells, a total amount of 1–5μg of mRNA, and 0.2–1.8μmol of complexed crRNA:tracrR or sgRNA. For the 4D Nucleofector, the conditions for both deaminase-MCP and nCas9-UGI-UGI (synthesized by Trilink) were EO-115 in a 20μl cuvette at 500k, a total amount of 1–5μg of mRNA, and 0.2–1.8μmol of complexed crRNA:tracrR or sgRNA (Horizon Discovery). After electroporation, the cells were transferred to Immunocult XT medium with 100U IL-2, 100U IL-7, and 100U IL-15 (STEMCELL Technologies) and cultured at 37°C and 5% CO2 for 48–72 hours. Activation of CD3+ T cells By using Dynabeads Human T Activator CD3 / CD28 beads (Thermofisher) in a 1:1 bead-to-cell ratio, T cells were activated and 100 U / ml of IL-2 (ST) was administered at 37°C and 5% CO2. Cells were cultured for 48 hours in Immunocult XT medium (STEMCELL Technologies) in the presence of EMCELL Technologies and 1x penicillin / streptomycin (Thermofisher). After activation, the beads were removed by placing the cells on a magnet and returning them to the culture medium. Flow cytometry T cell identity and QC were confirmed by CD3 antibody staining (Biolegend). T cell activation was confirmed by CD25 staining. Phenotypic gene KO:TRAC was confirmed by CD3 and TCRab antibody staining (Biolegend), and B2M was confirmed by B2M antibody (Biolegend); any phenotypic data were percentage changes relative to the reference substance in viable cells, only when confirmed by DAPI staining (BD Bioscience). Genome DNA analysis Genomic DNA was released from lysed cells 48–72 hours after electroporation. The target gene locus was amplified by PCR, and the product was then sent to Sanger sequencing (Genewiz). The data was analyzed using proprietary in-house software.

[0268] [Table 9-1]

[0269] [Table 9-2]

[0270] [Table 9-3]

[0271] [Table 9-4]

[0272] [Table 9-5]

[0273] [Table 9-6]

[0274] The list of guide examples designs both sgRNA and crRNA forms that can produce functional knockouts using the illustrated base editing techniques. This list includes guides specific to the introduction of immature stop codons and splicing disruption sites, which were generated using proprietary in-house software.

[0275] Example 5 Base editing efficiency of modified RNA scaffolds with crRNA, tracrRNA, and sgRNA. RNA synthesis All crRNAs, tracrRNAs, and sgRNAs were synthesized using either 2'-acetoxyethyl orthoester (2'-ACE) or 2'-tert-butyldimethylsilyl (2'-TBDMS) protection chemistry. Chemical modifications such as those mentioned were included, including two 2'-O-methylnucleotides and two phosphorothioate bonds (2xMS modification) at the 5' end of crRNAs and the 3' end of tracrRNAs. 2'OMe is represented by m, and phosphorothioates by *. RNA oligos were 2'-deprotected / desalted and purified by either high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE). Prior to transfection, the oligos were resuspended in 10 mM Tris buffer at pH 7.5. The gene sites targeted by each cRNA are (A)CR0118_PDCD1, (B)CR0107_PDCD1, (C)CR0057-TRAC_EX3, (D)CR0151_CD2, (E)Site2, (F)CR0121_PDCD1, and (G)CR0165_CIITA, as shown in Figures 11A-G; (A)CR0151_CD2, (B)CR0121_PDCD1, and (C)CR0165_CIITA, as shown in Figures 12A-C; and (A)TRAC_22550571, (B)PDCD1_241852953, and (C)CTNNB1, as shown in Figures 13A-C. The sgRNA target site sequences for base editing are listed in Table 5.

[0276] Transfection Cells stably transfected with U2OS nCas9 were transfected with DharmaFECT Duo, 25 nM synthetic crRNA:tracrRNA, and 200 ng of (a) rAPOBEC or (b) hAID mRNA. Cells were harvested at 72 hours.

[0277] Cell processing Cells were lysed at 56°C for 30 minutes in 100 μL of buffer containing proteinase K (Thermo Scientific, #FEREO0492), RNase A (Thermo Scientific, #FEREN0531), and Phusion HF buffer (Thermo Scientific, #F-518L), followed by thermal inactivation at 95°C for 5 minutes. This cell lysate was used to prepare 200-400 nucleotide PCR amplicons extending to the region containing the base editing site. The unpurified PCR amplicons were subjected to Sanger sequencing using Genewiz.

[0278] Editorial Analysis Base editing efficiency was calculated from AB1 files using the Chimera analysis tool fitted with the open-source tool BEAT. Chimera identifies editing efficiency by first subtracting background noise to define expected variability in the sample. This allows for evaluation of editing efficiency without the need to normalize to control for the sample. After this, Chimera uses the central absolute deviation (MAD) method to remove all outliers from the noise and then evaluates the editing efficiency of the base editor over the length of a 20 bp input guide sequence. Figure 11 shows the relative editing efficiency in base editing systems incorporating a single copy of either the C-5 or F-5 MS2 variant at the 3' end of the tracrRNA. Data are shown for the following crRNAs: a) (A) CR0118_PDCD1, (B) CR0107_PDCD1, (C) CR0057-TRAC_EX3, (D) CR0151_CD2, (E) Site2, (F) CR0121_PDCD1, and (G) CR0165_CIITA. The percentage of C-to-T editing detected indicates that the C-5 and F-5 variants provide similar levels of base editing at all loci examined, and furthermore, the editing window is ambiguous between the two MS2 variants. Figure 12 shows the following cr RNA: For (A) CR0151_CD2, (B) CR0121_PDCD1, and (C) CR0165_CIITA, the tracrRNA has C-5 or F-5 at its 3' end. Figure 13 shows the relative editing efficiency in base editing systems incorporating a single copy of either MS2 variant. Figure 13 shows the level of base editing with chemically synthesized 1xMS2_3'sgRNAs(C-5) or 1xMS2_3'_7bp-extended_US sgRNAs(C-5) which include a 7-base pair extension of the repeat:anti-repeat upper stem. Figure 14 demonstrates that when the amount of MCP deaminase is reduced to 20 ng, the higher affinity F-5 MS2 tracrRNA results in a higher percentage of C-to-T editing compared to C-5 MS2. Synthetic 1xMS2 tracrRNA-aptamer sequence used in Example 5

[0279] [ka]

[0280] [ka]

[0281] 2'OMe is represented by m, and phosphorothioates are represented by *. RNA scaffold-mediated recruitment system protein sequence (2 xUGI):

[0282] [ka]

[0283] [ka]

Claims

1. (a) tracrRNA; and (b) RNA motif with extended sequence, RNA scaffold containing this.

2. The RNA scaffold according to claim 1, further comprising a crRNA containing a guide RNA sequence.

3. The RNA scaffold according to claim 1 or 2, comprising one or more modifications.

4. The RNA scaffold according to claim 1 or 2, wherein an RNA motif is ligated to the 3' end of tracrRNA via a linker.

5. The RNA scaffold according to claim 4, wherein the linker is single-stranded RNA or a chemical bond.

6. The RNA scaffold according to any one of claims 2 to 5, wherein a tracrRNA is fused to a crRNA containing a guide RNA sequence that forms a single RNA molecule.

7. The RNA scaffold according to any one of claims 2 to 6, wherein the tracrRNA and the crRNA containing the guide RNA sequence are synthesized as separate RNA molecules.

8. The RNA scaffold according to any one of claims 2 to 7, wherein tracrRNA hybridizes to crRNA via a repeat:anti-repeat region.

9. Repeat: The RNA scaffold according to claim 8, wherein the anti-repeat region is extended.

10. The RNA scaffold according to claim 9, wherein the repeat:anti-repeat region includes an extended upper stem comprising the full length of 20 to 26 nucleotides.

11. The RNA scaffold according to claim 10, wherein the upper stem of the repeat:anti-repeat region comprises the full length of 22 nucleotides when synthesized as a single RNA molecule.

12. The RNA scaffold according to claim 10, wherein the upper stem of the repeat:anti-repeat region comprises the full length of 25 nucleotides when synthesized as two separate RNA molecules.

13. An RNA scaffold according to any one of claims 1 to 12, comprising one or more RNA motifs.

14. The RNA scaffold according to claim 13, wherein one or more RNA motifs include one or more modifications.

15. The RNA scaffold according to claim 14, wherein one or more modifications are located at the 5' and / or 3' ends of one or more RNA motifs.

16. The RNA scaffold according to claim 14, wherein one or more modifications are substitutions of an A base at position 10 to a 2-aminopurine (2AP).

17. The RNA scaffold according to claim 16, wherein 2-aminopurine (2AP) is 2'-deoxy-2-aminopurine or 2'-ribose-2-aminopurine.

18. The RNA scaffold according to claim 3, wherein one or more modifications are made to the backbone and / or sugar moiety of the RNA scaffold.

19. The RNA scaffold according to any one of claims 1 to 18, wherein the extended sequence of the RNA motif is a double-strand extension.

20. The RNA scaffold according to any one of claims 1 to 19, wherein the extended sequence of the RNA motif contains 2 to 24 nucleotides, and the recruiting RNA motif has a total length of 23 to 45 nucleotides.

21. The RNA scaffold according to any one of claims 5 to 20, wherein the single-stranded RNA linker comprises 0 to 10 nucleotides, preferably 2 to 6 nucleotides.

22. The RNA scaffold according to any one of claims 13 to 21, wherein one or more RNA motifs are bound to an aptamer-binding molecule.

23. The RNA scaffold according to any one of claims 13 to 22, wherein one or more RNA motifs are selected from the following aptamers: MS2, Ku, PP7, SfMu, and Sm7.

24. The RNA scaffold according to claim 22 or 23, wherein the MS2 aptamer binds to the MCP protein.

25. The RNA scaffold according to claim 23, wherein the MS2 aptamer is wild-type MS2, mutant MS2, or a variant thereof.

26. The RNA scaffold according to claim 25, wherein mutant MS2 is a C-5, F-5 hybrid and / or an F-5 mutant.

27. The RNA scaffold according to any one of claims 1 to 26, wherein the RNA motif mobilizes an effector module.

28. The RNA scaffold according to claim 27, wherein the effector module includes an RNA-binding domain capable of binding to an RNA motif and an effector domain.

29. The RNA scaffold according to claim 28, wherein the effector domain is selected from reporters, tags, molecules, proteins, microparticles, and nanoparticles.

30. The RNA scaffold according to claim 28, wherein the effector domain is a DNA modifying enzyme.

31. The RNA scaffold according to claim 30, wherein the DNA modifying enzyme is selected from AID, CDA, ABOBEC1, ABOBEC3A, ABOBEC3B, ABOBEC3C, ABOBEC3D, ABOBEC3F, or other ABOBEC family enzymes, ADA, ADAR family enzymes, or tRNA adenosine deaminase.

32. An RNA scaffold according to any one of claims 1 to 31, comprising a sequence selected from the sequences in Table 4.

33. The RNA scaffold according to any one of claims 1 to 32, wherein the RNA motif has a sequence selected from sequence numbers 21 to 24.

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