Synthetic self-replicating RNA vectors encoding CRISPR proteins and uses thereof

Abstract

A synthetic self-replicating RNA vector encoding a CRISPR protein and uses thereof are provided. A synthetic, non-infectious, self-replicating RNA vector encoding a CRISPR protein is provided. The various self-replicating RNA vectors comprise a sequence encoding a plurality of non-structural replicating complex proteins from an alpha virus and a sequence encoding a CRISPR protein. Also provided are methods for genome editing in which the synthetic self-replicating RNA vector is transfected into a cell along with at least one corresponding guide RNA.

Description

This application is a divisional application of an invention application with a filing date of May 13, 2020, a Chinese national application number of 202080050982.9, and an invention name of “Synthetic self-replicating RNA vector encoding CRISPR protein and its use”.

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 847,032 (filing date May 13, 2019), the entire contents of which are incorporated herein in their entirety.

[0002] Sequence Listing This application contains a sequence listing, which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on May 13, 2020 is named P19-084_WO-PCT_SL.txt and is 77,524 bytes in size.

[0003] field The present disclosure relates to synthetic self-replicating RNA vectors encoding CRISPR proteins, wherein the synthetic self-replicating RNA vectors can be transfected into cells together with corresponding guide RNAs for use in genome editing methods.

[0004] background The recent development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) CRISPR / Cas systems as genome editing tools has provided unprecedented ease and simplicity for the modification of site-specific endonucleases for eukaryotic genome modification. Many delivery systems have been developed to deliver CRISPR to eukaryotic cells. However, lentiviral, retroviral, and plasmid systems carry the risk of integrating CRISPR coding sequences into the host cell genome. Therefore, there is a need for a CRISPR gene delivery system that provides robust expression of CRISPR proteins and avoids the risk of genomic integration.

[0005] Overview In various aspects of the present disclosure, self-replicating RNA vectors encoding CRISPR proteins are provided.

[0006] One aspect of the present disclosure provides a self-replicating RNA vector comprising sequences encoding a variety of nonstructural replication complex proteins from an alpha virus and sequences encoding a CRISPR protein. The CRISPR protein may be a type II Cas9 protein, a type V Cas12 protein, a type VI Cas13 protein, a CasX protein, or a CasY protein. In certain embodiments, the CRISPR protein can be Streptococcus pyogenes Cas9, Francisella novicida Cas9, Staphylococcus aureus Cas9, Streptococcus thermophilus Cas9, Streptococcus pasteurianus Cas9, Campylobacter jejuni Cas9, Neisseria meningitis Cas9, Neisseria cinerea Cas9, Francisella novicida Cas12, Acidaminococcus sp. Cas12, Lachnospiraceae bacterium ND2006Cas12, Leptotrichia wadei Cas13a, Leptotrichia shahii) Cas13a, Prevotella sp. P5-125 Cas13, or Ruminococcus flavefaciens Cas13d. In a specific iteration, the CRISPR protein is Streptococcus pyogenes Cas9 or Staphylococcus aureus Cas9.

[0007] In some cases, the sequence encoding the CRISPR protein may contain at least one nucleotide insertion, deletion and / or substitution, resulting in the CRISPR protein having altered catalytic activity, improved target site specificity and / or reduced off-target effects. The CRISPR protein may have double-stranded cleavage activity, may cleave one strand of a double-stranded sequence, or may not have any cleavage activity.

[0008] The CRISPR protein can also be linked to at least one heterologous domain. For example, the CRISPR protein can be linked to at least one nuclear localization signal. The CRISPR protein can also be linked to at least one fluorescent protein, at least one chromatin regulatory motif, at least one epigenetic modification domain, at least one transcriptional regulatory domain, at least one RNA aptamer binding domain, or a combination thereof.

[0009] The sequences of the self-replicating RNA vector encoding the plurality of nonstructural replication complex proteins from an alphavirus may be derived from Aura virus, Babanki virus, Barmah Forest virus, Bebaru virus, Buggy Creek virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus, Mayaro virus, Middelburg virus, Mucambo virus, Ndumu virus, Pixuna virus, O'nyong-nyong virus, Ross River virus, Sagiyama virus virus), Semliki Forest virus, Sindbis virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, or Whataroa virus. These various sequences and their derivatives are known and can be found in the scientific literature, which is hereby incorporated by reference in its entirety. In a specific embodiment, the sequences encoding the various non-structural replication complex proteins are from Venezuelan equine encephalitis virus. See, for example, Yoshioka et al. (Cell Stem Cell 13, 246-254, August 1, 2013) and / or Petrakova et al. (J. Virology; vol. 72, no. 12, June 2005, p. 7597-7608), each of which is hereby incorporated by reference in its entirety. The self-replicating RNA vector may further comprise a sequence encoding a selectable marker and / or a sequence encoding an interferon response inhibitor.

[0010] Another aspect of the present disclosure provides a complex comprising the self-replicating RNA vector described above and at least one guide RNA engineered to complex with a CRISPR protein encoded by the self-replicating RNA vector.

[0011] The present disclosure also provides a eukaryotic cell or cell line comprising the self-replicating RNA vector disclosed herein.

[0012] Further aspects of the present disclosure include a plasmid vector encoding a self-replicating RNA vector.

[0013] Yet another aspect of the present disclosure provides a method for targeted genome editing. The method comprises introducing a self-replicating RNA vector disclosed herein and at least one guide RNA modified to complex with a CRISPR protein encoded by the self-replicating RNA vector into a eukaryotic cell.

[0014] Other aspects and iterations of the disclosure are described in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0016] Figure 1A Rendering Display Simplicon TM Schematic representation of the structure of the cloning vector and each of the VEE-Cas9 RNAs described herein.

[0017] Figure 1B Agarose gel electrophoresis showing VEE-Cas9 RNA. Lane 1: RNA marker (200-6000 bases); Lane 2: eSpCas9 RNA; Lane 3: eSp-Cas9-GFP RNA; Lane 4: Cas9-GFP RNA.

[0018] Figure 1C Images showing GFP (TagGFP2) expression in VEE-Cas9 transfected cells. VEE-Cas9 RNA and B18R-E3L RNA were co-transfected into HFFs on day 1, and GFP expression was captured using fluorescence microscopy on day 2.

[0019] Figure 1D Western blots of Cas9 proteins are presented. Lane 1: control; Lane 2: eSpCas9; Lanes 3 and 4: eSpCas9-GFP2; Lanes 5 and 6: Cas9-GFP2.

[0020] Figure 2AImages showing GFP expression at day 2 after HFFs were co-transfected with VEE-Cas9-TagGFP2 and a synthetic 1-component or 2-component gRNA targeting K-Ras simultaneously or sequentially.

[0021] Figure 2B The efficiency of genome editing after co-transfection or sequential transfection with VEE-Cas9 RNA and gRNA is presented. Cells were harvested on day 3 for co-transfection (simultaneously), on day 4 for sequential transfection, and on day 6 (after completion of puromycin selection). Indels were detected with the Guide-iT Mutation Detection Kit. + / - indicates resolvase treatment. Cleavage efficiency (%) is presented below the gel image.

[0022] Figure 2C Images showing GFP expression in human iPSC cell lines at day 2 following sequential transfection with VEE-Cas9-TagGFP2 and 1-component K-Ras gRNA (25 nM).

[0023] Figure 2D Presented in Figure 2C Figure 2. Genome editing efficiency in human iPSCs shown in Figure 2. Cells were harvested on day 5 (3 days after gRNA transfection) and day 7 (after completion of puromycin selection). Indels were detected using the Guide-iT Mutation Detection Kit. + / - indicates resolvase treatment. Cleavage efficiency (%) is presented below the gel image.

[0024] Figure 3A Shown is the efficiency of genome editing in HFFs transfected with VEE-Cas9 (S-Cas9) and B18R-E3L RNA or infected with lentiviral Cas9 (LV-Cas9) and then selected for 7 days with puromycin (0.8 μg / mL) or blasticidin (4 μg / mL), respectively. Cells were passaged on day 7 and transfected with 1-component K-Ras or EMX-1 sgRNA on day 9. Cells were collected on day 11 and analyzed for genome editing. + / - indicates resolvase treatment. % shows cleavage efficiency.

[0025] Figure 3B Presented are Guava flow cytometry analyses of GFP expression at day 4 in HEK293T cells co-transfected with Cas9 plasmid and K-Ras gRNA plasmid or co-transfected with VEE-Cas9-TagGFP2 RNA, B18R-E3L RNA, and 1-component K-Ras-gRNA.

[0026] Figure 3C Show in Figure 3B Efficiency of genome editing in cells as described in . + / - indicates resolvase treatment. % shows cleavage efficiency.

[0027] Figure 4A Presented are GFP expression in HFF cells generated by puromycin selection for one month following transfection with VEE-Cas9-TagGFP2, and genome editing following co-transfection with a 1-component K-Ras sgRNA as analyzed by Guava flow cytometry.

[0028] Figure 4B Shown is GFP expression in HEK293T cells generated by puromycin selection for one month following transfection with VEE-Cas9-TagGFP2, and genome editing following co-transfection with 1-component EMX-1 sgRNA as analyzed by Guava flow cytometry.

[0029] Figure 4C Comparison of genome editing in HFFs sequentially transfected with VEE-eSpCas9, VEE-eSpCas9-TagGFP2, or VEE-Cas9-TagGFP2 and 1-component EMX-1 gRNA. Cells were harvested on day 5 and analyzed for genome editing.

[0030] Figure 4D Comparison of genome editing in HEK293T cells sequentially transfected with VEE-Cas9-TagGFP2 or VEE-eSpCas9-TagGFP2, B18R-E3L RNA, and 1-component EMX-1 gRNA. Cells were harvested on day 4 and analyzed for genome editing.

[0031] Figure 5A Presented are images of GFP or RFP expression in HEK293T cells transfected with VEE-Cas9-D10A-TagGFP2 and VEE-Cas9-D10A-TagRFP at day 1 and day 3, respectively.

[0032] Figure 5B Images are presented for GFP or RFP expression after puromycin selection at day 14. GFP expression was also analyzed using Guava flow cytometry.

[0033] Figure 5C Comparison of genome editing in HEK 293T cells sequentially transfected with VEE-Cas9-D10A-TagGFP2, VEE-Cas9-D10A-TagRFP, or VEE-Cas9-TagGFP2 on day 1 and EMX-1 sgRNA-1, 9, or both on day 2. Cells were harvested on day 5 and analyzed for genome editing.

[0034] Figure 5DComparison of genome editing in HEK 293T cell lines expressing VEE-Cas9-D10A-TagGFP2, VEE-Cas9-D10A-TagRFP, or VEE-Cas9-TagGFP2. Each cell line was transfected with EMX-1 sgRNA-1 and / or -9 on day 1. Cells were harvested on day 4 and analyzed for genome editing.

[0035] Fig. 6A Rab11-BamHI oligonucleotide insertion by Cas9 cleavage in U2OS or HEK293T cells shown in the pool. U2OS or HEK 293T cells were transfected with VEE-Cas9-TagGFP2 or VEE-Cas9-D10A-GFP2 on day 1 and sequentially with sgRNA and Rab11-BamHI oligonucleotide on day 2. Cells were collected on day 5 and analyzed for BamHI insertion. Gel images show BamHI digestion resulting from Rab-11-BamHI oligonucleotide insertion at the cleavage site of VEE-Cas9-TagGFP2 (left) or VEE-Cas9-D10A-GFP2 (right).

[0036] Figure 6B Rab11-BamHI oligonucleotide insertion is shown at the Cas9 cleavage site in isolated clones. U2OS or human iPSCs were transfected with VEE-Cas9-GFP2 on day 1 and sgRNA and Rab11-BamHI oligonucleotide sequentially on day 2. Cells were selected with puromycin and clones were isolated for analysis. Gel image shows BamHI digestion resulting from Rab-11-BamHI oligonucleotide insertion at the VEE-Cas9-TagGFP2 cleavage site.

[0037] Figure 6C GFP fragment insertion is shown at the Cas9 cleavage site. HEK293 cells were sequentially transfected with VEE-Cas9-RFP on day 1 and sgRNA (for GAPDH localization) and GFP fragment on day 2. GFP expression was captured on day 5.

[0038] Details The present disclosure provides a synthetic, non-infectious self-replicating RNA vector based on a d virus, in which the sequence encoding the structural viral protein has been removed and replaced with a CRISPR protein sequence encoding. The self-replicating RNA vector is a single-stranded RNA that simulates cell mRNA with a 5' cap and a poly (A) tail. In certain embodiments, the self-replicating RNA vector does not utilize a DNA intermediate, and therefore there is no risk of CRISPR sequence genomic integration. The self-replicating RNA vector allows the CRISPR protein to be robustly expressed in multiple generations of cells. The co-transfection of cells with corresponding guide RNAs allows targeted genome editing. Due to dilution and degradation, the expression level of CRISPR decreases over time. Also provided herein is a method for introducing CRISPR proteins into cells together with corresponding guide RNAs for genome editing using a self-replicating RNA vector.

[0039] (I) RNA vector encoding CRISPR protein One aspect of the present disclosure provides a synthetic self-replicating RNA vector encoding a CRISPR protein. The self-replicating RNA vector comprises a sequence that ensures that the RNA vector is replicated in several generations of cells and that a heterologous protein sequence (e.g., at least one CRISPR protein) is translated. The self-replicating RNA vector is based on a modified alpha virus encoding a variety of replication complex proteins, but wherein the viral structural gene has been removed and replaced with a sequence encoding at least one CRISPR protein. Therefore, after entering the cell, RNA serves as a template for the translation of viral replication complex proteins and CRISPR proteins. The viral replication complex proteins form a replication complex that allows the RNA vector to be further replicated in the cytoplasm of the cell. The replicated RNA cannot be recombined with the cell DNA, and therefore there is no risk of integrating the CRISPR sequence into the cell genome.

[0040] (a) Synthetic self-replicating RNA The synthetic self-replicating RNA (or replicon) contains all sequence elements required for translating the encoded protein and replicating the RNA vector. In particular, the replicon is based on a modified alpha virus in which the nonstructural replicase gene is maintained and the structural gene (required for making infectious particles) is removed. In various embodiments, the modified alpha virus can be derived from Aola virus, Babanken virus, Bama Forest virus, Bebaru virus, Boji River virus, Chikungunya virus, Eastern Equine Encephalitis virus, Everglades virus, Fort Morgan virus, Geta virus, Highland J virus, Kezerageqi virus, Mayaro virus, Middleburg virus, Mukambu virus, Ndumau virus, Pixona virus, Ao Rongrong virus, Ross River virus, Lushan virus, Semliki Forest virus, Sindbis virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus or Vodaro River virus. These various sequences and their derivatives are known and can be found in the scientific literature, which is incorporated herein by reference in its entirety. In certain embodiments, the synthetic self-replicating RNA is based on a modified Venezuelan equine encephalitis (VEE) virus in which the structural genes have been removed. See, e.g., Yoshioka et al. (Cell Stem Cell 13, 246-254, August 1, 2013) and / or Petrakova et al. (J. Virology; vol. 72, no. 12, June 2005, p. 7597-7608), each of which is hereby incorporated by reference in its entirety.

[0041] The self-replicating RNA comprises sequences encoding a plurality of non-structural replication complex proteins. In a particular embodiment, the synthetic self-replicating RNA may encode four non-structural replication complex proteins (i.e., nsP1, nsP2, nsP3, nsP4). The non-structural replication complex proteins may be encoded by a single open reading frame (ORF).

[0042] The self-replicating RNA vector further comprises a sequence encoding at least one CRISPR protein described in detail in section (I)(b) below.

[0043] In general, the self-replicating RNA vector comprises a 5' cap, a 5' untranslated region (UTR) at the 5' end and a 3' UTR and a poly A tail at the 3' end. The self-replicating RNA vector generally comprises a promoter upstream of the sequence encoding the CRISPR protein. The upstream promoter may be a 26S subgenomic promoter.

[0044] In some embodiments, the self-replicating RNA vector may further comprise a sequence encoding at least one selective marker and / or a sequence encoding an interferon response inhibitor. Non-limiting examples of suitable selective markers include puromycin, geneticin, neomycin, hygromycin B, blastidinin S, etc. Examples of suitable interferon response inhibitors include, but are not limited to, vaccinia virus protein E3L, vaccinia virus protein B18R, influenza virus protein NS1, or lymphocytic choriomeningitis virus nucleoprotein.

[0045] The various protein coding sequences may be separated by an internal ribosome entry sequence (IRES) or a sequence encoding a 2A peptide. Non-limiting examples of suitable 2A peptides include the thosea asigna virus 2A peptide or T2A, the foot-and-mouth disease virus 2A peptide or F2A, the equine rhinitis A virus 2A peptide or E2A, and the porcine Teschovirus-1 2A peptide or P2A.

[0046] In a specific embodiment, the self-replicating RNA vector may be based on a modified Venezuelan equine encephalitis (VEE) virus and may comprise, from 5' to 3': a 5' cap, a 5' UTR, sequences encoding the four nonstructural replicases from VEE, a promoter, sequences encoding a CRISPR protein, an optional IRES, an optional sequence encoding an E3L protein, an optional IRES, an optional sequence encoding a selectable marker, an alphavirus 3' UTR, and a poly A tail (see Figure 1A ).

[0047] (b) CRISPR protein The self-replicating RNA vector also comprises a sequence encoding at least one CRISPR protein. CRISPR proteins that provide adaptive immunity to invading nucleic acids are present in various bacteria and archaea. In various embodiments, the CRISPR protein may be a type II Cas9 protein, a type V Cas12 (formerly known as Cpf1) protein, a type VI Cas13 (formerly known as C2cd) protein, a CasX protein, or a CasY protein.

[0048] The CRISPR protein can be from Acaryochloris spp., Acetohalobium spp., Acidaminococcus spp., Acidithiobacillus spp., Acidothermus spp., Akkermansia spp., Alicyclobacillus spp., Allochromatium spp., Ammonifex spp., Anabaena spp., Arthrospira spp., Bacillus spp., Bifidobacterium spp., Burkholderiales spp., Caldicelulosiruptor spp.), Campylobacter spp., Candidatus spp., Clostridium spp., Corynebacterium spp., Crocosphaera spp., Cyanothece spp., Deltaproteobacterium spp., Exiguobacterium spp., Finegoldia spp., Francisella spp., Ktedonobacter spp., Lachnospiraceae spp., Lactobacillus spp., Leptotrichia spp., Lyngbya spp. spp.), Marinobacter spp., Methanohalobium spp., Microscilla spp., Microcoleus spp., Microcystis spp., Mycoplasma spp., Natranaerobius spp., Neisseria spp., Nitratifractor spp., Nitrosococcus spp., Nocardiopsis spp., Nodularia spp., Nostoc spp., Oenococcus spp., Oscillatoria spp., Parasutterella spp., Pelotomaculum spp., Petrotoga spp., Planctomyces spp., Polaromonas spp., Prevotella spp., Pseudoalteromonas spp. spp.), Ralstonia spp., Ruminococcus spp., Staphylococcus spp., Streptococcus spp., Streptomyces spp., Streptosporangium spp., Synechococcus spp., Thermosipho spp., Verrucomicrobia spp., or Wolinella spp. These various CRISPR protein sequences and their derivatives are known and can be found in the scientific literature, which is hereby incorporated by reference herein. .

[0049] In some embodiments, the CRISPR protein can be Streptococcus pyogenes Cas9, Francisella novicida Cas9, Staphylococcus aureus Cas9, Streptococcus thermophilus Cas9, Streptococcus pasteurianus Cas9, Campylobacter jejuni Cas9, Neisseria meningitidis Cas9, Neisseria griseus Cas9, Francisella novicida Cas12, Acidaminococcus species Cas12, Lachnospiraceae bacteria ND2006 Cas12a, Weidenpilus Cas13a, Sachsenpilus Cas13a, Prevotella species P5-125Cas13, Ruminococcus chrysogenum Cas13d, 6Proteobacteria CasX, Planctomyces CasX or Candidatus CasY. In specific embodiments, the CRISPR protein is Streptococcus pyogenes Cas9 or Staphylococcus aureus Cas9.

[0050] CRISPR proteins can be wild-type or naturally occurring proteins. Wild-type CRISPR proteins generally contain two nuclease domains, such as Cas9 proteins containing RuvC and HNH domains, each of which cleaves one strand of a double-stranded sequence. CRISPR proteins also contain domains that interact with guide RNAs (e.g., REC1, REC2) or domains that interact with RNA / DNA heteroduplexes (e.g., REC3), and domains that interact with pre-spacer adjacent motifs (PAMs) (i.e., PAM-interaction domains).

[0051] Alternatively, the CRISPR protein can be modified or engineered to have altered activity, specificity and / or stability. For example, the CRISPR protein can be engineered to include one or more modifications / mutations (i.e., substitutions, deletions and / or insertions of at least one amino acid). The modified CRISPR protein can have altered catalytic (nuclease) activity, improved target site specificity, reduced off-target effects, altered PAM specificity, increased stability, and the like.

[0052] In various embodiments, the CRISPR protein may be a nuclease (i.e., cleaving both chains of a double-stranded nucleotide sequence or cleaving a single-stranded nucleotide sequence). In other embodiments, the CRISPR protein may be a nickase, which cleaves one chain of a double-stranded sequence. The nickase may be modified by inactivating a nuclease domain of the CRISPR protein. For example, the RuvC domain of the Cas9 protein may be inactivated by mutations (such as D10A, D8A, E762A, and / or D986A), or the HNH domain of the Cas9 protein may be inactivated by mutations (such as H840A, H559A, N854A, N856A, and / or N863A) (with reference to the numbering system of Streptococcus pyogenes Cas9SpyCas9) to produce a Cas9 nickase (e.g., nCas9). Comparable mutations in other CRISPR proteins may produce nickases (e.g., nCas12). Inactivating both nuclease domains produces a CRISPR protein without cleavage activity, that is, a catalytically inactive or nuclease-dead protein (e.g., dCas9, dCas12, etc.).

[0053] CRISPR proteins can also be modified by one or more amino acid substitutions, deletions, and / or insertions to have improved targeting specificity, improved fidelity, altered PAM specificity, reduced off-target effects, and / or increased stability. Non-limiting examples of one or more mutations that improve targeting specificity, improve fidelity, and / or reduce off-target effects include N497A, R661A, Q695A, K810A, K848A, K855A, Q926A, K1003A, R1060A, and / or D1135E (with reference to the numbering system of SpyCas9).

[0054] The RNA vector sequence encoding the CRISPR protein can be codon optimized for efficient translation into protein in the target eukaryotic cell. Codon optimization programs can be obtained as free software or from commercial sources. In a specific embodiment, the sequence encoding the CRISPR protein can be codon optimized for efficient expression in human cells.

[0055] Optional heterologous domain In some embodiments, CRISPR protein can be modified to include at least one heterologous domain, that is, CRISPR protein can be connected to one or more heterologous domains. Heterologous domains can be nuclear localization signals (NLS), cell penetration domains, marker domains (such as fluorescent proteins), chromatin regulatory motifs, epigenetic modification domains (such as deaminase domains, histone acetyltransferase domains, etc.), transcriptional regulatory domains, RNA aptamer binding domains or non-CRISPR nuclease domains. In the case where two or more heterologous domains are fused with CRISPR protein, the two or more heterologous domains can be the same, or they can be different. One or more heterologous domains can be connected to the N-terminus, C-terminus, internal position or a combination thereof of CRISPR protein. The connection can be direct (via chemical bonds), or the connection can be indirect (via one or more joints). Suitable joints are known in the art. In certain embodiments, the connection can be through a 2A peptide sequence.

[0056] In some embodiments, the one or more heterologous domains may be a nuclear localization signal (NLS). Non-limiting examples of nuclear localization signals include PKKKRKV (SEQ ID NO: 1), PKKKRRV (SEQ ID NO: 2), KRPAATKKAGQAKKKK (SEQ ID NO: 3), YGRKKRRQRRR (SEQ ID NO: 4), RKKRRQRRR (SEQ ID NO: 5), PAAKRVKLD (SEQ ID NO: 6), RQRRNELKRSP (SEQ ID NO: 7), VSRKRPRP (SEQ ID NO: 8), PPKKARED (SEQ ID NO: 9), PQPKKKPL (SEQ ID NO: 10), SALIKKKKKMAP (SEQ ID NO: 11), PKQKKRK (SEQ ID NO: 12), RKLKKKIKKL (SEQ ID NO: 13), REKKKFLKRR (SEQ ID NO: 14), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 15), RKCLQAGMNLEARKTKK (SEQ ID NO: 16), ID NO: 16), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 17) and RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 18).

[0057] In other embodiments, the one or more heterologous domains may be cell penetrating domains. Examples of suitable cell penetrating domains include, but are not limited to, GRKKRRQRRRPPQPKKKRKV (SEQ ID NO: 19), PLSSIFSRIGDPPKKKRKV (SEQ ID NO: 20), GALFLGWLGAAGSTMGAPKKKRKV (SEQ ID NO: 21), GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 22), KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 23), YARAAARQARA (SEQ ID NO: 24), THRLPRRRRRR (SEQ ID NO: 25), GGRRARRRRRR (SEQ ID NO: 26), RRQRRTSKLMKR (SEQ ID NO: 27), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 28), KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 29), and RQIKIWFQNRRMKWKK (SEQ ID NO: 30). ID NO: 30).

[0058] In alternative embodiments, the one or more heterologous domains may be marker domains. The marker domains include fluorescent proteins and purification or epitope tags. Suitable fluorescent proteins include, but are not limited to, green fluorescent proteins (e.g., GFP, eGFP, GFP-2, tagGFP, turboGFP, Emerald, Azami Green, monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., BFP, EBFP, EBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-monomer, HcRed-tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), orange fluorescent protein (e.g., mOrange, mKO, Kusabira-Orange, monomeric Kusabira-Orange, mTangerine, tdTomato), or a combination thereof. The fluorescent protein may comprise tandem repeats of one or more fluorescent proteins (e.g., Suntag). Non-limiting examples of suitable purification or epitope tags include 6xHis (SEQ ID NO: 40), HA, GST, Myc, SAM, etc. Non-limiting examples of heterologous fusions that facilitate detection or enrichment of CRISPR complexes include streptavidin (Kipriyanov et al., Human Antibodies, 1995, 6(3):93-101.), avidin (Airenne et al., Biomolecular Engineering, 1999, 16(1-4):87-92), monomeric forms of avidin (Laitinen et al., Journal of Biological Chemistry, 2003, 278(6):4010-4014), peptide tags that facilitate biotinylation during recombinant production (Cull et al., Methods in Enzymology, 2000, 326:430-440).

[0059] In yet other embodiments, the one or more heterologous domains may be chromatin regulatory motifs (CMMs). Non-limiting examples of CMMs include peptides derived from high mobility group (HMG) proteins (e.g., HMGB1, HMGB2, HMGB3, HMGN1, HMGN2, HMGN3a, HMGN3b, HMGN4, and HMGN5 proteins) that interact with nucleosomes, central globular domains of histone H1 variants (e.g., histone H1.0, H1.1, H1.2, H1.3, H1.4, H1.5, H1.6, H1.7, H1.8, H1.9, and H.1.10), or chromatin remodeling complexes (e.g., SWI / SNF (Switch / Sucrose Not Fermented), ISWI (Switch Simulated ( I mitation SWI The CMMs may also be derived from topoisomerases, helicases, or viral proteins. The sources of CMMs may and will vary. CMMs may be derived from humans, animals (i.e., vertebrates and invertebrates), plants, algae, or yeast.

[0060] In yet other embodiments, the one or more heterologous domains may be epigenetic modifying domains. Non-limiting examples of suitable epigenetic modification domains include those having DNA deamination (e.g., cytidine deaminase, adenosine deaminase, guanosine deaminase), DNA methyltransferase activity (e.g., cytosine methyltransferase), DNA demethylase activity, DNA amination, DNA oxidation activity, DNA helicase activity, histone acetyltransferase (HAT) activity (e.g., a HAT domain derived from the E1A binding protein p300), histone deacetylase activity, histone methyltransferase activity, histone demethylase activity, histone kinase activity, histone phosphatase activity, histone ubiquitin ligase activity, histone deubiquitinating activity, histone adenylating activity, histone deadenylating activity, histone SUMOylating activity, histone deSUMOylating activity, histone ribosylating activity, histone deribosylating activity, histone myristoylation activity, histone demyristoylation activity, histone citrullination activity, histone alkylating activity, histone dealkylating activity, or histone oxidizing activity. In particular embodiments, the epigenetic modification domain can comprise cytidine deaminase activity, adenosine deaminase activity, histone acetyltransferase activity, or DNA methyltransferase activity.

[0061] In other embodiments, the one or more heterologous domains may be transcriptional regulatory domains (i.e., transcriptional activation domains or transcriptional repressor domains). Suitable transcriptional activation domains include, but are not limited to, herpes simplex virus VP16 domain, VP64 (i.e., four tandem copies of VP16), VP160 (i.e., ten tandem copies of VP16), NFκB p65 activation domain (p65), Epstein-Barr virus R trans-acting factor (Rta) domain, VPR (i.e., VP64+p65+Rta), p300-dependent transcriptional activation domain, p53 activation domains 1 and 2, heat shock factor 1 (HSF1) activation domain, Smad4 activation domain (SAD), cAMP response element binding protein (CREB) activation domain, E2A activation domain, nuclear factor of activated T cells (NFAT) activation domain, or a combination thereof. Non-limiting examples of suitable transcriptional repressor domains include Kruppel associated box (KRAB) repressor domains, Mxi repressor domains, inducible cAMP early repressor (ICER) domains, YY1 glycine-rich repressor domains, Sp1-like repressors, E (spl) repressors, IκB repressors, Sin3 repressors, methyl-CpG binding protein 2 (MeCP2) repressors, or combinations thereof. Transcriptional activation or transcriptional repressor domains can be genetically fused to the Cas9 protein, or bound via non-covalent protein-protein, protein-RNA or protein-DNA interactions.

[0062] In a further embodiment, the one or more heterologous domains may be RNA aptamer binding domains (Konermann et al., Nature, 2015, 517(7536):583-588; Zalatan et al., Cell, 2015, 160(1-2):339-50). Examples of suitable RNA aptamer protein domains include MS2 coat protein (MCP), PP7 phage coat protein (PCP), μ phage Com protein, λ phage N22 protein, stem-loop binding protein (SLBP), fragile X mental retardation syndrome-related protein 1 (FXR1), derived from bacteriophages (such as AP205, BZ13, f1, f2, fd, fr, ID2, JP34 / GA, JP501, JP34, JP500, KU1, M11, M12, MX1, NL95, PP7, Qβ, R17, SP-β, TW18, TW19 and VK) proteins, fragments thereof or derivatives thereof.

[0063] In other embodiments, the one or more heterologous domains may be non-CRISPR nuclease domains. Suitable nuclease domains may be obtained from any endonuclease or exonuclease. Non-limiting examples of endonucleases from which nuclease domains may be derived include, but are not limited to, restriction endonucleases and homing endonucleases. In some embodiments, the nuclease domain may be derived from a II-S type restriction endonuclease. II-S type endonucleases cleave DNA at sites that are usually a few base pairs away from the recognition / binding site, and therefore have separable binding and cleavage domains. These enzymes are generally monomers that associate to form dimers briefly to cleave each strand of DNA at staggered positions. Non-limiting examples of suitable II-S type endonucleases include BfiI, BpmI, BsaI, BsgI, BsmBI, BsmI, BspMI, FokI, MboII, and SapI. In some embodiments, the nuclease domain may be a FokI nuclease domain or a derivative thereof. II-S type nuclease domains can be modified to promote dimerization of two different nuclease domains. For example, the cleavage domain of FokI can be modified by mutating certain amino acid residues. As a non-limiting example, amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of the FokI nuclease domain are targets for modification. In a particular embodiment, the FokI nuclease domain may comprise a first FokI half-domain (comprising Q486E, I499L, and / or N496D mutations) and a second FokI half-domain (comprising FA90K, I538K, and / or H537R mutations).

[0064] (II) Complex containing a self-replicating RNA vector and a guide RNA Another aspect of the present disclosure comprises a complex comprising any of the self-replicating RNA vectors described in section (I) above and at least one guide RNA, wherein the guide RNA is designed to complex with a CRISPR protein encoded by the self-replicating RNA vector.

[0065] The guide RNA interacts with the CRISPR protein and the target sequence in the target nucleic acid and guides the CRISPR protein to the target sequence. There is no sequence restriction on the target sequence, except that the sequence is adjacent to the pre-spacer adjacent motif (PAM) sequence. The CRISPR protein can recognize different PAM sequences. For example, the PAM sequences for Cas9 protein include 5'-NGG, 5'-NGGNG, 5'-NNAGAAW, 5'-NNNNGATT and 5-NNNNRYAC, while the PAM sequences for Cas12 protein include 5'-TTN and 5'-TTTV, where N is defined as any nucleotide, R is defined as G or A, W is defined as A or T, Y is defined as C or T and V is defined as A, C or G. In general, the Cas9 PAM is located at the 3' of the target sequence, while the Cas12 PAM is located at the 5' of the target sequence.

[0066] Guide RNAs are engineered to complex with specific CRISPR proteins. Generally, guide RNAs include (i) CRISPR RNA (crRNA) containing a guide sequence at the 5' end that hybridizes with a target sequence, and (ii) a trans-acting crRNA (tracrRNA) sequence that interacts with the CRISPR protein. The crRNA guide sequence of each guide RNA is different (i.e., sequence-specific). The tracrRNA sequence is generally the same in guide RNAs designed to complex with specific CRISPR proteins.

[0067] The crRNA guide sequence is designed to hybridize with the target sequence (i.e., the pre-spacer sequence) in the target sequence. In general, the complementarity between crRNA and the target sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. In a specific embodiment, the complementarity is complete (i.e., 100%). In various embodiments, the length range of the crRNA guide sequence may be from about 15 nucleotides to about 25 nucleotides. For example, the crRNA guide sequence length may be about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In a specific embodiment, the crRNA length is about 19, 20, or 21 nucleotides. In one embodiment, the crRNA guide sequence has a length of 20 nucleotides.

[0068] The guide RNA comprises a repetitive sequence that forms at least one stem-loop structure that interacts with the CRISPR protein, and a 3' sequence that generally maintains a single strand. Each loop and stem length may be different. For example, the length of the loop may range from about 3 to about 10 nucleotides, and the length of the stem may range from about 6 to about 20 base pairs. The stem may comprise one or more protrusions of 1 to about 10 nucleotides. The length of the single-stranded 3' region may be different. The tracrRNA sequence in the guide RNA is generally based on the sequence of the wild-type tracrRNA that interacts with the wild-type CRISPR protein. The wild-type sequence may be modified to promote secondary structure formation, increase secondary structure stability, promote expression in eukaryotic cells, and the like. For example, one or more nucleotide changes may be introduced into the guide RNA coding sequence. The tracrRNA sequence length may range from about 50 nucleotides to about 300 nucleotides. In various embodiments, the tracrRNA can range in length from about 50 to about 90 nucleotides, from about 90 to about 110 nucleotides, from about 110 to about 130 nucleotides, from about 130 to about 150 nucleotides, from about 150 to about 170 nucleotides, from about 170 to about 200 nucleotides, from about 200 to about 250 nucleotides, or from about 250 to about 300 nucleotides.

[0069] In some embodiments, the guide RNA can be a single molecule (e.g., a single guide RNA (sgRNA) or a 1-component sgRNA), wherein the crRNA sequence is linked to the tracrRNA sequence. In some embodiments, the guide RNA can be two separate molecules (e.g., a 2-component gRNA). The first molecule comprises a crRNA comprising a 3' sequence (comprising from about 6 to about 20 nucleotides) capable of base pairing with the 5' end of the second molecule, wherein the second molecule comprises a tracrRNA comprising a 5' sequence (comprising from about 6 to about 20 nucleotides) capable of base pairing with the 3' end of the first molecule.

[0070] In some embodiments, the tracrRNA sequence of the guide RNA can be modified to include one or more aptamer sequences (Konermann et al., Nature, 2015, 517(7536):583-588; Zalatan et al., Cell, 2015, 160(1-2):339-50). Suitable aptamer sequences include those that bind to adapter proteins selected from MCP, PCP, Com, SLBP, FXR1, AP205, BZ13, f1, f2, fd, fr, ID2, JP34 / GA, JP501, JP34, JP500, KU1, M11, M12, MX1, NL95, PP7, Qβ, R17, SP-β, TW18, TW19, VK, fragments thereof or derivatives thereof. Those skilled in the art will appreciate that the length of the aptamer sequence may vary.

[0071] In other embodiments, the guide RNA may further comprise at least one detectable marker. The detectable marker may be a fluorophore (e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluors, Halo tags or suitable fluorescent dyes), a detection label (e.g., biotin, digoxigenin, etc.), quantum dots or gold particles.

[0072] The guide RNA may comprise standard ribonucleotides and / or modified ribonucleotides. In some embodiments, the guide RNA may comprise standard or modified deoxyribonucleotides. In embodiments where the guide RNA is enzymatically synthesized (i.e., in vivo or in vitro), the guide RNA generally comprises standard ribonucleotides. In embodiments where the guide RNA is chemically synthesized, the guide RNA may comprise standard or modified ribonucleotides and / or deoxyribonucleotides. Modified ribonucleotides and / or deoxyribonucleotides include base modifications (e.g., pseudouridine, 2-thiouridine, N6-methyladenosine, etc.) and / or sugar modifications (e.g., 2'-O-methyl, 2'-fluoro, 2'-amino, locked nucleic acid (LNA), etc.). The backbone of the guide RNA may also be modified to include phosphorothioate bonding, borane phosphate bonding, or peptide nucleic acid.

[0073] (III) Eukaryotic cells Another aspect of the present disclosure comprises a eukaryotic cell or cell line comprising any one of the self-replicating RNA vectors described in section (I) above. That is, the eukaryotic cell or cell line has been transfected with one of the self-replicating RNA vectors. In some embodiments, the eukaryotic cell or cell line may further comprise at least one guide RNA engineered to complex with a CRISPR protein encoded by the RNA vector.

[0074] The eukaryotic cell or cell line can be a human cell, a non-human mammalian cell, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, or a unicellular eukaryotic organism. Examples of suitable eukaryotic cells are described in detail in section (V)(c) below. The eukaryotic cell can be in vitro, ex vivo, or in vivo.

[0075] (IV) Plasmid vector encoding self-replicating RNA A further aspect of the present disclosure provides a plasmid vector encoding the self-replicating RNA described in section (I) above. In particular, the plasmid vector comprises a sequence encoding a non-structural replication complex protein from an alpha virus, a sequence encoding a CRISPR protein, and additional viral sequences (such as 5'UTR, subgenomic promoter and 3'UTR), an optional selectable marker sequence, an optional interferon inhibitor sequence, an optional IRES, etc.

[0076] In general, the plasmid vector encoding self-replicating RNA is a DNA vector. The sequence encoding self-replicating RNA can be operably connected to a promoter sequence recognized by a bacteriophage RNA polymerase for in vitro RNA synthesis. For example, the promoter sequence can be a T7, T3 or SP6 promoter sequence, or a variant of a T7, T3 or SP6 promoter sequence. The promoter sequence can be a wild type, or it can be modified for more efficient or effective expression. The plasmid vector can further include at least one transcription termination sequence, and at least one replication origin and / or a selective marker sequence (e.g., an antibiotic resistance gene) for value-added in bacterial cells. The plasmid vector can be derived from pUC, pBR322, pET, pBluescript or its variants. Additional information about vectors and their use can be found in “Current Protocols in Molecular Biology” Ausubel et al., John Wiley & Sons, New York, 2003 or in “Molecular Cloning: A Laboratory Manual” Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001.

[0077] Following in vitro synthesis of self-replicating RNA, the RNA can be purified, 5' capped, and polyadenylated using standard procedures or commercially available kits.

[0078] (V) Methods for genome editing Further aspects of the present disclosure include methods for genome editing in eukaryotic cells. In general, the method includes introducing any one of the self-replicating RNA vectors described in section (I) above and at least one guide RNA modified to be complexed with a CRISPR protein encoded by the RNA vector into a target eukaryotic cell. The method may also include introducing the complex described in section (II) above into a eukaryotic cell. The CRISPR system includes a CRISPR protein and a guide RNA.

[0079] In embodiments where the CRISPR protein comprises nuclease or nickase activity, genome editing may comprise replacement of at least one nucleotide, deletion of at least one nucleotide, and / or insertion of at least one nucleotide. In some iterations, the method comprises introducing a CRISPR system comprising nuclease activity or two CRISPR systems comprising nickase activity into a eukaryotic cell without introducing a donor polynucleotide, causing one or more CRISPR systems to introduce double-strand breaks in the target site of the target sequence, and introducing at least one nucleotide change (i.e., insertion / deletion) by double-strand break repair of the cellular DNA repair process, thereby inactivating the sequence (i.e., gene knockout). In other iterations, the method comprises introducing a CRISPR system comprising nuclease activity or two CRISPR systems comprising nickase activity and a donor polynucleotide into a eukaryotic cell, causing one or more CRISPR systems to introduce double-strand breaks in the target site of the target sequence, and repairing double-strand breaks by the cellular DNA repair process results in the insertion or exchange of sequences in the donor polynucleotides into the target site of the target sequence (i.e., gene correction or gene knock-in).

[0080] In embodiments in which the CRISPR protein comprises epigenetic modification activity or transcriptional regulation activity, genome editing may comprise conversion of at least one nucleotide in the chromosomal sequence in or near the target site (i.e., base editing), modification of at least one nucleotide in or near the target site, modification of at least one histone in or near the target site, and / or transcriptional changes in or near the target site.

[0081] (a) Introduced into cells As described above, the method comprises introducing at least one self-replicating RNA vector described in section (I) above, at least one guide RNA modified to complex with a CRISPR protein encoded by the RNA vector, and an optional donor polynucleotide into a eukaryotic cell. The molecule can be introduced into the target cell by various means.

[0082] For example, a self-replicating RNA vector, a guide RNA, and an optional donor polynucleotide can be transfected into a target cell. Suitable transfection methods include nuclear transfection (or electroporation), calcium phosphate-mediated transfection, cationic polymer transfection (e.g., DEAE-dextran or polyethyleneimine), viral transduction, virosome transfection, virion transfection, liposome transfection, cationic liposome transfection, immunoliposome transfection, non-liposomal lipid transfection, dendrimer transfection, heat shock transfection, magnetofection, lipofection, gene gun delivery, impalefection, sonoporation, optical transfection, and nucleic acid uptake enhanced by proprietary agents. Transfection methods are well known in the art (see, for example, "Current Protocols in Molecular Biology" Ausubel et al., John Wiley & Sons, New York, 2003 or "Molecular Cloning: A Laboratory Manual" Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, third edition, 2001). In other embodiments, the molecule can be introduced into the cell by microinjection. For example, the molecule can be injected into the cytoplasm or nucleus of the target cell. The amount of each molecule introduced into the cell can vary, but those skilled in the art are familiar with means of determining the appropriate amount.

[0083] The various molecules can be introduced into the cell simultaneously or sequentially. For example, a self-replicating RNA vector and a guide RNA can be introduced simultaneously. Alternatively, one can be introduced first, and then the other can be introduced into the cell subsequently.

[0084] In general, cells are maintained under conditions suitable for cell growth and / or maintenance. Suitable cell culture conditions are well known in the art and are described, for example, in Santiago et al., Proc. Natl. Acad. Sci. USA, 2008, 105: 5809-5814; Moehle et al., Proc. Natl. Acad. Sci. USA, 2007, 104: 3055-3060; Urnov et al., Nature, 2005, 435: 646-651; and Lombardo et al., Nat. Biotechnol., 2007, 25: 1298-1306. Those skilled in the art will appreciate that methods for culturing cells are known in the art and can and will vary depending on the cell type. In all cases, routine optimization can be used to determine the best technique for a particular cell type.

[0085] (b) Optional donor polynucleotide In embodiments where the CRISPR protein comprises nuclease or nickase activity, the method may further comprise introducing at least one donor polynucleotide into the cell. The donor polynucleotide may be single-stranded or double-stranded, linear or circular, and / or RNA or DNA. In some embodiments, the donor polynucleotide may be a vector, such as a plasmid vector.

[0086] The donor polynucleotide comprises at least one donor sequence. In some aspects, the donor sequence of the donor polynucleotide may be a modified version of an endogenous or native chromosome sequence. For example, the donor sequence may be substantially identical to a portion of a chromosome sequence at or near the sequence targeted by the CRISPR system, but comprises a change in at least one nucleotide. Therefore, after integration or exchange with the native sequence, the sequence at the targeted chromosome position comprises a change in at least one nucleotide. For example, the change may be one or more nucleotide insertions, one or more nucleotide deletions, one or more nucleotide replacements, or a combination thereof. As a result of the integration of the modified sequence "gene correction", the cell may produce a modified gene product from the targeted chromosome sequence.

[0087] In other aspects, the donor sequence of the donor polynucleotide can be an exogenous sequence. As used herein, an "exogenous" sequence refers to a sequence that is not native to the cell, or a sequence of different positions in the cell genome in its native position. For example, the exogenous sequence can include a protein coding sequence that can be operably connected to an exogenous promoter control sequence, so that after being integrated into the genome, the cell can express the protein encoded by the sequence integrated. Alternatively, the exogenous sequence can be integrated into the chromosomal sequence, causing its expression to be regulated by an endogenous promoter control sequence. In other iterations, the exogenous sequence can be a transcription control sequence, another expression control sequence, an RNA coding sequence, etc. As described above, the integration of an exogenous sequence into the chromosomal sequence is referred to as "knock-in".

[0088] As will be appreciated by those skilled in the art, the length of the donor sequence can and will vary. For example, the length of the donor sequence can vary from a few nucleotides to hundreds of nucleotides to hundreds of thousands of nucleotides.

[0089] Typically, the donor sequence in the donor polynucleotide is flanked by an upstream sequence and a downstream sequence that have substantial sequence identity to the sequences located upstream and downstream of the sequence targeted by the CRISPR system, respectively. Because of these sequence similarities, the upstream and downstream sequences of the donor polynucleotide allow homologous recombination between the donor polynucleotide and the targeted chromosomal sequence, so that the donor sequence can be integrated into (or exchanged with) the chromosomal sequence.

[0090] As used herein, an upstream sequence refers to a nucleic acid sequence that shares basic sequence identity with a chromosomal sequence upstream of a sequence targeted by a CRISPR system. Similarly, a downstream sequence refers to a nucleic acid sequence that shares basic sequence identity with a chromosomal sequence downstream of a sequence targeted by a CRISPR system. As used herein, the phrase "basic sequence identity" refers to a sequence having at least about 75% sequence identity. Therefore, the upstream and downstream sequences in the donor polynucleotide may have about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with a sequence upstream or downstream of the target sequence. In exemplary embodiments, the upstream and downstream sequences in the donor polynucleotide may have about 95% or 100% sequence identity to the chromosomal sequence upstream or downstream of the sequence targeted by the CRISPR system.

[0091] In some embodiments, the upstream sequence shares basic sequence identity with a chromosomal sequence located immediately upstream of the sequence targeted by the CRISPR system. In other embodiments, the upstream sequence shares basic sequence identity with a chromosomal sequence located within about one hundred (100) nucleotides upstream of the target sequence. Thus, for example, the upstream sequence may share basic sequence identity with a chromosomal sequence located about 1 to about 20, about 21 to about 40, about 41 to about 60, about 61 to about 80, or about 81 to about 100 nucleotides upstream of the target sequence. In some embodiments, the downstream sequence shares basic sequence identity with a chromosomal sequence located immediately downstream of the sequence targeted by the CRISPR system. In other embodiments, the downstream sequence shares basic sequence identity with a chromosomal sequence located within about one hundred (100) nucleotides downstream of the target sequence. Thus, for example, the downstream sequence may share basic sequence identity with a chromosomal sequence located about 1 to about 20, about 21 to about 40, about 41 to about 60, about 61 to about 80, or about 81 to about 100 nucleotides downstream of the target sequence.

[0092] The length range of each upstream or downstream sequence can be from about 20 nucleotides to about 5000 nucleotides. In some embodiments, the upstream and downstream sequences can include about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800 or 5000 nucleotides. In a specific embodiment, the length range of the upstream and downstream sequences can be from about 50 to about 1500 nucleotides.

[0093] (c) Cell type Various eukaryotic cells are suitable for use in the methods disclosed herein. For example, the cell may be a human cell, a non-human mammal cell, a non-mammalian vertebrate cell, an invertebrate cell, an insect cell, a plant cell, a yeast cell, or a unicellular eukaryotic organism. In some embodiments, the cell may be a primary cell isolated directly from a specific tissue. In other embodiments, the cell may be a cell line cell. In other embodiments, the cell may be an embryo of one cell. For example, a non-human mammal embryo, including rats, hamsters, rodents, rabbits, cats, dogs, sheep, pigs, cattle, horses, and primates. In other embodiments, the cell may be a stem cell, such as an embryonic stem cell, an ES-like stem cell, a fetal stem cell, an adult stem cell, an induced pluripotent stem cell, and the like. In one embodiment, the stem cell is not a human embryonic stem cell. In addition, stem cells may include those cells made by the techniques disclosed in WO2003 / 046141 (incorporated herein in its entirety) or Chung et al. (Cell Stem Cell, 2008, 2: 113-117). In various embodiments, the cell can be in vitro (i.e., in culture), ex vivo (i.e., in a tissue isolated from an organism), or in vivo (i.e., within an organism). In exemplary embodiments, the cell is a mammalian cell or a mammalian cell line. In specific embodiments, the cell is a human cell or a human cell line.

[0094] Non-limiting examples of suitable mammalian cells or cell lines include human embryonic kidney cells (HEK293, HEK293T); human cervical carcinoma cells (HELA); human lung cells (W138); human liver cells (Hep G2); human U2-OS osteosarcoma cells, human A549 cells, human A431 cells, and human K562 cells; Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells; mouse myeloma NS0 cells, human primary fibroblasts, human foreskin fibroblasts, mouse embryonic fibroblast 3T3 cells (NIH3T3), mouse B lymphoma A20 cells; mouse melanoma B16 cells; mouse myoblast C2C12 cells; mouse myeloma SP2 / 0 cells; mouse embryonic mesenchymal C3H-10T1 / 2 cells; mouse Cancer CT26 cells, mouse prostate DuCuP cells; mouse mammary gland EMT6 cells; mouse liver cancer Hepa1c1c7 cells; mouse myeloma J5582 cells; mouse epithelial MTD-1A cells; mouse myocardial MyEnd cells; mouse kidney RenCa cells; mouse pancreatic RIN-5F cells; mouse melanoma X64 cells; mouse lymphoma YAC-1 cells; rat glioblastoma 9L cells; rat B lymphoma RBL cells; rat neuroblastoma B35 cells; rat hepatoma cell (HTC); buffalo rat liver BRL 3A cells; canine kidney cells (MDCK); canine mammary gland (CMT) cells; rat osteosarcoma D17 cells; rat monocyte / macrophage DH82 cells; monkey kidney SV-40 transformed fibroblasts (COS7) cells; monkey kidney CVI-76 cells; African green monkey kidney (VERO-76) cells. An extensive listing of mammalian cell lines can be found in the catalog of the American Type Culture Collection (ATCC, Manassas, VA).

[0095] (VI) Application The compositions and methods disclosed herein can be used in a variety of therapeutic, diagnostic, industrial and research applications. In some embodiments, the disclosure can be used to modify any target chromosome sequence in a cell, animal or plant to construct a gene function model and / or study gene function, study target genetic or epigenetic conditions, or study biochemical pathways involved in various diseases or conditions. For example, a transgenic organism that constructs a disease or condition model can be created in which the expression of one or more nucleic acid sequences associated with the disease or condition is altered. Disease models can be used to study the effects of mutations on organisms, study the development and / or progression of a disease, study the effects of pharmaceutically active compounds on a disease, and / or evaluate the efficacy of potential gene therapy strategies.

[0096] In other embodiments, the compositions and methods can be used to perform efficient and cost-effective functional genomic screens, which can be used to study the function of genes involved in specific biological processes and how any changes in gene expression can affect biological processes, or can be used to perform saturation or deep scanning mutagenesis of genomic loci in conjunction with cell phenotypes. For example, saturation or deep scanning mutagenesis can be used to determine the critical minimal features and discrete vulnerabilities of functional elements required for gene expression, drug resistance, and disease reversal.

[0097] In further embodiments, the compositions and methods disclosed herein can be used in diagnostic tests to determine the presence of a disease or condition and / or to determine treatment options. Examples of suitable diagnostic tests include detection of specific mutations in cancer cells (such as specific mutations in EGFR, HER2, etc.), detection of specific mutations associated with specific diseases (such as trinucleotide repeats, mutations in beta globin associated with sickle cell disease, specific SNPs, etc.), detection of hepatitis, detection of viruses (such as Zika), and the like.

[0098] In other embodiments, the compositions and methods disclosed herein can be used to correct gene mutations associated with specific diseases or conditions, such as correcting globin gene mutations associated with sickle cell disease or thalassemia, correcting mutations in adenosine deaminase genes associated with severe combined immunodeficiency (SCID), reducing the expression of HTT (the causative gene for Huntington's disease), or correcting mutations in rhodopsin genes to treat retinitis pigmentosa. Such modifications can be performed in ex vivo cells.

[0099] In yet other embodiments, the compositions and methods disclosed herein can be used to produce crops with improved traits or enhanced resistance to environmental stress. The disclosure can also be used to produce livestock or production animals with improved traits. For example, pigs have many characteristics that make them attractive as biomedical models, particularly in regenerative medicine or xenotransplantation.

[0100] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs. The following references provide the technician with common definitions of many terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (Second Edition 1994); The Cambridge Dictionary of Science and Technology (Walker, 1988); The Glossary of Genetics, Fifth Edition, R. Rieger et al., Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise indicated, the following terms have the meanings ascribed thereto.

[0101] When introducing elements of the present disclosure or the preferred embodiments thereof, the articles "a", "an", "the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0102] When used in relation to a numerical value such as x, the term "about" means x ± 5%.

[0103] As used herein, the term "complementary" or "complementarity" refers to the association of double-stranded nucleic acids by base pairing via specific hydrogen bonds. Base pairing can be standard Watson-Crick base pairing (e.g., 5'-AGTC-3' pairs with the complementary sequence 3'-TCAG-5'). Base pairing can also be Hoogsteen or reverse Hoogsteen hydrogen bonding. Complementarity is usually measured with respect to the duplex region and therefore does not include, for example, overhangs. If only some (e.g., 70%) bases are complementary, the complementarity between the two chains in the duplex region can be partial and can be expressed as a percentage (e.g., 70%). Non-complementary bases are "mismatched". If all bases in the duplex region are complementary, complementarity can also be complete (i.e., 100%).

[0104] As used herein, the term "CRISPR / Cas system" or "Cas9 system" refers to a complex comprising a Cas9 protein (ie, a nuclease, a nickase, or a catalytically dead protein) and a guide RNA.

[0105] As used herein, the term "endogenous sequence" refers to a chromosomal sequence native to a cell.

[0106] As used herein, the term "exogenous" refers to a sequence that is not native to the cell, or a chromosomal sequence whose native location in the genome of the cell is at a different chromosomal location.

[0107] The term "expression" about a gene or polynucleotide refers to the transcription of a gene or polynucleotide, and, where appropriate, to the translation of an mRNA transcript to a protein or polypeptide. Therefore, as is clear from the context, the expression of a protein or polypeptide is the result of the transcription and / or translation of an open reading frame.

[0108] As used herein, "gene" refers to a DNA region encoding a gene product (including exons and introns), as well as all DNA regions that regulate the production of a gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, a promoter sequence, a terminator, a translation regulatory sequence (such as a ribosome binding site and an internal ribosome entry site), an enhancer, a silencer, an insulator, a boundary element, an origin of replication, a matrix binding site, and a locus control region.

[0109] The term "heterologous" refers to an entity that is not endogenous or native to the target cell. For example, a heterologous protein refers to a protein that is derived from or originally derived from an exogenous source (such as an exogenously introduced nucleic acid sequence). In some cases, the heterologous protein is not normally produced by the target cell.

[0110] The term "nickase" refers to an enzyme that cleaves one strand of a double-stranded nucleic acid sequence (i.e., nicks the double-stranded sequence). For example, a nuclease having double-stranded cleavage activity can be modified by mutation and / or deletion to function as a nickase and cleave only one strand of a double-stranded sequence.

[0111] As used herein, the term "nuclease" refers to an enzyme that cleaves both strands of a double-stranded nucleic acid sequence or cleaves a single-stranded nucleic acid sequence.

[0112] The terms "nucleic acid" and "polynucleotide" refer to deoxyribonucleotide or ribonucleotide polymers, which are linear or circular structures, and are in single-stranded or double-stranded form. For the purposes of this disclosure, these terms should not be interpreted as limitations on polymer length. The term may include known analogs of natural nucleotides, as well as nucleotides modified in base, sugar and / or phosphate moieties (such as thiophosphate backbones). In general, analogs of a particular nucleotide have the same base pairing specificity, i.e., analogs of A will pair with T bases.

[0113] The term "nucleotide" refers to a deoxyribonucleotide or a ribonucleotide. Nucleotides may be standard nucleotides (i.e., adenosine, guanosine, cytidine, thymidine, and uridine), nucleotide isomers, or nucleotide analogs. Nucleotide analogs refer to nucleotides having modified purine or pyrimidine bases or modified ribose moieties. Nucleotide analogs may be naturally occurring nucleotides (e.g., inosine, pseudouridine, etc.) or non-naturally occurring nucleotides. Non-limiting examples of modifications on the sugar or base portion of a nucleotide include the addition (or removal) of acetyl, amino, carboxyl, carboxymethyl, hydroxyl, methyl, phosphoryl, and thiol groups, as well as replacement of carbon and nitrogen atoms in the base with other atoms (e.g., 7-deazapurine). Nucleotide analogs also include dideoxynucleotides, 2'-O-methyl nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholinos.

[0114] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues.

[0115] The terms "target sequence" and "target site" are used interchangeably to refer to a specific sequence in a target nucleic acid (e.g., chromosomal DNA or cellular RNA) to which a CRISPR system is targeted, as well as the site at which the CRISPR system modifies the nucleic acid or a protein associated with the nucleic acid.

[0116] Techniques for determining nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA of a gene and / or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this manner. In general, identity refers to the exact correspondence of nucleotides to nucleotides or amino acids to amino acids of two polynucleotide or polypeptide sequences, respectively. Two or more sequences (polynucleotides or amino acids) can be compared by determining their percent identity. Whether nucleic acid or amino acid sequence, the percent identity of two sequences is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence and multiplied by 100. Approximate alignments of nucleic acid sequences are provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981). This algorithm can be applied to amino acid sequences using a scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 suppl. 3: 353-358, National Biomedical Research Foundation, Washington, DC, USA, and standardized by Gribskov, Nucl. Acids Res., 14 (6): 6745-6763 (1986). An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the "BestFit" utility application. Other programs suitable for calculating percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST using default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; chain = two; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sorting mode = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + Swiss protein + Spupdate + PIR. The details of these programs can be found on the GenBank website.

[0117] As various changes could be made in the above-described cells and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and the examples given below shall be interpreted as illustrative and not in a limiting sense. Example

[0118] The following examples illustrate certain aspects of the disclosure.

[0119] Example 1. Construction of self-replicating Cas9 RNA vector A synthetic, polycistronic, self-replicating RNA encoding Cas9 was generated based on a modified Venezuelan equine encephalitis (VEE) virus in which the structural genes had been removed (i.e., Simplicon TM Cloning vector E3L; MilliporeSigma). pVAV-Cas9-2A-GFP plasmid (encoding wild-type SpCas9), CMV-eSpCas9-2A-GFP plasmid and SpCas9-Blasticidin Lenti plasmid were used as templates to PCR amplify cDNAs of Cas9-T2A-TagGFP2, eSpCas9-T2A-GFP2 and eSpCas9, respectively. eSpCas9 is a modified version of wild-type SpCas9 that is modified to enhance on-target fidelity without losing cleavage efficiency (Slaymaker et al., Science. 2016, 351(6268): 84-8). Each cDNA was then cloned into Simplicon TM The NdeI / NotI sites of the vector were respectively named T7-VEE-Cas9-TagGFP2 (SEQ ID NO: 31), T7-VEE-eSpCas9-TagGFP2 (SEQ ID NO: 32) and T7-VEE-eSpCas9 (SEQ ID NO: 33). The schematic diagram of each VEE-Cas9 RNA is shown in Figure 1A Displayed in.

[0120] For RNA synthesis, the VEE-Cas9 plasmid and the B18R-E3L plasmid were each linearized by digestion with MluI and BamHI, respectively, to generate templates for RNA synthesis. RNA synthesis and 5′-capping were performed at 37°C using the RiboMAX Large-Scale RNA Production System-T7 (Promega) kit. Reagent AG (Trilink) for 2 hours. For B18R-E3L RNA synthesis, an additional ~150 bases of poly(A) tail were added by poly(A) polymerase (CELLSCRIPT) at 37°C for 30 minutes. After purification and precipitation with 2.5M ammonium acetate, RNA was resuspended in RNA storage solution (Ambion) at a concentration of 1 μg / μl and stored at -80°C. VEE-Cas9 RNA was analyzed by agarose gel electrophoresis ( Figure 1B). The VEE-Cas9 RNA band showed the strongest intensity at the predicted size (~15 kb) with the fewest degradation bands.

[0121] Example 2. Transfection of self-replicating Cas9 RNA These VEE-Cas9 RNAs were co-transfected into cells along with B18R-E3L RNA, which inhibits the interferon (IFN) response induced by RNA transfection and replication.

[0122] Human foreskin fibroblasts (HFF) and HEK293T cells were cultured in DMEM containing 10% FBS, MEM non-essential amino acids (NEAA), pyruvate, penicillin and streptomycin. Cells were passaged one day before transfection, so their confluence was 30-60% on the day of transfection. Each VEE-Cas9 RNA was co-transfected with B18R-E3L RNA at a 1:1 ratio (1 μg each for 1 well of a 6-well plate) using Lipofectamine MessengerMax transfection reagent (Thermofisher) into the cells.

[0123] like Figure 1C As shown in , the expression of TagGFP2 (GFP) was observed in cells transfected with Cas9-TagGFP2 and eSpCas9-TagGFP2, and the expression of each Cas9 protein was confirmed by Western blotting ( Figure 1D ). These data show that self-replicating RNA technology allows the expression of Cas9 in human cells.

[0124] Example 3. Non-integrating Cas9 genome editing Next, VEE-Cas9 RNA was used in combination with chemically synthesized gRNA to examine editing of the targeted genome. Commercially available 2-component synthetic RNA (crRNA:tracrRNA complex) and 1-component synthetic single gRNA (sgRNA) targeting K-Ras and EMX-1 were purchased from Thermofisher Scientific. The crRNA sequences including the PAM sequences (underlined) of the K-Ras and EMX-1 targets were 5′TAGTTGGAGCTGGTGGCGT AGG (SEQ ID NO: 34) and 5′GAGTCCGAGCAGAAGAAGAA GGG (SEQ ID NO: 35).

[0125] First, HFF cells were co-transfected with VEE-Cas9-TagGFP2, B18R-E3L RNA (as described above) together with 1-component gRNA or 2-component gRNA. Cells were harvested 2-6 days after transfection, and indels were detected using the Guide-iT Mutation Detection Kit (Clontech). The efficiency of DNA cleavage was determined using the ChemiDoc TM The target regions of K-Ras (340 bp) and EMX-1 (410 bp) genes were amplified by PCR using the following primer sets: 5′-GATACACGTCTGCAGTCAACTG (SEQ ID NO: 36) / 5′-GCATATTACTGGTGCAGGACC (SEQ ID NO: 37) and 5′-GCCTGAGTGTTGAGGCCCCA (SEQ ID NO: 38) / 5′-GTCCCTCTGTCAATGGCGGC (SEQ ID NO: 39), respectively.

[0126] like Figure 2A As shown in , GFP expression was observed using either the 1-component or 2-component gRNA, although GFP expression was reduced in cells transfected with the 2-component gRNA. On day 3 (two days post-transfection), more than 15% lysis was observed in cells transfected with the 1-component sgRNA, whereas no lysis was detected in cells transfected with the 2-component gRNA ( Figure 2B Puromycin selection was performed to remove Cas9-negative cells, and lysis efficiency increased in cells transfected with the 1-component sgRNA, but no lysis was detected in cells transfected with the 2-component gRNA ( Figure 2B , day 6).

[0127] To increase editing efficiency, cells were transfected sequentially with VEE-Cas9 RNA and gRNA. For this experiment, VEE-Cas9 and B18R-E3L RNA were co-transfected on day 1, and gRNA was then transfected on day 2 using Lipofectamine RNAiMax transfection reagent (Thermofisher). Figure 2B As shown in , higher efficiency of genome editing was obtained using sequential transfection of either 1-component or 2-component gRNAs on day 4 (two days after gRNA transfection), and a further increase in efficiency was obtained after puromycin selection (day 6). Two different amounts of gRNA were tested (25 nM and 50 nM), but no significant differences were observed ( Figure 2B ).

[0128] Next, VEE-Cas9 genome editing was tested in human iPSC cell lines by a sequential transfection approach using a 1-component sgRNA. Epithelial-1 iPSCs or PBMC-iPSCs (CD34+ cord blood iPSCs) were cultured in laminin-coated wells in the presence of mTeSRTM-1 medium (Stemcell Technologies). iPSC cells were transfected as described above. GFP expression was obtained in both human iPSC cell lines ( Figure 2C ), and genome editing efficiencies ranged from 16-32% in both cell lines ( Figure 2D ).

[0129] Example 4. Comparison of self-replicating Cas9 RNA with other expression systems The genome editing efficiency between VEE-Cas9 RNA and lentiviral Cas9 was compared. The lentiviral vector has a blasticidin selection marker. Therefore, lentiviral Cas9 (LV-Cas9) (at MOI=3) or VEE-Cas9-TagGFP2 (S-Cas9) was introduced into HFFs, and the cells were then selected for one week with blasticidin (4μg / mL) or puromycin (0.8μg / mL), respectively. After selection, both cells were passaged for 1-component sgRNA transfection the next day. Similar genome editing efficiencies were observed for both Cas9 expression methods for K-Ras targets (35% and 36%, respectively) and EMX-1 targets (47% and 40%, respectively). Figure 3A ).

[0130] Next, VEE-Cas9 RNA was compared to plasmid Cas9 encoding Cas9-TagGFP2. HEK293T cells were co-transfected with plasmid Cas9 and plasmid encoding K-Ras-gRNA (full DNA component), or HEK293T cells were co-transfected with VEE-Cas9-TagGFP2, B18R-E3L RNA, and 1-component K-Ras sgRNA (full RNA component). Figure 3B As shown in , transfection of either the full DNA component or the full RNA component resulted in similar percentages of GFP-positive cells (65-74%). The genome editing efficiency of both methods was also similar (20-29%) ( Figure 3C ). These data show that genome editing with VEE-Cas9 RNA is comparable to that provided by lentiviral and plasmid Cas9 expression tools.

[0131] Example 5. Long-term expression of self-replicating Cas9 RNA VEE-Cas9-TagGFP2 RNA and B18R-E3L RNA were co-transfected into HFF or HEK293T cells, and the cells were maintained under puromycin selection in the presence of B18R protein. After one month and four cell passages, HFF cells were co-transfected with 1-component K-Ras sgRNA. Figure 4A As shown in , about 40% of HFF cells were GFP positive and a genome editing efficiency of about 10% was obtained. After one month and eight cell passages, HEK293T was co-transfected with 1-component EMX-1 sgRNA. It was found that about 81% of HEK293T cells were GFP positive and the genome editing efficiency was about 26% ( Figure 4B ). These data indicate that VEE-Cas9 RNA allows the generation of Cas9-expressing cell lines without manipulation of the host cell genome and that the cell lines can be used for targeted genome editing.

[0132] The genome editing efficiencies of the three VEE-Cas9 RNA vectors prepared in Example 1 were compared. Figure 4C and 4D As shown in , in HFF and HEK293T cells, eSpCas9, eSpCas9-TagGFP2 and Cas9-TagGFP2 achieved similar genome editing efficiencies (e.g., 5-12% in HFF and 20-26% in HEK293T cells).

[0133] Example 6. Genome editing using self-replicating D10A-Cas9 RNA The D10A mutation on the Cas9 protein results in single-strand cleavage rather than double-strand cleavage. Therefore, it is believed that it performs genome editing with less off-target cleavage and is useful for precise genome editing. To examine the feasibility of D10A-Cas9 using self-replicating RNA, we generated a new construct of Cas9 with a D10A point mutation. Figure 5A The expression of D10A-Cas9-TagGFP2 and D10A-Cas9-TagRFP in 293T cells at days 1 and 3 was shown, and a Cas9-expressing cell line (293T) was generated ( Figure 5B ). To test the usability of D10A-Cas9, two sgRNAs (sgRNA-1 and 9) at the EMX1 locus were transfected to generate double-strand breaks. Figure 5C As shown in , genome editing was observed when two sgRNAs were transfected into Cas9-D10A mutant expressing cells, whereas no genome editing was observed when one sgRNA was transfected. The same results were observed in the D10A-Cas9 cell line ( Figure 5D). These data show that self-replicating D10A-Cas9 can be used for precise genome editing.

[0134] Example 7. Insertion of DNA oligonucleotides and GFP DNA fragments using self-replicating Cas9 RNA Next, we tested DNA insertion at the Cas9 cleavage site. First, we inserted a DNA oligonucleotide with a BamHI restriction enzyme site (BamHI oligonucleotide) into the Rab11 locus. Self-replicating Cas9 or D10A-Cas9 was transfected on day 1, and then the BamHI oligonucleotide was co-transfected with the sgRNA. Cells were collected for analysis three days after sgRNA transfection, or clones were isolated after puromycin selection. Fig. 6A As shown in , by digesting the PCR product with BamHI at the Rab11A locus, BamHI oligonucleotide insertion was observed in both Cas9 and D10A-Cas9 cleaved samples. Cell clones were also isolated and examined for BamHI oligonucleotide insertion. Figure 6B As shown in , BamHI oligonucleotides were detected in 4 of 6 iPSC clones and in 8 of 11 U2OS cell clones. Secondly, we inserted the TagGFP2 fragment at the GAPDH locus. HEK293 cells were transfected with self-replicating Cas9-TagRFP on day 1, and then the PCR-amplified GFP fragment and sgRNA were co-transfected on day 2. GFP-positive cells were observed 3 days after sgRNA transfection ( Figure 6C ). These data suggest that self-replicating Cas9 can be used for DNA insertion for genome editing.

Claims

1. A method for targeted genome editing, the method comprising introducing into a eukaryotic cell a self-replicating RNA vector and at least one guide RNA engineered to complex with a CRISPR protein encoded by the self-replicating RNA vector, the self-replicating RNA vector comprising sequences encoding multiple non-structural replication complex proteins from an alphavirus and sequences encoding CRISPR proteins, wherein the vector is based on a modified Venezuelan equine encephalitis (VEE) virus and comprises from 5' to 3': a 5' cap, a 5' UTR, sequences encoding four non-structural replication complex proteins from the VEE virus, a promoter, sequences encoding the CRISPR proteins, an optional IRES, an optional sequence encoding a selectable marker, an alphavirus 3' UTR, and a poly A tail; and sequentially introducing into the eukaryotic cell a plasmid encoding an interferon response inhibitor.

2. The method of claim 1, wherein the CRISPR protein is a type II Cas9 protein, a type V Cas12 protein, a type VI Cas13 protein, a CasX protein, or a CasY protein, and wherein the CRISPR protein is Streptococcus pyogenes Cas9, Francisella novicida Cas9, Staphylococcus aureus Cas9, Streptococcus thermophilus Cas9, Streptococcus pasteurianus Cas9, Campylobacter jejuni Cas9, Neisseria meningitidis Cas9, Neisseria griseus Cas9, Francisella novicida Cas12, Acidaminococcus species Cas12, Lachnospiraceae bacteria ND2006 Cas12, Leptotrichia wadeii Cas13a, Leptotrichia saboriae Cas13a, Prevotella species P5-125 Cas13, or Ruminococcus xanthophylla Cas13d.

3. The method of claim 2, wherein the CRISPR protein is Streptococcus pyogenes Cas9 or Staphylococcus aureus Cas9.

4. The method of any one of claims 1 to 3, wherein the sequence encoding the CRISPR protein comprises at least one nucleotide insertion, deletion and / or substitution, such that the CRISPR protein has altered catalytic activity, improved target site specificity and / or reduced off-target effects.

5. The method of any one of claims 1 to 4, wherein the CRISPR protein is a nuclease, a nickase, or has no cleavage activity.

6. The method of any one of claims 1 to 5, wherein the CRISPR protein is linked to at least one nuclear localization signal.

7. The method of any one of claims 1 to 6, wherein the CRISPR protein is linked to at least one fluorescent protein, at least one chromatin regulatory motif, at least one functional domain, or a combination thereof.

8. The method of claim 7, wherein the at least one functional domain is an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain.

9. The method of any one of claims 1 to 8, wherein the sequence encoding the CRISPR protein is codon-optimized for expression in human cells.

10. A eukaryotic cell or cell line comprising a self-replicating RNA vector and produced by the method of any one of claims 1 to 9.

11. The eukaryotic cell or cell line of claim 10, further comprising at least one guide RNA engineered to complex with the CRISPR protein encoded by the self-replicating RNA vector.

12. The method of any one of claims 1 to 9, wherein the self-replicating RNA vector is encoded by a plasmid vector further comprising a T7 or SP6 promoter for in vitro transcription.

13. The method of any one of claims 1 to 9, further comprising introducing at least one donor polynucleotide into the cell.

14. The method of claim 13, wherein the eukaryotic cell is a human cell.

15. The method of any one of claims 1 to 9, wherein the interferon response inhibitor is E3L.

16. The method of claim 15, wherein the plasmid encoding the interferon response inhibitor encodes B18R-E3L RNA.

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