Split gene editing system and uses thereof

Abstract

The present disclosure relates to split gene editing systems and uses thereof, including polypeptides, vectors, cells, compositions, kits, and methods thereof.

Description

Technical Field

[0001] This disclosure relates to fission gene editing systems and their uses, including peptides, vectors, cells, compositions, kits, and methods thereof.

[0002] sequence list

[0003] This application contains a sequence list XML file named “Seq list.xml”, which is 361 KB in size and was created on November 17, 2023. The information contained in the sequence list is incorporated herein by reference. Background Technology

[0004] CRISPR-Cas9 is a novel genome editing tool that, guided by guide RNA (gRNA), cuts at target sites in the genome, causing double-strand breaks (DSBs). DSBs induce cells to initiate non-homologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms. Combined with a donor template containing homologous arms, HDR can be used to achieve precise insertion or deletion of DNA sequences, but its efficiency is relatively low. Base editors (BEs) developed based on Cas9 nickases and deaminases can achieve four types of base transitions, including C→T, G→A, A→G, and T→C. However, their editing window and capabilities are limited, and their off-target rate is high. Moreover, for other mutation types that account for approximately 70% of known pathogenic mutations, effective genome editing tools are still lacking.

[0005] David Liu and his team have developed a novel gene-editing tool called PrimeEditor (PE). PE enables precise insertions, deletions, and all 12 types of base conversions without requiring DSB or donor template DNA. In the initial version of PE (PE1), Cas9n-H840A, capable of single-strand cutting, was used, with its C-terminus fused to Moloney murine leukemia virus reverse transcriptase (M-MLV), along with a lead editing guide RNA (pegRNA) with an extension sequence at its 3' end containing a reverse transcriptase template (RT template) and a primer binding site (PBS) targeting the editing site. PE1 enables single-base substitutions and short sequence insertions and deletions, but its editing efficiency is relatively low. Typically, only <5% of the target alleles are converted to the desired sequence. To improve the editing efficiency of PE1, five mutations were introduced into M-MLV to enhance its thermostability, reaction persistence, and affinity for DNA and RNA substrates, and to inhibit the RNase H activity of M-MLV. This is the second-generation lead editing system (PE2). Compared to PE1, PE2 improves editing efficiency in human cells by 1.6 to 5.1 times. Both PE1 and PE2 rely on endogenous cellular repair processes to replace the unedited strand with the edited strand in the template. By introducing a single guide RNA (sgRNA) targeting the edited strand into PE2, a nick is created on the unedited strand, promoting this replacement process and thus improving editing efficiency; this system is called PE3. By optimizing the binding site of the sgRNA, higher editing efficiency can be achieved while maintaining a relatively low rate of insertions and deletions (indels); this system is called PE3b. Furthermore, it has been reported that 3′ end degradation of pegRNA affects editing efficiency. To address this, researchers have added different RNA protection structures to the 3′ end of pegRNA, developing engineered pegRNAs such as epigRNA and xr-pegRNA. Subsequent studies have found that the cell's DNA mismatch repair (MMR) mechanism inhibits PE gene editing and leads to additional indels. Based on these findings, PE systems expressing the dominant-negative mismatch repair protein MLH1 (MLH1dn) were developed, namely PE4 (PE2+MLH1dn) and PE5 (PE3+MLH1dn). By utilizing MLH1dn to inhibit MMR, gene editing efficiency can be improved.Building upon the different versions of the aforementioned PE system, a further optimized PEmax system can be obtained by employing a codon-optimized PE2 protein, introducing multiple mutations in SpCas9, and adjusting the length and position of the peptide linker between Cas9n-H840A and reverse transcriptase. Furthermore, combining different versions of the PE system with serine recombinases yields the Twin PE and PASTE systems, enabling precise insertion and deletion of large sequences in the genome, as well as chromosomal inversions and translocations.

[0006] Given its diverse editing types, high specificity, and low off-target rate, gene editing (PE) holds immense potential in gene editing therapy. Efficient and precise delivery of PE to its target site in vivo is crucial for its effectiveness. Viral vectors are widely used delivery tools in gene therapy. Target genes can be introduced into recipient cells via viruses for disease treatment. This approach has been applied in cell, animal models, and clinical settings. Commonly used viral vectors include, but are not limited to, adenoviruses, lentiviruses, and adeno-associated viruses (AAVs). AAVs exhibit relatively low immunogenicity and can be injected directly into the target site. Therefore, AAVs are a safe and effective delivery tool and have been widely used in clinical gene therapy.

[0007] However, the size of a PE (polyurea) is approximately 6.5 kb, far exceeding the loading capacity of an AAV (4.8 kb). This obstacle in PE delivery limits its further clinical application. Therefore, there is still a need in the field for highly efficient PE delivery methods. Summary of the Invention

[0008] This disclosure provides compositions and methods for delivering gene-editing systems (e.g., leader editors (PE) and base editors (BE)) into cells in a split form. In some embodiments, the gene-editing system is split into two parts and packaged separately in two vectors, such as two AAV vectors. After transfection of cells with the two vectors, the two parts can recombine via RNA trans-splicing or peptide-mediated trans-splicing to form a complete mRNA or protein, which then performs gene-editing function. In some embodiments, the gene-editing system is split into more than two parts and packaged separately in more than two vectors.

[0009] This disclosure provides multiple cleavage sites in the Cas9 protein or its variants. In some embodiments, the cleavage sites identified in this disclosure are located between the second and third amino acids of any one of SEQ ID NO: 1-10 and 12 of the Cas9 protein or its variants. In some embodiments, the cleavage site is located between the first and second amino acids of SEQ ID NO: 11, wherein the first two N-terminal amino acids of the C-terminal portion of the cleaved Cas9 protein or its variants are replaced by cysteine ​​and phenylalanine, or cysteine ​​and tryptophan. In some embodiments, the cleavage sites are 212-213, 269-270, 309-310, 367-368, 459-460, 554-555, 940-941, 959-960, 1023-1026, 1039-1040, 1087-1088, and 1115-1116.

[0010] The cleavage sites disclosed herein can also be used in combination. For example, in some embodiments, the Cas9 protein or variants thereof cleave into three parts at two cleavage sites disclosed herein. This disclosure also discloses a variety of novel combinations of cleavage sites with integrins.

[0011] In one aspect, this disclosure provides a polynucleotide comprising a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises an N-terminal portion of a full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof.

[0012] In one aspect, this disclosure provides a polynucleotide comprising a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises a C-terminal portion of a full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of the full-length Cas9 protein or variant thereof in SEQ ID NO: 1-10.

[0013] In one aspect, this disclosure provides a polynucleotide comprising a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises a C-terminal portion of a full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or variant thereof.

[0014] In one aspect, this disclosure provides a polynucleotide comprising a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant thereof comprises a portion of a full-length Cas9 protein or variant thereof between a first cleavage site and a second cleavage site, wherein the first cleavage site is upstream of the second cleavage site, wherein at least one of the two cleavage sites is selected from the site between the second and third amino acids of any one of SEQ ID NO: 1-10 and 12, and the site between the first and second amino acids of SEQ ID NO: 11; wherein, if the first cleavage site is located between the first and second amino acids of SEQ ID NO: 11, the first two N-terminal amino acids of the truncated Cas9 protein or variant thereof are replaced by cysteine ​​and phenylalanine, or cysteine ​​and tryptophan. In some embodiments, both splitting sites are selected from the site between the second and third amino acids of any one of SEQ ID NO: 1-10 and 12, and the site between the first and second amino acids of SEQ ID NO: 11; wherein, if the first splitting site is located between the first and second amino acids of SEQ ID NO: 11, the first two amino acids at the N-terminus of the truncated Cas9 protein or its variants are replaced by cysteine ​​and phenylalanine, or cysteine ​​and tryptophan.

[0015] In some embodiments, the polynucleotide described herein further comprises a sequence encoding an N-terminal portion of an inteptide, said inteptide being linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof.

[0016] In some embodiments, the integrin in the polynucleotide described herein is selected from Mxe-GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the integrin in the polynucleotide described herein is Rma.

[0017] In some implementations, the polynucleotides described herein further comprise guide RNA or a sequence encoding it.

[0018] In some embodiments, the polynucleotide described herein further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter.

[0019] In some embodiments, the polynucleotide described herein further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase. In some embodiments, the reverse transcriptase is M-MLV reverse transcriptase or a functional variant thereof. In some embodiments, the gene-editing enzyme is a deaminase. In some embodiments, the protein having enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methyltransferase or a functional fragment thereof.

[0020] In some embodiments, the polynucleotide further comprises a sequence encoding a 2A peptide (P2A).

[0021] In some embodiments, the polynucleotide further comprises a sequence encoding a nuclear localization signal (NLS).

[0022] In some embodiments, the Cas9 protein or a variant thereof is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein or a variant thereof is Cas9n-H840A (SEQ ID NO: 23), eSp-Cas9 (SEQ ID NO: 17), HiFi-Cas9 (SEQ ID NO: 18), Lz3-Cas9 (SEQ ID NO: 19), SpCas9-NG (SEQ ID NO: 20), Sniper-Cas9 (SEQ ID NO: 21), SuperFiCas9 (SEQ ID NO: 22), or dCas9 (SEQ ID NO: 24).

[0023] In one aspect, this disclosure provides a vector comprising the polynucleotides described herein.

[0024] In some embodiments, the vector described herein is a viral vector. In some embodiments, the vector described herein is a plasmid. Viral vectors may include, but are not limited to, adenovirus vectors, lentiviral vectors, retroviral vectors, and adeno-associated virus vectors. Suitable vectors, promoters, and enhancer elements are known in the art; many vectors, promoters, and enhancer elements for generating recombinant constructs are commercially available.

[0025] In some embodiments, the vector described herein is selected from adenovirus, lentivirus, and adeno-associated virus (AAV). In some embodiments, the vector described herein is AAV.

[0026] In one aspect, this disclosure provides a composition comprising the polynucleotides described herein.

[0027] In some embodiments, the compositions described herein comprise a first polynucleotide and a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or a variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or a variant thereof; and wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or a variant thereof.

[0028] In some embodiments of the compositions described herein, the first polynucleotide further comprises a sequence encoding an N-terminal portion of an inteptide, said inteptide being linked to a C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the first polynucleotide further comprises one or more guide RNAs or sequences encoding them. In some embodiments, at least one of said guide RNAs comprises a reverse transcription template. In some embodiments, the first polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the first polynucleotide further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, said enzymatically active protein is a reverse transcriptase or a functional fragment thereof. In some embodiments, said reverse transcriptase is Moroni mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0029] In some embodiments of the compositions described herein, the second polynucleotide further comprises a sequence encoding a C-terminal portion of an inteptide, said inteptide C-terminus being linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the second polynucleotide further comprises one or more guide RNAs or sequences encoding them. In some embodiments, at least one of said guide RNAs comprises a reverse transcription template. In some embodiments, the second polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the second polynucleotide further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, said enzymatically active protein is a reverse transcriptase or a functional fragment thereof. In some embodiments, said reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0030] In some embodiments, the compositions described herein comprise a first polynucleotide and a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and, wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or variant thereof; and The first polynucleotide further comprises a sequence encoding the N-terminal portion of an inteptide, said N-terminal portion being linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The second polynucleotide further comprises a sequence encoding the C-terminal portion of an inteptide, said inteptide C-terminus being linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The N-terminal portion and the C-terminal portion of the inteptide form a full-length inteptide.

[0031] In some embodiments, the inteptide is selected from Mxe-GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma.

[0032] In some embodiments of the compositions described herein, at least one of the polynucleotides comprises guide RNA or a sequence encoding it.

[0033] In some embodiments of the compositions described herein, at least one of the polynucleotides comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein having enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methyltransferase or a functional fragment thereof.

[0034] In one aspect, this disclosure provides a composition comprising the carrier described herein.

[0035] In some embodiments, the compositions described herein comprise a first carrier and a second carrier, the first carrier comprising a first polynucleotide and the second carrier comprising a second polynucleotide, wherein the first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the N-terminal portion of a full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof being divided into two parts at a cleavage site, wherein the cleavage site is located between the second and third amino acids of any sequence of SEQ ID NO: 1-10 of the full-length Cas9 protein or a variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or a variant thereof; and

[0036] The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or a variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or a variant thereof; and wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or a variant thereof.

[0037] In some embodiments of the compositions described herein, the first polynucleotide further comprises a sequence encoding an N-terminal portion of an inteptide, said inteptide being linked to a C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the first polynucleotide further comprises one or more guide RNAs or sequences encoding them. In some embodiments, at least one of said guide RNAs comprises a reverse transcription template. In some embodiments, the first polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the first polynucleotide further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, said enzymatically active protein is a reverse transcriptase or a functional fragment thereof. In some embodiments, said reverse transcriptase is Moroni mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0038] In some embodiments of the compositions described herein, the second polynucleotide further comprises a sequence encoding a C-terminal portion of an inteptide, said inteptide C-terminus being linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the second polynucleotide further comprises one or more guide RNAs or sequences encoding them. In some embodiments, at least one of said guide RNAs comprises a reverse transcription template. In some embodiments, the second polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the second polynucleotide further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, said enzymatically active protein is a reverse transcriptase or a functional fragment thereof. In some embodiments, said reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0039] In some embodiments, the compositions described herein comprise a first carrier and a second carrier, the first carrier comprising a first polynucleotide and the second carrier comprising a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and, wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or variant thereof; and The first polynucleotide further comprises a sequence encoding the N-terminal portion of an inteptide, said N-terminal portion being linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The second polynucleotide further comprises a sequence encoding the C-terminal portion of an inteptide, said inteptide C-terminus being linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The N-terminal portion and the C-terminal portion of the inteptide form a full-length inteptide.

[0040] In some embodiments, the inteptide is selected from Mxe-GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma.

[0041] In some embodiments of the compositions described herein, at least one of the polynucleotides comprises guide RNA or a sequence encoding it.

[0042] In some embodiments of the compositions described herein, at least one of the polynucleotides comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein having enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methyltransferase or a functional fragment thereof.

[0043] In some embodiments of the compositions described herein, at least one of the carriers is an AAV carrier. In some embodiments of the compositions described herein, both carriers are AAV carriers.

[0044] In some embodiments of the compositions described herein, the Cas9 protein or a variant thereof is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein or a variant thereof is Cas9n-H840A (SEQ ID NO: 23), eSp-Cas9 (SEQ ID NO: 17), HiFi-Cas9 (SEQ ID NO: 18), Lz3-Cas9 (SEQ ID NO: 19), SpCas9-NG (SEQ ID NO: 20), Sniper-Cas9 (SEQ ID NO: 21), SuperFiCas9 (SEQ ID NO: 22), or dCas9 (SEQ ID NO: 24).

[0045] In one aspect, this disclosure provides a cell that comprises the polynucleotides described herein.

[0046] In some implementations, the cells described herein comprise a first polynucleotide and a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or a variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or a variant thereof; and wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or a variant thereof.

[0047] In some embodiments of the cell described herein, the first polynucleotide further comprises a sequence encoding an N-terminal portion of an inteptide, said N-terminal portion being linked to a C-terminus encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the first polynucleotide further comprises one or more guide RNAs or sequences encoding them. In some embodiments, at least one of the guide RNAs comprises a reverse transcription template. In some embodiments, the first polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the first polynucleotide further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0048] In some embodiments of the cells described herein, the second polynucleotide further comprises a sequence encoding a C-terminal portion of an inteptide, said inteptide C-terminus being linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the second polynucleotide further comprises one or more guide RNAs or sequences encoding them. In some embodiments, at least one of the guide RNAs comprises a reverse transcription template. In some embodiments, the second polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the second polynucleotide further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0049] In some implementations, the cells described herein comprise a first polynucleotide and a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and, wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or variant thereof; and The first polynucleotide further comprises a sequence encoding the N-terminal portion of an inteptide, said N-terminal portion being linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The second polynucleotide further comprises a sequence encoding the C-terminal portion of an inteptide, said inteptide C-terminus being linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The N-terminal portion and the C-terminal portion of the inteptide form a full-length inteptide.

[0050] In some embodiments, the inteptide is selected from Mxe-GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma.

[0051] In some embodiments, at least one of the polynucleotides comprises a guide RNA or a sequence encoding it. In some embodiments, the guide RNA is a pegRNA containing a reverse transcription template. In some embodiments, at least one of the polynucleotides comprises a pegRNA and an sgRNA, the sgRNA targeting the edited strand of the gene editing system and introducing a nick on the unedited strand, for example, in the PE3 system. In some embodiments, the polynucleotide containing the guide RNA further comprises a U6 promoter. In some embodiments, the first polynucleotide comprises a pegRNA, the second polynucleotide comprises an sgRNA, and each polynucleotide further comprises a U6 promoter.

[0052] In some embodiments of the cells described herein, at least one of the polynucleotides comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the enzymatically active protein is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the enzymatically active protein is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methyltransferase or a functional fragment thereof.

[0053] In one aspect, this disclosure provides a cell comprising the vector described herein.

[0054] In some embodiments, the cell described herein comprises a first carrier and a second carrier, the first carrier comprising a first polynucleotide and the second carrier comprising a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or a variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or a variant thereof; and wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or a variant thereof.

[0055] In some embodiments of the cell described herein, the first polynucleotide further comprises a sequence encoding an N-terminal portion of an inteptide, said N-terminal portion being linked to a C-terminus encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the first polynucleotide further comprises one or more guide RNAs or sequences encoding them. In some embodiments, at least one of the guide RNAs comprises a reverse transcription template. In some embodiments, the first polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the first polynucleotide further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0056] In some embodiments of the cells described herein, the second polynucleotide further comprises a sequence encoding a C-terminal portion of an inteptide, said inteptide C-terminus being linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the second polynucleotide further comprises one or more guide RNAs or sequences encoding them. In some embodiments, at least one of the guide RNAs comprises a reverse transcription template. In some embodiments, the second polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the second polynucleotide further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0057] In some embodiments, the cell described herein comprises a first carrier and a second carrier, the first carrier comprising a first polynucleotide and the second carrier comprising a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and, wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or variant thereof; and The first polynucleotide further comprises a sequence encoding the N-terminal portion of an inteptide, said N-terminal portion being linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The second polynucleotide further comprises a sequence encoding the C-terminal portion of an inteptide, said inteptide C-terminus being linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The N-terminal portion and the C-terminal portion of the inteptide form a full-length inteptide.

[0058] In some embodiments, the inteptide is selected from Mxe-GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma.

[0059] In some embodiments, at least one of the polynucleotides comprises a guide RNA or a sequence encoding it. In some embodiments, the guide RNA is a pegRNA containing a reverse transcription template. In some embodiments, at least one of the polynucleotides comprises a pegRNA and an sgRNA, the sgRNA targeting the edited strand of the gene editing system and introducing a nick on the unedited strand, for example, in the PE3 system. In some embodiments, the polynucleotide containing the guide RNA further comprises a U6 promoter. In some embodiments, the first polynucleotide comprises a pegRNA, the second polynucleotide comprises an sgRNA, and each polynucleotide further comprises a U6 promoter.

[0060] In some embodiments, at least one of the polynucleotides comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein having enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methyltransferase or a functional fragment thereof.

[0061] In some embodiments of the cells described herein, at least one vector is an AAV vector. In some embodiments of the cells described herein, both vectors are AAV vectors.

[0062] In some embodiments of the cells described herein, the Cas9 protein or a variant thereof is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein or a variant thereof is Cas9n-H840A (SEQ ID NO: 23), eSp-Cas9 (SEQ ID NO: 17), HiFi-Cas9 (SEQ ID NO: 18), Lz3-Cas9 (SEQ ID NO: 19), SpCas9-NG (SEQ ID NO: 20), Sniper-Cas9 (SEQ ID NO: 21), SuperFiCas9 (SEQ ID NO: 22), or dCas9 (SEQ ID NO: 24).

[0063] In one aspect, this disclosure provides a kit comprising the polynucleotides described herein.

[0064] In some implementations, the kit described herein comprises a first polynucleotide and a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or a variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or a variant thereof; and wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or a variant thereof.

[0065] In some embodiments of the kit described herein, the first polynucleotide further comprises a sequence encoding an N-terminal portion of an inteptide, said inteptide being linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the first polynucleotide further comprises one or more guide RNAs or sequences encoding them. In some embodiments, at least one of said guide RNAs comprises a reverse transcription template. In some embodiments, the first polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the first polynucleotide further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, said enzymatically active protein is a reverse transcriptase or a functional fragment thereof. In some embodiments, said reverse transcriptase is Moroni mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0066] In some embodiments of the kit described herein, the second polynucleotide further comprises a sequence encoding a C-terminal portion of an inteptide, said inteptide C-terminus being linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the second polynucleotide further comprises one or more guide RNAs or sequences encoding them. In some embodiments, at least one of said guide RNAs comprises a reverse transcription template. In some embodiments, the second polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the second polynucleotide further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, said enzymatically active protein is a reverse transcriptase or a functional fragment thereof. In some embodiments, said reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0067] In some implementations, the kit described herein comprises a first polynucleotide and a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and, wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or variant thereof; and The first polynucleotide further comprises a sequence encoding the N-terminal portion of an inteptide, said N-terminal portion being linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The second polynucleotide further comprises a sequence encoding the C-terminal portion of an inteptide, said inteptide C-terminus being linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The N-terminal portion and the C-terminal portion of the inteptide form a full-length inteptide.

[0068] In some embodiments, the inteptide is selected from Mxe-GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma.

[0069] In some embodiments of the kit described herein, at least one of the polynucleotides comprises a guide RNA or a sequence encoding it. In some embodiments, the guide RNA is a pegRNA containing a reverse transcription template. In some embodiments, at least one of the polynucleotides comprises both pegRNA and sgRNA, the sgRNA targeting the edited strand of the gene editing system and introducing a nick on the unedited strand, for example, in the PE3 system. In some embodiments, the polynucleotide containing the guide RNA further comprises a U6 promoter. In some embodiments, the first polynucleotide comprises pegRNA, the second polynucleotide comprises sgRNA, and each polynucleotide further comprises a U6 promoter.

[0070] In some embodiments of the kit described herein, at least one of the polynucleotides comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the enzymatically active protein is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the enzymatically active protein is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methyltransferase or a functional fragment thereof.

[0071] In one aspect, this disclosure provides a kit comprising the vector described herein.

[0072] In some embodiments, the kit described herein comprises a first carrier and a second carrier, the first carrier comprising a first polynucleotide and the second carrier comprising a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or a variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or a variant thereof; and wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or a variant thereof.

[0073] In some embodiments of the kit described herein, the first polynucleotide further comprises a sequence encoding an N-terminal portion of an inteptide, said inteptide being linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the first polynucleotide further comprises one or more guide RNAs or sequences encoding them. In some embodiments, at least one of said guide RNAs comprises a reverse transcription template. In some embodiments, the first polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the first polynucleotide further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, said enzymatically active protein is a reverse transcriptase or a functional fragment thereof. In some embodiments, said reverse transcriptase is Moroni mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0074] In some embodiments of the kit described herein, the second polynucleotide further comprises a sequence encoding a C-terminal portion of an inteptide, said inteptide C-terminus being linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the second polynucleotide further comprises one or more guide RNAs or sequences encoding them. In some embodiments, at least one of said guide RNAs comprises a reverse transcription template. In some embodiments, the second polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the second polynucleotide further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, said enzymatically active protein is a reverse transcriptase or a functional fragment thereof. In some embodiments, said reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0075] In some embodiments, the kit described herein comprises a first carrier and a second carrier, the first carrier comprising a first polynucleotide and the second carrier comprising a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and, wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or variant thereof; and The first polynucleotide further comprises a sequence encoding the N-terminal portion of an inteptide, said N-terminal portion being linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The second polynucleotide further comprises a sequence encoding the C-terminal portion of an inteptide, said inteptide C-terminus being linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The N-terminal portion and the C-terminal portion of the inteptide form a full-length inteptide.

[0076] In some embodiments, the inteptide is selected from Mxe-GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma.

[0077] In some embodiments of the kit described herein, at least one of the polynucleotides comprises a guide RNA or a sequence encoding it. In some embodiments, the guide RNA is a pegRNA containing a reverse transcription template. In some embodiments, at least one of the polynucleotides comprises both pegRNA and sgRNA, the sgRNA targeting the edited strand of the gene editing system and introducing a nick on the unedited strand, for example, in the PE3 system. In some embodiments, the polynucleotide containing the guide RNA further comprises a U6 promoter. In some embodiments, the first polynucleotide comprises pegRNA, the second polynucleotide comprises sgRNA, and each polynucleotide further comprises a U6 promoter.

[0078] In some embodiments of the kit described herein, at least one of the polynucleotides comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the enzymatically active protein is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the enzymatically active protein is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methyltransferase or a functional fragment thereof.

[0079] In some embodiments of the kit described herein, at least one vector is an AAV vector. In some embodiments of the kit described herein, both vectors are AAV vectors.

[0080] In some embodiments of the kit described herein, the Cas9 protein or a variant thereof is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein or a variant thereof is Cas9n-H840A (SEQ ID NO: 23), eSp-Cas9 (SEQ ID NO: 17), HiFi-Cas9 (SEQ ID NO: 18), Lz3-Cas9 (SEQ ID NO: 19), SpCas9-NG (SEQ ID NO: 20), Sniper-Cas9 (SEQ ID NO: 21), SuperFiCas9 (SEQ ID NO: 22), or dCas9 (SEQ ID NO: 24).

[0081] In one aspect, this disclosure provides a method for altering a target nucleic acid in a cell, the method comprising delivering the composition or kit described herein into the cell.

[0082] In one aspect, this disclosure provides a method for altering a target nucleic acid in a cell, the method comprising: Provide the cells with a first polynucleotide encoding the N-terminal portion of the Cas9 protein or a variant thereof. Provide the cells with a second polynucleotide encoding the remaining C-terminal portion of the Cas9 protein or a variant thereof. Guide RNA is provided to the cells, the guide RNA containing a spacer sequence complementary to the target nucleic acid. The cells are capable of RNA trans-splicing and / or peptide-mediated protein trans-splicing, and the cells express the complete Cas9 protein or a variant thereof; The splitting site is located between the second and third amino acids of any of the full-length Cas9 protein or its variants in SEQ ID NO: 1-10; or, the splitting site is located between the first and second amino acids of the full-length Cas9 protein or its variants in SEQ ID NO: 11, and the first two amino acids at the N-terminus of the second polynucleotide are replaced by cysteine ​​and phenylalanine, or cysteine ​​and tryptophan.

[0083] In some embodiments of the methods described herein, the first polynucleotide further comprises a sequence encoding the N-terminal portion of an inteptide, the N-terminal portion of which is linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof, and the second polynucleotide further comprises a sequence encoding the remaining C-terminal portion of the inteptide, the remaining C-terminal portion of which is linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma.

[0084] In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0085] In some embodiments, at least one of the polynucleotides further comprises a guide RNA or a sequence encoding it. In some embodiments of the methods described herein, the guide RNA further comprises a reverse transcription template.

[0086] In some embodiments of the methods described herein, the first polynucleotide is provided by a first carrier and the second polynucleotide is provided by a second carrier.

[0087] In some embodiments of the method described herein, the first vector and the second vector are viral vectors.

[0088] In some embodiments of the methods described herein, the viral vector is selected from adenovirus, lentivirus, and adeno-associated virus (AAV).

[0089] In some embodiments of the methods described herein, the Cas9 protein or a variant thereof is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein or its variants are Cas9n-H840A (SEQ ID NO: 23), eSp-Cas9 (SEQ ID NO: 17), HiFi-Cas9 (SEQ ID NO: 18 or 98), Lz3-Cas9 (SEQ ID NO: 19 or 97), SpCas9-NG (SEQ ID NO: 20), Sniper-Cas9 (SEQ ID NO: 21 or 99), SuperFiCas9 (SEQ ID NO: 22 or 100), HypaCas9 (SEQ ID NO: 93), Cas9HF1 (SEQ ID NO: 94), evoCas9 (SEQ ID NO: 95), SpCas9 (SEQ ID NO: 96), eSpCas9(1.1) (SEQ ID NO: 101), xCas9 (SEQ ID NO: 102), SpGCas9 (SEQ ID NO: 103), SpRYCas9 (SEQ ID NO: 104), Cas9-NG (SEQ ID NO: 20), eSpCas9(SEQ ID NO: 102), eSpCas9(SEQ ID NO: 103), eSpCas9(SEQ ID NO: 104), eSpCas9(SEQ ID NO: 105), eSpCas9(SEQ ID NO: 106), eSpCas9(SEQ ID NO: 107), eSpCas9(SEQ ID NO: 108), eSpCas9(SEQ ID NO: 109 ... Cas9-VRQR (SEQ ID NO: 106), Cas9-VRER (SEQ ID NO: 107), or dCas9 (SEQ ID NO: 24).

[0090] In some embodiments of the methods described herein, the target nucleic acid is altered in vivo.

[0091] In some implementations, the methods described herein are performed in vivo. Attached Figure Description

[0092] Figure 1 is a schematic diagram of Split-PE. Figure 1aThe diagram shows 12 cleavage sites: 212-213, 269-270, 309-310, 367-368, 459-460, 554-555, 940-941, 959-960, 1023-1026, 1039-1040, 1087-1088, and 1115-1116. The numbers 212, 269, 309, 367, 459, 554, 940, 959, 1023, 1039, 1087, and 1115 refer to the cleavage sites 212-213, 269-270, 309-310, 367-368, 459-460, 554-555, 940-941, 959-960, 1023-1026, 1039-1040, 1087-1088, and 1115-1116, respectively. For all figures, NLS represents the nuclear localization sequence; RuvC represents the endonuclease domain; BH represents the bridging helix; REC represents the recognition domain; HNH represents the His-Asn-His endonuclease domain; PI represents the domain that interacts with the adjacent motif (PAM) of the prespacer sequence; and RT represents the reverse transcriptase (e.g., M-MLV, engineered Moroni mouse leukemia virus reverse transcriptase (D200N+L603W+T330P+T306K+W313F)). Figure 1b This illustrates intron-mediated protein trans-splicing to form the complete protein PE. For all figures, intron-C represents the C-terminal portion of the intron, and intron-N represents the remaining N-terminal portion of the same intron.

[0093] Figure 2 is a schematic diagram of pegRNA design. Figure 2a The G→T single base substitution in the third exon region of the endogenous site EMX1, where the PBS region is 15 nt and the RT template is 13 nt; Figure 2b The G→A single base substitution at the endogenous APP site, where the PBS region is 13 nt and the RT template is 18 nt; Figure 2c The C→T single base substitution at the endogenous CETP site, where the PBS region is 13 nt and the RT template is 15 nt; Figure 2d The G→T single base substitution at the endogenous site VEGFA, where the PBS region is 13 nt and the RT template is 14 nt; Figure 2e The G→A single base substitution of the endogenous site ALDOB, where the PBS region is 13 nt and the RT template is 14 nt; Figure 2f The G→A single base substitution at the endogenous site SLC30A8, where the PBS region is 13 nt and the RT template is 15 nt; Figure 2gThe endogenous site MECP2 undergoes a C→T single-base substitution, with the PBS region being 13 nt and the RT template being 17 nt; Figure 2h The GTA tribase insertion at the endogenous site RNF2, where the PBS region is 15 nt and the RT template is 17 nt; Figure 2i The endogenous site HEK3 undergoes CTT tribase insertion, with the PBS region being 13 nt and the RT template being 13 nt; Figure 2j The C-base insertion at the endogenous site ANGPTL3, where the PBS region is 14 nt and the RT template is 11 nt; Figure 2k The A-base insertion at the endogenous site TTR, where the PBS region is 15 nt and the RT template is 11 nt; Figure 2l The endogenous site FANCF shows a G→T single-base substitution, with the PBS region being 14 nt and the RT template being 17 nt. In each subfigure, the RT template is indicated in bold, and the introduced edited sequence is highlighted in black within the RT template and marked in white font.

[0094] Figure 3 This illustrates the single-base substitution efficiency of transient transfection with Split-PE dual plasmids. Plasmids containing Split-PE plasmids with different cleavage sites were transfected into HEK293T cells to achieve G→T single-base substitution, targeting exon 3 of EMX1 in the genome. The horizontal axis represents the cleavage site of the Split-PE plasmids used in the experiment and the inteins used. Specifically, these are Split-PE-Rma-1024 (control), Split-PE-Rma-959, Split-PE-Rma-1039, Split-PE-Rma-1087, Split-PE-Rma-1115, Split-PE-Npu-1023-CF, Split-PE-Npu-1023-CW, Split-PE-Npu-1039-CF, and Split-PE-Npu-1039-CW. In all figures, Rma and Npu represent two different inteins, and the numbers indicate the cleavage site. CF indicates that the first two amino acids at the N-terminus of the C-terminus of the Split-PE were replaced with cysteine ​​and phenylalanine; CW indicates that the first two amino acids at the N-terminus of the C-terminus of the Split-PE were replaced with cysteine ​​and tryptophan. The vertical axis represents the proportion of correct edits or indels obtained through high-throughput sequencing analysis. An indel refers to an unexpected base change that occurs at the EMX1 editing site, such as a base insertion or deletion.

[0095] Figure 4This illustrates the single-base substitution efficiency of Split-PE double AAV transfection. HEK293T cells were transfected with AAV2 packaged with Split-PE, which targets exon 3 of EMX1 in the genome to achieve G→T single-base substitution. The horizontal axis represents the mitotic site and inteins of the Split-PE used in the experiment. Specifically, they are Split-PE-Rma-1024 (control), Split-PE-Rma-959, Split-PE-Rma-1039, Split-PE-Rma-1087, Split-PE-Rma-1115, Split-PE-Npu-1023-CF, Split-PE-Npu-1023-CW, Split-PE-Npu-1039-CF, and Split-PE-Npu-1039-CW. Rma and Npu are two different inteins, and the numbers indicate the mitotic site. CF indicates that the first two amino acids at the N-terminus of the C-terminus of the Split-PE were replaced with cysteine ​​and phenylalanine; CW indicates that the first two amino acids at the N-terminus of the C-terminus of the Split-PE were replaced with cysteine ​​and tryptophan. The vertical axis represents the proportion of correct edits or indels obtained through high-throughput sequencing analysis. Indel refers to... EMX1 Unexpected base substitutions occurring at the editing site, such as base insertions or deletions.

[0096] Figure 5 This is a flowchart illustrating the transient transfection of the Split-PE dual plasmid into HEK293T cells. The two parts of Split-PE, Split-PE-N and Split-PE-C, were constructed into two plasmids containing inverted terminal repeats (ITRs), respectively. HEK293T cells were resuscitated 36 hours prior to transfection. Cells were seeded in 24-well plates 12 hours prior to transfection. When the cell density reached 60%-70%, the plasmids (3 μg) were transfected into the HEK293T cell line using Lipo3000, with Split-PE-N and Split-PE-C plasmids added at a 1:1 molar ratio. This point was recorded as hour 0. Six hours post-transfection, 500 μL of complete culture medium was added to each well. Cells were cultured for another 72 hours post-transfection, at which point cells were harvested and genomic DNA was extracted for library sequencing analysis.

[0097] Figure 6This is a flowchart illustrating the delivery of Split-PE to HEK293T cells via dual AAV delivery. The two fractions of Split-PE, Split-PE-N and Split-PE-C, were packaged into two separate AAV2 viral particles. HEK293T cells were resuscitated 36 hours prior to viral infection. Cells were seeded in 24-well plates 12 hours prior to viral infection. When cell density reached 60%-70%, cells were infected with AAV2 virus at a 1:1 ratio, with a total MOI of 1. 10 6 Record this time as 0 hours. 24 hours post-infection, add 500 μL of complete culture medium to each well. Continue culturing cells until 72 hours post-infection, replacing 1 mL of complete culture medium in each well. Continue culturing until 168 hours post-infection, harvest cells and extract genomic DNA.

[0098] Figure 7 This is a schematic diagram of two ITR-containing plasmids, Split-PE-N and Split-PE-C, which contain Split-PE-Rma-1024. The PE2 editing tool split the plasmid into two parts, Split-PE-N and Split-PE-C, at the splitting site 1024-1025, using Rma as the integrity. Split-PE-Rma-1024 serves as the control group. In all figures, CMV and U6 represent promoters; WPRE represents a post-transcriptional regulatory element of marmot hepatitis virus, which enhances gene expression; and pegRNA represents the leader editor guide RNA. Figure 7-13 and Figure 16 In the Split-PE-N, the N-terminal portion of the Cas9 protein is contained, and the Split-PE-C contains the C-terminal portion of the Cas9 protein, the linker, and the reverse transcriptase (RT-RNaseH).

[0099] Figure 8 This is a schematic diagram of two ITR-containing plasmids, Split-PE-N and Split-PE-C, which contain Split-PE-Rma-959. The PE2 editing tool split the plasmid into two parts, Split-PE-N and Split-PE-C, at the splitting site 959-960, using the integrity Rma.

[0100] Figure 9 is a schematic diagram of two ITR-containing plasmids, Split-PE-N and Split-PE-C, which contain Split-PE-Rma-1039. The PE2 editing tool split the plasmid into two parts, Split-PE-N and Split-PE-C, at the splitting site 1039-1040, using the integrity Rma.

[0101] Figure 10This is a schematic diagram of two ITR-containing plasmids, Split-PE-N and Split-PE-C, which contain Split-PE-Rma-1087. The PE2 editing tool split the plasmids into two parts, Split-PE-N and Split-PE-C, at the splitting site 1087-1088, using the integrity Rma.

[0102] Figure 11 This is a schematic diagram of two ITR-containing plasmids, Split-PE-N and Split-PE-C, which contain Split-PE-Rma-1115. The PE2 editing tool split the plasmid into two parts, Split-PE-N and Split-PE-C, at the splitting site 1115-1116, using the integrity Rma.

[0103] Figure 12 This is a schematic diagram of two ITR-containing plasmids, Split-PE-N and Split-PE-C, which contain Split-PE-Npu-1023-CF. The PE2 editing tool split the plasmid into two parts, Split-PE-N and Split-PE-C, at the splitting site 1023-1026-CF, using the integument Npu.

[0104] Figure 13 This is a schematic diagram of two ITR-containing plasmids, Split-PE-N and Split-PE-C, which contain Split-PE-Npu-1023-CW. The PE2 editing tool split the plasmids into two parts, Split-PE-N and Split-PE-C, at the splitting site 1023-1026-CW, using the integument Npu.

[0105] Figure 14a This demonstrates the single-base substitution efficiency of transient transfection with the Split-PE double plasmid. The plasmid containing Split-PE was transfected into HEK293T cells to achieve single-base substitution efficiency in the genome. FANCF The target is a G→T single-base substitution. The horizontal axis represents the Split-PE cleavage site and integrins used in the experiment. The vertical axis represents the correct editing ratio obtained through high-throughput sequencing analysis. Indel refers to... FANCF Unexpected base substitutions occurring at the editing site, such as base insertions or deletions. Figure 14b The insertion efficiency of transient transfection with the Split-PE double plasmid is shown. The plasmid containing Split-PE was transfected into HEK293T cells to obtain the desired genomic value. HEK3Genomic sites are the target sites for CTT nucleotide insertion. The horizontal axis represents the splitting site of the Split-PE used in the experiment and the integrins used. The vertical axis represents the correct editing ratio obtained through high-throughput sequencing analysis. Indel refers to... HEK3 Unexpected insertions and deletions occurring at edit sites.

[0106] Figure 15 This demonstrates the single-base substitution efficiency of transient transfection with the Split-PE double plasmid. The plasmid containing Split-PE was transfected into HEK293T cells to achieve single-base substitution efficiency in the genome. EMX1 Exon 3 was the target site for achieving a G→T single-base substitution. The horizontal axis represents the splitting site of Split-PE used in the experiment and the inpeptides used. Specifically, these are Split-PE-Rma-212, Split-PE-Rma-269, Split-PE-Rma-309, Split-PE-Rma-367, Split-PE-Rma-459, Split-PE-Rma-554, and Split-PE-Rma-940. The vertical axis represents the proportion of correct edits or indels obtained through high-throughput sequencing analysis. Indels refer to... EMX1 Unexpected base substitutions occurring at the editing site, such as base insertions or deletions.

[0107] Figure 16 This is a schematic diagram of the ITR-containing plasmids Split-PE-N and Split-PE-C, which contain Split-PE-Rma-1105. The PE2 editing tool split the plasmids into two parts, Split-PE-N and Split-PE-C, at the splitting site 1105-1106, using Rma as the integrity.

[0108] Figure 17 This is a schematic diagram of an ITR-containing plasmid containing multiple segments of PE3-based Split-PE-Rma-212. Cas9H840A splits at cleavage sites 212-213, using Rma as the integument. The U6 promoter drives the transcription of epigRNA and gRNA (for nick generation). The epigRNA and gRNA are linked at the DNA level via tRNA, which is processed by an endogenous tRNA processing enzyme, releasing the epigRNA and gRNA as two separate molecules. Peptide 2A (P2A) links the truncated Cas9 protein (Cas9H840A - N-terminus (1-212)) to reverse transcriptase (RT-RNaseH).

[0109] Figure 18This is a schematic diagram of an ITR-containing plasmid containing multiple segments of PE3-based Split-PE-Rma-269. Cas9H840A splits at cleavage sites 269-270, using Rma as the integument. The U6 promoter drives the transcription of epigRNA and gRNA (for nick generation). EpigRNA and gRNA are linked at the DNA level via tRNA, which is processed by an endogenous tRNA processing enzyme, releasing epigRNA and gRNA as two separate molecules. Peptide 2A (P2A) links the truncated Cas9 protein (Cas9H840A - N-terminus (1-269)) to reverse transcriptase (RT-RNaseH).

[0110] Figure 19 This is a schematic diagram of an ITR-containing plasmid containing multiple segments of PE3-based Split-PE-Rma-309. Cas9H840A splits at cleavage sites 309-310, using Rma as the integument. Transcription of the epegRNA and guide RNA is driven by two U6 promoters. The 2A peptide (P2A) connects the truncated Cas9 protein (Cas9H840A-N-terminus (1-309)) and reverse transcriptase (RT-RNaseH).

[0111] Figure 20 This is a schematic diagram of an ITR-containing plasmid containing multiple segments of PE3-based Split-PE-Rma-367. Cas9H840A splits at cleavage sites 367-368, using Rma as the integument. Transcription of the epegRNA and guide RNA is driven by two U6 promoters. The 2A peptide (P2A) connects the truncated Cas9 protein (Cas9H840A - N-terminus (1-367)) and reverse transcriptase (RT-RNaseH).

[0112] Figure 21 This is a schematic diagram of an ITR-containing plasmid containing multiple segments of PE3-based Split-PE-Rma-459. Cas9H840A splits at cleavage sites 459-460, using Rma as the integrity. epegRNA and gRNA are linked at the DNA level via tRNA, which will be processed by an endogenous tRNA processing enzyme, releasing gRNA and epigRNA as two separate molecules. Peptide 2A (P2A) links a truncated Cas9 protein (Cas9H840A - N-terminus (1-459)) to reverse transcriptase (RT-RNase H).

[0113] Figure 22This is a schematic diagram of an ITR-containing plasmid containing multiple segments of PE3-based Split-PE-Rma-554. Cas9H840A splits at the cleavage site 554-555, using Rma as the integrity. epegRNA and gRNA are linked at the DNA level via tRNA, which will be processed by an endogenous tRNA processing enzyme, releasing gRNA and epigRNA as two separate molecules. Peptide 2A (P2A) links a truncated Cas9 protein (Cas9H840A - N-terminus (1-554)) to reverse transcriptase (RT-RNaseH).

[0114] Figure 23 This is a schematic diagram of an ITR-containing plasmid containing multiple segments of PE3-based Split-PE-Rma-940. Cas9H840A splits at cleavage sites 940-941, using Rma as the integument. Transcription of the epegRNA and guide RNA is driven by two U6 promoters.

[0115] Figure 24 This is a multiple sequence alignment diagram of different Cas9n variants, in which the 12 splitting sites reported in this application are marked. Each vertical line represents a splitting site, with the sequence before the vertical line forming the Cas9n-N portion and the sequence after the vertical line forming the Cas9n-C portion. Figure 24 The 12 cleavage sites marked in the middle are: (1) 212-213 (IL SA), (2) 269-270 (KD) TY), (3) 309-310 (VN) TE), (4) 367-368 (GA) SQ), (5) 459-460 (GN) SR), (6) 554-555 (FK) TN), (7) 940-941 (MN) TK), (8) 959-960 (LK) SK), (9) 1023-1026 (A) KSE), (10) 1039-1040 (FY SN), (11) 1087-1088 (VL SM), (12) 1115-1116 (RN SD). (" "The position corresponding to the vertical line indicates the splitting point." Figure 24The sequences of the Cas9n variants from top to bottom are: SEQ ID NO: 93 (HypaCas9), SEQ ID NO: 94 (Cas9HF1), SEQ ID NO: 95 (evoCas9), SEQ ID NO: 96 (SpCas9), SEQ ID NO: 97 (LZ3Cas9), SEQ ID NO: 98 (HiFi Cas9), SEQ ID NO: 99 (Sniper-Cas9), SEQ ID NO: 100 (SuperFi-Cas9), SEQ ID NO: 101 (eSpCas9(1.1)), SEQ ID NO: 102 (xCas9), SEQ ID NO: 103 (SpGCas9), SEQ ID NO: 104 (SpRYCas9), SEQ ID NO: 105 (Cas9-NG), SEQ ID NO: 106 (Cas9-VRQR), and SEQ ID NO: 107 (Cas9-VRER).

[0116] Figure 25 In order to be in APP , CETP , VEGFA , ALDOB , SLC30A8 , MECP2 and FANCF The efficiency of single-base substitution during Split-ePE3 dual plasmid transient transfection at the site.

[0117] Figure 26 The insertion efficiency of Split-ePE3 dual plasmid transient transfection was measured in the experiment. RNF2 , HEK3 , ANGPTL3 and TTR The site is used.

[0118] Figure 27 This is a schematic diagram of two ITR-containing plasmids, Split-PE-N and Split-PE-C, which contain Split-PE-Npu-1024. The PE3 editing tool split the plasmid into two parts, Split-PE-N and Split-PE-C, at the splitting site 1024-1028-CFN, using the integument Npu.

[0119] Figure 28 This is a schematic diagram of an ITR-containing plasmid containing multiple segments of PE3-based Split-PE-Rma-1024. Cas9H840A splits at cleavage sites 1024-1025, using Rma as the integument. Transcription of the pegRNA and guide RNA is driven by two U6 promoters.

[0120] Figure 29 This is a schematic diagram of an ITR-containing plasmid containing multiple segments of PE3-based Split-PE-Rma-1105. Cas9H840A splits at cleavage sites 1105-1106, using Rma as the integrity. Transcription of the pegRNA and guide RNA is driven by two U6 promoters.

[0121] Figure 30 This is a schematic diagram of an ITR-containing plasmid containing multiple segments of PE3-based Split-PE-Rma-1115. Cas9H840A splits at cleavage sites 1115-1116, using Rma as the integrity. Transcription of the pegRNA and guide RNA is driven by two U6 promoters. Detailed Implementation

[0122] definition

[0123] All publications cited in this specification are incorporated herein by reference in their entirety. In the event of any discrepancy or inconsistency between the content of cited references and this disclosure, this disclosure shall prevail.

[0124] To facilitate understanding of the principles of this disclosure, the following description is made with reference to the embodiments shown in the accompanying drawings and using specific language. However, this does not imply limitation of the scope of this disclosure.

[0125] In this disclosure, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. While any methods and materials similar to or equivalent to those described and used herein may be used to practice this disclosure, preferred methods and materials are described herein. Therefore, the terminology defined herein will be more fully described by referring to the entire specification.

[0126] As used herein, the singular terms “a,” “an,” and “the” include the plural references unless the context clearly indicates otherwise.

[0127] Unless otherwise stated, nucleic acids are written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the direction from the amino terminus to the carboxyl terminus.

[0128] It should be understood that this disclosure is not limited to the specific methodologies, protocols, and reagents described, as these methodologies, protocols, and reagents may vary depending on the specific context in which they are used.

[0129] Unless the context requires otherwise, the terms “comprise,” “comprises,” and “comprising,” or similar terms, are intended to indicate a non-exclusive inclusion, such that the recorded list of elements or features includes not only those elements described or listed, but also other elements or features not listed or described.

[0130] Normalization algorithms can be used to align polynucleotide sequences. These algorithms insert gaps in the compared sequences in a normalized and reproducible manner to optimize the alignment between sequences, thus enabling more meaningful comparisons. A commonly used and freely available suite of sequence alignment algorithms is provided by the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) (Altschul, SF et al. (1990) J. Mol. Biol. 215:403-410), which is available from multiple sources, including the NCBI in Bethesda, Maryland, and online at http: / / www.ncbi.nlm.nih.gov / BLAST / .

[0131] As used herein, the term "variant" refers to a modified form of an object, including wild-type, naturally occurring, or artificially mutated forms. In some implementations, the variant has the same or similar functionality as the original object.

[0132] As used herein, the terms “truncated,” “truncate,” or “truncation,” in the context of proteins, refer to the removal of at least one amino acid from the N-terminus and / or C-terminus of a protein. In some embodiments, a truncated protein may have an additional amino acid added at the truncated site.

[0133] As used herein, the term "full-length" protein refers to the original form of a protein or protein variant. Proteins or protein variants that have not been truncated are full-length proteins.

[0134] As used herein, in some embodiments, the protein splitting site is denoted as “nm”, where n and m are each integers. “Split site nm” indicates that the protein splits at a position between the nth and mth amino acids (counted from the N-terminus to the C-terminus). In most embodiments, mn = 1, in which case the protein splits between the nth and mth amino acids. In some embodiments, mn > 1. In this case, the protein splits between the nth and (n+1)th amino acids, and at least one of the (n+1)th to (m-1)th amino acids is removed or replaced with another amino acid, preferably Cys. For example, splitting sites 1115-1116 indicate that the protein splits between the 1115th and 1116th amino acids. For example, splitting sites 1023-1026 indicate that the protein splits between the 1023rd and 1024th amino acids, and at least one of the 1024th and 1025th amino acids is removed or replaced with another amino acid. For example, the split site 1023-1026-CF indicates that the protein is split between the 1023rd and 1024th amino acids, and the 1024th and 1025th amino acids are replaced by cysteine ​​and phenylalanine, respectively.

[0135] In some embodiments, the protein cleavage site is described with reference to a short sequence fragment in the protein. For example, in some embodiments, the cleavage site is "the site between the second and third amino acids in SEQ ID NO: 1 of the protein". The cleavage site can be determined by first locating the sequence fragment in the amino acid sequence of the protein (in this example, SEQ ID NO: 1 is "ILSA"), and then locating the second and third amino acids in that sequence (L and S, respectively, in this example). The amino acid numbering of the cleavage site may vary depending on the amino acid sequence of the protein. For example, when the protein is eSp-Cas9 as shown in SEQ ID NO: 17, the cleavage site in the above example could also be represented as 212-213.

[0136] As used herein, "split prime editor" or "Split-PE" refers to a modified form of prime editor (PE) in which the Cas9 protein and / or reverse transcriptase (RT or RTase) are delivered to the cell in the form of multiple segments. In some embodiments, the segments are designed to fit the delivery capacity of a delivery tool, such as the delivery capacity of an AAV vector. After delivery to the cell, the segments can be further assembled to form a functional prime editor. The prime editor in a Split-PE can be any form of PE, such as PE1, PE2, PE3, or PE4. In some embodiments of the prime editor, the Cas9 protein is linked to the RTase via a linker. In some embodiments of the prime editor, the Cas9 protein is fused to the RTase. In some embodiments, the Cas9 protein and the RTase are separate from each other. In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9) or a variant thereof.

[0137] Delivery of the split-type gene editing system

[0138] Viral vectors are widely used delivery vectors in gene therapy. Commonly used viral vectors include, but are not limited to, adenovirus, lentivirus, and adeno-associated virus (AAV). AAV has relatively low immunogenicity and can be injected directly into the target site. Therefore, AAV is a safe and effective delivery vector and has been widely used clinically in gene therapy.

[0139] AAV is a non-enveloped virus that can be engineered to deliver DNA to target cells. AAV consists of a protein coat surrounding and protecting a small, single-stranded DNA genome approximately 4.8 kilobases (kb) long. Belonging to the Parvoviridae family, AAV relies on co-infection with other viruses, primarily adenoviruses, to replicate. Its single-stranded genome contains three genes: Rep (replication), Cap (capsid), and aap (assembly). These genes produce at least nine gene products through the use of three promoters, different translation initiation sites, and differential splicing. These coding sequences are flanked by inverted terminal repeats (ITRs), which are essential for genome replication and packaging. The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40), which are essential for viral genome replication and packaging, while Cap expression produces viral capsid proteins (VP; VP1 / VP2 / VP3), which form the protective outer capsid of the viral genome and are also actively involved in cell binding and internalization. It is estimated that the viral capsid consists of 60 proteins arranged in an icosahedral structure, with a molar ratio of capsid proteins of 1:1:10 (VP1:VP2:VP3). The aap gene encodes an assembly activation protein (AAP) in another reading frame overlapping with the cap gene. This nucleoprotein is thought to provide a scaffold function in capsid assembly. In AAV2, AAP is essential for nucleolar localization of the VP protein and capsid assembly, while subnuclear localization of AAP varies in 11 other serotypes studied recently, and it is not essential in AAV4, AAV5, and AAV11. Recombinant AAV (rAAV) lacks viral DNA and is essentially a protein-based nanoparticle engineered to cross the cell membrane and ultimately be transported to the nucleus to deliver its DNA payload. In the absence of the Rep protein, transgenes flanking the ITR encoded in rAAV can form persistent circular tandem structures in the nucleus of transduced cells and exist in free form. Because the recombinant free DNA does not integrate into the host genome, it eventually becomes diluted over time with repeated cell division. This ultimately leads to the loss of transgenes and transgene expression, with the rate of transgene loss depending on the turnover rate of the transduced cells. These properties make rAAV suitable for certain gene therapy applications.

[0140] Eleven AAV serotypes have been identified to date, with AAV2 being the most thoroughly studied and widely used. These serotypes differ in their tropism, i.e., the cell types they infect, making AAV a very useful system for preferentially transducing specific cell types. Table 1 summarizes the tropism of AAV serotypes and shows the optimal serotypes for organ-specific transduction.

[0141] Table 1

[0142] In some embodiments, the AAV used herein is a self-complementary AAV (scAAV). Because viruses rely on cellular DNA replication mechanisms to synthesize complementary strands, transgene expression can be delayed. To overcome this rate-limiting step, scAAV contains a complementary sequence that can spontaneously anneal after infection, thereby eliminating the need for host cell DNA synthesis.

[0143] The primary consideration in the rational design of rAAV vectors is the packaging size of the expression cassette to be placed between the two ITRs. It is generally considered that a size of less than 5 kb, including the viral ITR, is sufficient. Attempting to generate rAAV vectors containing expression cassettes larger than 5 kb leads to a significant reduction in viral yield or transgenic recombination (truncation). Therefore, large coding sequences, such as those in the PE or BE systems, cannot be effectively packaged in a single AAV vector.

[0144] This disclosure provides compositions and methods for delivering gene editing systems (e.g., PE and BE) into cells in a split form. In some embodiments, the gene editing system is split into two parts and packaged into two vectors (e.g., two AAV vectors). After transfection of cells with said two vectors, the two parts can be reassembled into a complete mRNA or protein by RNA transsplicing or peptide-mediated transsplicing, subsequently exerting gene editing function. In some embodiments, the gene editing system is split into more than two parts, such as 3, 4, 5, or 6 parts.

[0145] RNA trans-splicing is the process of precisely joining RNA exons from two discontinuous primary transcripts. Separate RNA precursor molecules provide different portions of mature mRNA, which are joined together through spliceosome-mediated processing. This differs from normal or cis-splicing, where the joined exons originate from the same primary transcript. Spliceosome trans-splicing mainly includes two types: genic trans-splicing and leader sequence (SL) trans-splicing. In genic trans-splicing, portions of two different RNA transcripts are joined at the splice site. These exons can originate from different pre-mRNAs of the same gene, transcripts from different genes or intergenic regions, or even sequences transcribed from different chromosomes. In SL trans-splicing, a short exon is provided by the 5' end of a specific longer RNA molecule and ligated to or near the 5' end of the mRNA, thus becoming the first exon of that mRNA. SL exons can ligate to various pre-mRNAs, resulting in a variety of mature mRNA transcripts, all containing a common sequence at their 5' ends. Both SL trans-splicing and genotypic trans-splicing utilize the basic mechanism of the major spliceosome, a dynamic, large RNA / protein complex responsible for cis-splicing or intron removal. In cis-splicing, two exons of the same pre-mRNA are precisely ligated by coordinating the recognition and pairing of 5' (donor) and 3' (acceptor) splice sites, removing the intronic RNA sequence between the two exons. The spliceosome snRNP and various cofactors recognize the sequences on the pre-mRNA that define the splice site boundaries and catalyze two transesterification reactions.

[0146] Inteins are internal protein domains with autocatalytic activity that catalyze the self-splicing of two parts of a polypeptide, resulting in a full-length functional protein after translation. Intein-mediated protein trans-splicing is more efficient than RNA trans-splicing, thus enabling more efficient in vivo gene editing. Commonly used inteins include, but are not limited to, those derived from *Rhodophyton floccosum* (Marine Rhodophyton). Rhodothermus marinus Rma of DnaB and that derived from Nostoc punctata ( Nostoc punctiforme ) of Npu.

[0147] Inteins are genetic elements found in single-celled organisms that are transcribed and translated as internal polypeptide fragments within host proteins. During post-translational processes, inteins mediate their own excision from the precursor protein without leaving any amino acid modifications in the final protein product. Intein-mediated protein splicing requires no energy supply, exogenous host-specific proteases, or cofactors. Intein activity is environment-dependent; efficient trans-splicing requires certain peptide sequences around the linker site (called N- and C-extenes), with the most important being that the first residue of the C-terminal exoteric intein is an amino acid containing a thiol or hydroxyl group (Cys, Ser, or Thr). Split inteins are a subclass of inteins that express as two separate polypeptides at the ends of two host proteins and catalyze their trans-splicing, resulting in a larger single polypeptide. Inteptides, including splitting inteptides, are widely used in the field of biotechnology, including protein purification and labeling steps, as well as the reconstruction of widely used clustered regular-spaced short palindromic repeat sequences (CRISPR-Cas9) genome editing nucleases.

[0148] Different combinations of introns and cleavage sites enable efficient AAV delivery and gene editing in cell lines or mice. Previous studies have reported several cleavage sites in PE or BE, such as 573-574, 637-638, 674-675, 684-685, 729-730, 739-740, 1005-1006, or 1024-1025 sites in Cas9n or dCas9. However, gene editing systems delivered based on these cleavage sites have shown low editing efficiency, limiting their application in clinical treatment.

[0149] This disclosure provides several novel splitting sites and combinations thereof. In some embodiments, the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 and 12 of the Cas9 protein or its variants. In some embodiments, the splitting site is located between the first and second amino acids of SEQ ID NO: 11. In some embodiments, the splitting site is located between the first and second amino acids of SEQ ID NO: 11, and the first two N-terminal amino acids of the C-terminal portion of the split Cas9 protein are replaced by cysteine ​​and phenylalanine, or cysteine ​​and tryptophan. In some embodiments, the splitting site is any one of 212-213, 269-270, 309-310, 367-368, 459-460, 554-555, 940-941, 959-960, 1039-1040, 1023-1026, 1087-1088, and 1115-1116 of the Cas9 protein or its variants. This disclosure also discloses several novel combinations of cleavage sites and integrins. In some embodiments, the Cas9 protein or a variant thereof cleaves at more than one cleavage site, at least one of which is a novel cleavage site disclosed herein. Experiments have shown that delivering the Split-PE system using the identified cleavage sites achieves higher editing efficiency. For example, the Split-PE system was tested at the EMX1 gene. Experiments have shown that Split-PE achieves accurate base substitutions at the EMX1 gene in cells when delivered via plasmid or dual AAV. Compared to the previously reported 1024-1025 cleavage site (Split-PE-Rma-1024, control), the 1115-1116 cleavage site (Split-PE-RmA-1115), discovered for the first time in this disclosure, achieves 5.8-fold higher editing efficiency when delivered via dual AAV, comparable to that of the intact PE. Furthermore, the combination of the cleavage site and inteptide disclosed herein can be applied to other optimized versions of PE, such as PE3, PE4, PE5 and shortened PE, to improve editing efficiency.

[0150] Novel cleavage sites and novel combinations of cleavage sites with integrins can be applied to Cas proteins with high sequence homology to wild-type Cas9 proteins. Homology can be evaluated by aligning two amino acid sequences using the Basic Local Alignment Search (BLAST) algorithm provided by the National Center for Biotechnology Information (NCBI) (Altschul, SF et al. (1990) J. Mol. Biol. 215:403-410). After sequence alignment, those skilled in the art can identify cleavage sites in the Cas protein of interest that correspond to the cleavage sites disclosed herein. For example, the amino acids surrounding the cleavage site are often identical or similar. For example, the amino acid immediately following the cleavage site is often serine or threonine.

[0151] In some implementations, the Cas9 protein can be replaced by other types of Cas proteins, such as those from the V and VI families. For example, the cleavage site and its application also apply to the Cas13 protein.

[0152] In some embodiments, the splitting site and / or a combination thereof is applied to the PE system. In some embodiments, the PE system is a PE1 or PE2 system, comprising the Cas9 protein, reverse transcriptase, and a lead editing guide RNA (pegRNA). In some embodiments, the PE system is a PE3 system, comprising the Cas9 protein, reverse transcriptase, pegRNA, and a single guide RNA (sgRNA). The sgRNA targets the edited strand of the lead editor and introduces a cut on the unedited strand, thereby improving editing efficiency. In some embodiments, the pegRNA in the PE system is engineered pegRNA (epegRNA) containing a stable structure at its 3' end, such as a modified prequeosine1-1 riboswitching aptamer (evopreQ1) or a frameshift pseudoknot derived from Moloney murine leukemia virus (Nelson et al., 2021). PE systems containing eegRNA are sometimes referred to as "ePE".

[0153] In some embodiments, the PE system comprises a complete leader editor protein, which is either a Cas9 protein fused with reverse transcriptase or a Cas9 protein linked to reverse transcriptase via a linker. For adaptation to a vector, such as an AAV vector, the leader editor protein can be split into smaller parts at a splitting site within the Cas9 protein described herein.

[0154] In some embodiments of the PE system, the reverse transcriptase is untethered to the Cas9 protein (Liu et al., 2002). In some embodiments, the sequence encoding the reverse transcriptase is linked to the sequence encoding the Cas9 protein (or a truncated Cas9 protein) via a sequence encoding a 2A peptide (P2A), thereby allowing the reverse transcriptase and the Cas9 protein (or a truncated Cas9 protein) to be expressed as two separate proteins. The inclusion of the 2A peptide allows for a more flexible arrangement of the Cas9 protein (in whole or in part) with the reverse transcriptase. For example, in some embodiments of the Split-PE system, the reverse transcriptase is not located at the 3' end or the C-terminal portion of the Cas9 protein. Instead, the reverse transcriptase can be positioned with the N-terminal portion of the Cas9 protein, with the 2A peptide positioned between them. This flexible arrangement achieved by the 2A peptide allows for the use of split sites located outside the central region of the Cas9-RT fusion protein. See also Figure 17-22 In some carefully designed cases (see...) Figure 19 , Figure 20 and Figure 23 The two U6 promoters used for pegRNA and gRNA can be added to polynucleotides respectively.

[0155] 2A peptides, sometimes called 2A self-cleaving peptides, are a class of peptides 18-22 amino acids in length that can induce ribosome jumping during protein translation in cells. These peptides share a core sequence motif, DxExNPGP, and are present in a wide range of viral families. The term "self-cleaving" is not entirely accurate, as these peptides function by causing the ribosome to skip the peptide bond synthesis at the C-terminus of the 2A element, resulting in the separation of the 2A sequence terminus from its downstream peptide. The "cleavage" occurs between glycine and proline residues in the peptide. 2A peptides are widely used in molecular biology to clone multiple genes in a single vector, greatly facilitating the co-expression of multiple factors or multi-subunit complex proteins in basic and translational research.

[0156] polypeptide

[0157] This disclosure provides novel cleavage sites in Cas9 proteins or variants thereof. The cleavage sites disclosed herein are located between the second and third amino acids of any sequence in SEQ ID NO: 1-10 of a full-length Cas9 protein or variant thereof, and between the first and second amino acids of SEQ ID NO: 11 of a full-length Cas9 protein or variant thereof. In some embodiments, when the cleavage site is located between the first and second amino acids of SEQ ID NO: 11, the first two amino acids at the N-terminus of the C-terminal portion of the cleaved protein are replaced with cysteine ​​and phenylalanine, or cysteine ​​and tryptophan.

[0158] In some embodiments, the Cas9 protein or variants thereof undergo a single cleavage at any of the cleavage sites disclosed herein, thereby being split into two parts: an N-terminal portion and a C-terminal portion. This disclosure provides polynucleotides comprising sequences encoding either of these N-terminal or C-terminal portions.

[0159] In some embodiments, the Cas9 protein or its variants are cleaved more than once, with at least one cleavage site selected from the cleavage sites disclosed herein. In some embodiments, the Cas9 protein or its variants are cleaved twice, with both cleavage sites selected from the cleavage sites disclosed herein, thereby cleaving the Cas9 protein or its variants into three parts: an N-terminal part, a middle part, and a C-terminal part. This disclosure also provides polynucleotides comprising sequences encoding these middle parts located between two cleavage sites disclosed herein.

[0160] In one aspect, this disclosure provides a polynucleotide comprising a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant thereof comprises an N-terminal portion of a full-length Cas9 protein or variant thereof split into two parts at a split site, wherein the split site is located between the second and third amino acids of any sequence in SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof.

[0161] In some embodiments, the truncated Cas9 protein or its variants comprise the 212th, 269th, 309th, 367th, 456th, 554th, 940th, 959th, 1039th, 1087th, 1023rd, or 1115th amino acids starting from the N-terminus of the full-length Cas9 protein or its variants. In some embodiments, the truncated Cas9 protein or its variants comprise the N-terminus of the full-length Cas9 protein or its variants, specifically the first 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 299, 300. 301,302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316,317, 318, 319, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368,369, 370, 371, 372, 373, 374, 375, 376, 377, 446, 447, 448, 449, 450, 451,452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 1029, 1030, 1031, 1032, 1033, 1034,1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, or 1125 amino acids.

[0162] In one aspect, this disclosure provides a polynucleotide comprising a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises a C-terminal portion of a full-length Cas9 protein or a variant thereof that is split into two parts at a split site, wherein the split site is located between the second and third amino acids of any sequence in SEQ IDNO: 1-10 of the full-length Cas9 protein or a variant thereof.

[0163] In one aspect, this disclosure provides a polynucleotide comprising a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant thereof comprises a C-terminal portion of a full-length Cas9 protein or variant thereof split into two parts at a split site, wherein the split site is located between the third and fourth amino acids of SEQ ID NO: 11 in the full-length Cas9 protein or variant thereof; and wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or variant thereof.

[0164] In one aspect, this disclosure provides a polynucleotide comprising a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises a portion of the full-length Cas9 protein or variant thereof located between a first cleavage site and a second cleavage site, wherein the first cleavage site is upstream of the second cleavage site, wherein at least one of the two cleavage sites is selected from: a site between the second and third amino acids of any of the sequences SEQ ID NO: 1-10 and 12, and a site between the first and second amino acids of SEQ ID NO: 11; wherein, when the first cleavage site is located between the first and second amino acids of SEQ ID NO: 11, the first two amino acids at the N-terminus of the truncated Cas9 protein or variant thereof are replaced with cysteine ​​and phenylalanine, or cysteine ​​and tryptophan. In some embodiments, both splitting sites are selected from the following sites: the site between the second and third amino acids of any of the sequences in SEQ ID NO: 1-10 and 12, and the site between the first and second amino acids of SEQ ID NO: 11; wherein, when the first splitting site is located between the first and second amino acids of SEQ ID NO: 11, the first two amino acids at the N-terminus of the truncated Cas9 protein or its variants are replaced with cysteine ​​and phenylalanine, or cysteine ​​and tryptophan.

[0165] In some embodiments, the polynucleotide described herein further comprises a sequence encoding an N-terminal portion of an inteptide, said inteptide being linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof.

[0166] In some embodiments, the integrin in the polynucleotide described herein is selected from Mxe-GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the integrin in the polynucleotide described herein is Rma.

[0167] In some embodiments, the inteptide is Rma, wherein the N-terminal portion of the Rma inteptide is encoded by SEQ ID NO:13, and the C-terminal portion of the Rma inteptide is encoded by SEQ ID NO:14. In some embodiments, the inteptide is Npu, wherein the N-terminal portion of the Npu inteptide is encoded by SEQ ID NO:15, and the C-terminal portion of the Npu inteptide is encoded by SEQ ID NO:16.

[0168] The Mxe intipeptide derived from *Mycobacterium xenopi* GyrA, the Npu intipeptide derived from *Nostoc punctiforme* DnaE, ​​the Ssp DnaB intipeptide derived from *Synechocystis* sp. PCC6803, the Ssp GyrB intipeptide derived from *Synechocystis* sp. PCC6803, and the Rma intipeptide derived from *Rhodothermus marinus* DnaB are the most commonly used intipeptides for split-Cas9. The GyrA intipeptide derived from *Mycobacterium xenopi* (Mxe-GyrA) is a natural miniature intipeptide (198 aa) lacking a central intipeptide endonuclease domain. As a bacterial protein, it can be expressed at very high levels in *E. coli*. Previous studies have also shown that the splicing of the Mxe-GyrA intipeptide and the cleavage at the single splice junction can be regulated by temperature. The Mxe intein is the first intein used in the split-Cas9 strategy, splitting at positions 656-657 (i.e., Cas9 splits between amino acids 656 and 657), resulting in approximately 50% restoration of cleavage activity compared to full-length SpCas9. Previous studies have reported that the Npu intein functions at positions 573-574 and 637-638. Further research used the Rma intein with a splitting position at 713-714 to achieve in vivo genome editing of SpCas9. (Chen, Yuxi, et al. "Development of highly efficient dual‐AAV split adenosine base editor for in vivo genetherapy." Small Methods 4.9 (2020): 2000309.)

[0169] In some embodiments, the polynucleotide described herein further comprises a guide RNA or a sequence encoding said guide RNA. In some embodiments, the guide RNA is a pegRNA containing a reverse transcriptase template. In some embodiments, the pegRNA further comprises a primer binding site (PBS). In some embodiments, the guide RNA targets the edited strand of the gene editing system and introduces a nick on the unedited strand, for example in the PE3 system.

[0170] In some embodiments, the polynucleotide described herein further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is the U6 promoter. RNA polymerase III promoters can be used to transcribe short non-coding RNA molecules, such as guide RNAs. The U6 promoter and the H1 promoter are two commonly used RNA polymerase III promoters. The U6 promoter comprises a TATA-like cassette, a proximal sequence element (PSE), and an upstream distal sequence element (DSE) (Anderson et al., 2020; Gao et al., 2018). In some embodiments, the U6 promoter has the sequence of SEQ ID NO: 48.

[0171] In some embodiments, the polynucleotide described herein further comprises a sequence encoding a protein with enzymatic activity. In some embodiments, the protein with enzymatic activity is a reverse transcriptase. In some embodiments, the reverse transcriptase is M-MLV or a functional variant thereof. In some embodiments, the gene-editing enzyme is a deaminase. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methyltransferase or a functional fragment thereof.

[0172] Reverse transcriptase is an enzyme used to generate complementary DNA (cDNA) from RNA as a template; this process is called reverse transcription. In some implementations of prime editing, reverse transcriptase uses a reverse transcriptase template (RT template) in a prime editing guide RNA (pegRNA) to introduce the desired edit at the editing site.

[0173] "Cytidine deaminase" refers to enzymes that catalyze the hydrolysis and deamination of cytidine and deoxycytidine into uridine and deoxyuridine, respectively. For example, one family of cytidine deaminases is APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like). "Adenosine deaminase" refers to an enzyme in purine metabolism that catalyzes the irreversible deamination of adenosine and deoxyadenosine into inosine and deoxyinosine, respectively.

[0174] Methyltransferases, also known as methyltransferases, modify or protect a recognition sequence by adding a methyl group (-CH3) to an adenine or cytosine base. Examples of such methyltransferases include DNMT1, DNMT3a1, DNMT3a2, and DNMT3b. Reverse transcriptase is an RNA-dependent DNA polymerase. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a functional variant thereof. In some embodiments, the MMLV-RT has an amino acid sequence encoded by SEQ ID NO: 47.

[0175] In some embodiments, the polynucleotide further comprises a sequence encoding a 2A peptide (P2A). In some embodiments, the sequence encoding the 2A peptide is located between the sequence encoding the truncated Cas9 protein and the sequence encoding the protein with enzymatic activity. As described above, the 2A peptide can induce ribosome jumping during protein translation in the cell, allowing the two proteins encoded upstream and downstream to be expressed as two separate proteins. In some embodiments, the 2A peptide has an amino acid sequence encoded by SEQ ID NO: 49.

[0176] In some embodiments, the polynucleotide further comprises a sequence encoding a nuclear localization signal (NLS). In some embodiments, the sequence encoding the NLS is located at the 5' end and / or 3' end of the protein-coding sequence. In some embodiments, the polynucleotide comprises more than one sequence encoding an NLS, wherein the NLS may be the same or different. In some embodiments, the NLS is selected from BE4max1NLS (encoded by SEQ ID NO: 108), BPNLS (encoded by SEQ ID NO: 109), BP-SV40(BE4Max2)NLS (encoded by SEQ ID NO: 110), and BPNLS-alt (encoded by SEQ ID NO: 111).

[0177] In some embodiments, the polynucleotide comprises a promoter sequence upstream of the protein-coding sequence and a sequence encoding BE4max1NLS (SEQ ID NO: 108) located between the promoter sequence and the protein-coding sequence. In some embodiments, the protein-coding sequence encodes the N-terminal portion of a full-length Cas9 protein or a variant thereof, or the C-terminal portion of a full-length Cas9 protein or a variant thereof.

[0178] In some embodiments, the polynucleotide includes a sequence encoding BPNLS (SEQ ID NO: 109) located at the 3' end of the sequence encoding the N-terminal portion of the inteptide.

[0179] In some embodiments, the polynucleotide comprises a sequence encoding BP-SV40(BE4Max2)NLS (SEQ ID NO: 110) located at the 3' end of the sequence encoding the reverse transcriptase or at the 3' end of the protein-coding sequence. In some embodiments, the protein-coding sequence encodes the N-terminal portion of the full-length Cas9 protein or a variant thereof, or the C-terminal portion of the full-length Cas9 protein or a variant thereof.

[0180] In some embodiments, the polynucleotide comprises a sequence encoding BPNLS-alt (SEQ ID NO: 111) located at the 3' end of the sequence encoding the P2A.

[0181] Nuclear localization signals (NLSs) are typically short peptides that act as signal fragments mediating protein transport from the cytoplasm to the nucleus. NLSs can be recognized by corresponding nuclear transport proteins that interact with nuclear porins, thereby facilitating the passage of NLS-containing proteins through the nuclear pore complex to the nucleus. Several types of NLSs have been identified in the prior art (Lu et al., 2021). For example, classic NLSs include single-site NLSs (MP NLSs) and two-site NLSs (BP NLSs). MP NLSs are single clusters of 4-8 basic amino acids, typically containing four or more positively charged residues, namely arginine (R) or lysine (K). The characteristic motif of MPNLSs is usually defined as K(K / R)X(K / R), where X can be any residue. For example, the NLS of the SV40 large T antigen is 126PKKKRKV132, which contains five consecutive positively charged amino acids (KKKRK). BP NLS is characterized by two clusters containing 2-3 positively charged amino acids, separated by a linker region containing 9-12 amino acids, which contains several proline (P) residues

[16] . The common sequence can be represented as R / K(X)10-12KRXK. It is noteworthy that in BP NLS, the upstream and downstream amino acid clusters are interdependent and both are essential, jointly determining the protein's intracellular location. For example, the BP NLS at the C-terminus of nucleoplasmic proteins, with the sequence 155KRPAATKKAGQAKKKK170, can guide the protein into the nucleus. In addition, there are non-classical NLS, such as the "proline-tyrosine" class of PY-NLS. PY-NLS is characterized by a disordered structure of 20-30 amino acids, including an N-terminal hydrophobic or basic motif and a C-terminal R / K / H(X)2-5PY motif (where X2-5 represents any sequence of 2-5 residues).

[0182] In some embodiments, the Cas9 protein or a variant thereof is a Cas9 nickase (nCas9). In some embodiments of the polynucleotide described herein, the Cas9 protein or a variant thereof is Cas9n-H840A (SEQ ID NO: 23), eSp-Cas9 (SEQ ID NO: 17), HiFi-Cas9 (SEQ ID NO: 18), Lz3-Cas9 (SEQ ID NO: 19), SpCas9-NG (SEQ ID NO: 20), Sniper-Cas9 (SEQ ID NO: 21), SuperFiCas9 (SEQ ID NO: 22), or dCas9 (SEQ ID NO: 24).

[0183] In some embodiments, the Cas9 protein or its variants comprise the sequence of SEQ ID NO: 93 (HypaCas9), SEQ ID NO: 94 (Cas9HF1), SEQ ID NO: 95 (evoCas9), SEQ ID NO: 96 (SpCas9), SEQ ID NO: 97 (LZ3 Cas9), SEQ ID NO: 98 (HiFi Cas9), SEQ ID NO: 99 (Sniper-Cas9), SEQ ID NO: 100 (SuperFi-Cas9), SEQ ID NO: 101 (eSpCas9(1.1)), SEQ ID NO: 102 (xCas9), SEQ ID NO: 103 (SpGCas9), SEQ ID NO: 104 (SpRYCas9), SEQ ID NO: 105 (Cas9-NG), SEQ ID NO: 106 (Cas9-VRQR), or SEQ ID NO: 107 (Cas9-VRER).

[0184] In some embodiments, the Cas9 protein or its variants have an amino acid sequence that is at least 90% identical to the Cas9 protein or its variants described herein. In some embodiments, the Cas9 protein or its variants have an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 17-24 and 93-107.

[0185] In some embodiments of the polynucleotide described herein, the Cas9 protein or a variant thereof may be replaced by other Cas proteins that have high sequence homology with the wild-type Cas9 protein near the cleavage site disclosed herein.

[0186] The polynucleotides described herein can be obtained by methods known in the art. For example, they can be obtained from cloned DNA (e.g., a DNA library), through chemical synthesis, through cDNA cloning, or by cloning genomic DNA or fragments thereof purified from the desired cell. When polynucleotides are generated via recombination, any method known to those skilled in the art for identifying nucleic acids encoding the desired gene can be used. Modified or variant polynucleotides, including truncated forms of Cas proteins (e.g., Cas9 protein), can be engineered from wild-type polynucleotides using standard recombinant DNA methods. Polynucleotides can be cloned or isolated using any available method known in the art. Such methods include PCR amplification of nucleic acids and library screening, including nucleic acid hybridization screening, antibody-based screening, and activity-based screening.

[0187] Polynucleotide amplification methods, including polymerase chain reaction (PCR), can be used to isolate polynucleotides encoding desired proteins. PCR can be performed using any method or procedure known in the art. Exemplary methods include the use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp). Nucleic acids containing the target gene can be used as source material to amplify nucleic acid molecules encoding the desired polypeptide. For example, DNA and mRNA preparations, cell extracts, tissue extracts from suitable sources (e.g., testes, prostate, breast), fluid samples (e.g., blood, serum, saliva), and samples from healthy and / or diseased subjects can be used in amplification methods. Sources can be from any eukaryotic species, including but not limited to vertebrates, mammals, humans, pigs, cattle, cats, birds, horses, dogs, and other primates. Nucleic acid libraries can also be used as source material. Primers can be designed to amplify the desired polynucleotide. For example, primers can be designed based on the expressed sequence to generate the desired polynucleotide. Primers can also be designed based on the reverse translation of the polypeptide amino acid sequence. Degenerate primers can be used for amplification when needed. Oligonucleotide primers that hybridize to the 5' and 3' ends of the desired sequence can be used as primers for PCR amplification from nucleic acid samples. These primers can be used to amplify full-length polynucleotides or truncated sequences thereof. The resulting nucleic acid molecules can be sequenced to confirm that they encode the desired polypeptide.

[0188] carrier

[0189] In one aspect, this disclosure provides a vector comprising the polynucleotides described herein.

[0190] In some embodiments, the vector described herein is a viral vector. In some embodiments, the vector described herein is a plasmid. Viral vectors may include, but are not limited to, adenovirus vectors, lentiviral vectors, retroviral vectors, and adeno-associated virus vectors. Suitable vectors, promoters, and enhancer elements are known in the art; many are commercially available for constructing the recombinant constructs described herein.

[0191] In some embodiments, the vector described herein is selected from adenovirus, lentivirus, and adeno-associated virus (AAV). In some embodiments, the vector described herein is AAV.

[0192] Adeno-associated virus (AAV) belongs to the Parvoviridae family. The AAV genome consists of a linear single-stranded DNA molecule containing approximately 4.7 kb and comprises two major open reading frames (ORFs), encoding the non-structural protein Rep (replication) and the structural protein Cap (capsid), respectively. A second ORF, encoding the assembly activation protein (AAP), has also been identified within the cap gene. The DNA flanking the AAV coding region consists of two cis-acting inverted terminal repeat (ITR) sequences, approximately 145 nucleotides in length, containing interrupted palindromic sequences that fold to form energy-stable hairpin structures and serve as primers for DNA replication. In addition to participating in DNA replication, ITR sequences have been shown to be involved in viral DNA integration into the cellular genome, rescue from the host genome or plasmids, and packaging viral nucleic acids into mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. 158:97-129).

[0193] Vectors derived from AAV (i.e., recombinant AAV (rAVV) or AAV vectors) are suitable for delivering genetic material because: (i) they can infect (transduce) a wide range of non-dividing and dividing cell types, including myocytes and neurons; (ii) they do not contain viral structural genes, thereby reducing the host cell’s response to viral infection, such as an interferon-mediated response; (iii) wild-type viruses are considered non-pathogenic to humans; (iv) replication-deficient AAV vectors lack the rep gene compared to wild-type AAVs that can integrate into the host cell genome and usually persist in a free form, thereby reducing the risk of insertional mutations or genotoxicity; and (v) AAV vectors are generally considered to have relatively weak immunogenicity compared to other vector systems, and therefore do not trigger a significant immune response (see ii), thus allowing the vector DNA to persist and potentially enabling long-term expression of therapeutic transgenes. AAV vectors can also be produced and formulated at high titers and delivered via intra-arterial, intravenous, or intraperitoneal injection, enabling vector distribution and gene transport in large muscle regions in rodents (Goyenvalle et al., 2004; Fougerse et al., 2007; Koppanati et al., 2010; Wang et al., 2009) and dogs with a single injection.

[0194] Expression vectors containing the polynucleotides disclosed herein can be constructed using any method known to those skilled in the art for inserting DNA fragments into a vector. These methods can include in vitro recombinant DNA and synthetic techniques as well as in vivo (genetic) recombination. The polynucleotides disclosed herein can be operatively linked to regulatory sequences in the expression vector to ensure expression of the encoded protein. Such regulatory sequences may include, but are not limited to, leader or signal sequences, promoters (e.g., naturally associated or heterologous promoters), ribosome binding sites, enhancer or activator elements, translation initiation and termination sequences, and transcription initiation and termination sequences, and are selected to be compatible with the host cell chosen for expression of the encoded protein. Constitutive or inducible promoters known in the art are also included within the scope of this disclosure. The promoters may be naturally occurring promoters, heterozygous promoters combining elements of more than one promoter, or synthetic promoters. The expression construct may be present in a cell in free form, such as in plasmid form, or the expression construct may be inserted into a chromosome, such as into a gene locus. In some embodiments, the expression vector includes a selectable marker gene to allow selection of transformed host cells. Some embodiments include an expression vector comprising a nucleotide sequence encoding a variant polypeptide, the nucleotide sequence being operatively linked to at least one regulatory control sequence. Regulatory control sequences as used herein include promoters, enhancers, and other expression control elements. In some embodiments, the expression vector is designed based on the host cell to be transformed, the specific variant polypeptide to be expressed, the copy number of the vector, the ability to control the copy number, and / or the ability to control the expression of any other protein encoded by the vector (e.g., an antibiotic marker).

[0195] Examples of suitable mammalian promoters include, for example, promoters derived from the following genes: elongation factor 1α (EF1α) promoter, CAG promoter, hamster ubiquitin / S27a promoter (WO 97 / 15664), simian vacuolating virus 40 (SV40) early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, the long terminal repeat (LTR) region of Rous sarcoma virus (RSV), mouse mammary tumor virus promoter (MMTV), the long terminal repeat region of Moloney mouse leukemia virus, and the early promoter of human cytomegalovirus (CMV). Other examples of heterologous mammalian promoters include actin promoters, immunoglobulin promoters, or heat shock promoters. In another embodiment, the promoter for mammalian host cells can be derived from the genome of a virus, such as polyomavirus, fowlpox virus (UK 2,211,504, published July 5, 1989), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40). In a further embodiment, a heterologous mammalian promoter is used. Examples include actin promoters, immunoglobulin promoters, and heat shock promoters. Early and late promoters of SV40 can be readily obtained as SV40 restriction fragments, which also contain the SV40 viral origin of replication (Fiers et al., Nature 273:113-120 (1978)). The immediate early promoter of human cytomegalovirus can be readily obtained as a HindIII restriction endonuclease fragment (Greenaway et al., Gene 18:355-360 (1982)). The above literature is incorporated herein by reference in its entirety.

[0196] Viral vectors may contain nucleic acid molecules, including nucleic acid elements derived from viruses, which typically facilitate the transfer or integration of the nucleic acid molecules into the genome of a cell, or facilitate the transfer of the nucleic acid molecules into viral particles that mediate nucleic acid transfer. Viral particles typically include various viral components in addition to nucleic acids, and sometimes also include host cell components. Viral vectors may include, for example, viruses or viral particles capable of transferring nucleic acids into cells, or the transferred nucleic acids (e.g., naked DNA). Viral vectors and transfer plasmids may contain structural and / or functional genetic elements primarily derived from viruses. Retroviral vectors may contain viral vectors or plasmids containing structural and functional genetic elements or portions thereof primarily derived from retroviruses.

[0197] Composition

[0198] In one aspect, this disclosure provides a composition comprising the polynucleotides described herein.

[0199] In some embodiments, the compositions described herein comprise a first polynucleotide and a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or a variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or a variant thereof; and wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or a variant thereof.

[0200] In some embodiments of the compositions described herein, the first polynucleotide further comprises a sequence encoding the N-terminal portion of an intein, the N-terminal portion of which is linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the intein is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the intein is Rma. In some embodiments, the first polynucleotide further comprises one or more guide RNAs or a sequence encoding the guide RNA. In some embodiments, at least one guide RNA comprises a reverse transcriptase template. In some embodiments, the first polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the first polynucleotide further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moroni mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0201] In some embodiments of the compositions described herein, the second polynucleotide further comprises a sequence encoding a C-terminal portion of an inteptide, the C-terminal portion of which is linked to an N-terminus encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the second polynucleotide further comprises one or more guide RNAs or a sequence encoding the guide RNA. In some embodiments, at least one guide RNA comprises a reverse transcriptase template. In some embodiments, the second polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the second polynucleotide further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moroni mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0202] In some embodiments, the compositions described herein comprise a first polynucleotide and a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, at the N-terminus of the truncated Cas9 protein or variant thereof; and The first polynucleotide further comprises a sequence encoding the N-terminal portion of an inteptide, the N-terminal portion of which is linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The second polynucleotide further comprises a sequence encoding the C-terminal portion of an inteptide, the C-terminal portion of which is linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The N-terminal portion and the C-terminal portion of the inteptide form a full-length inteptide.

[0203] In some embodiments, the inteptide is selected from Mxe-GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma.

[0204] In some embodiments of the compositions described herein, at least one of the polynucleotides comprises a guide RNA or a sequence encoding the guide RNA. In some embodiments, the guide RNA is a pegRNA containing a reverse transcriptase template. In some embodiments, at least one of the polynucleotides comprises both pegRNA and sgRNA, the sgRNA targeting the edited strand of the gene editing system and introducing a nick on the unedited strand, for example, in the PE3 system. In some embodiments, the polynucleotide containing the guide RNA also contains a U6 promoter. In some embodiments, the first polynucleotide comprises pegRNA, the second polynucleotide comprises sgRNA, and each polynucleotide also contains a U6 promoter.

[0205] In some embodiments of the compositions described herein, at least one of the polynucleotides comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein having enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methyltransferase or a functional fragment thereof.

[0206] In one aspect, this disclosure provides compositions comprising the carrier described herein.

[0207] In some embodiments, the compositions described herein comprise a first carrier containing a first polynucleotide and a second carrier containing a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or a variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or a variant thereof; and wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, at the N-terminus of the truncated Cas9 protein or a variant thereof.

[0208] In some embodiments of the compositions described herein, the first polynucleotide further comprises a sequence encoding an N-terminal portion of an inteptide, the N-terminal portion of which is linked to a C-terminus encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the first polynucleotide further comprises one or more guide RNAs or a sequence encoding the guide RNA. In some embodiments, at least one guide RNA comprises a reverse transcriptase template. In some embodiments, the first polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the first polynucleotide further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moroni mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0209] In some embodiments of the compositions described herein, the second polynucleotide further comprises a sequence encoding a C-terminal portion of an inteptide, the C-terminal portion of which is linked to an N-terminus encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the second polynucleotide further comprises one or more guide RNAs or a sequence encoding the guide RNA. In some embodiments, at least one guide RNA comprises a reverse transcriptase template. In some embodiments, the second polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the second polynucleotide further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moroni mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0210] In some embodiments, the compositions described herein comprise a first carrier containing a first polynucleotide and a second carrier containing a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, at the N-terminus of the truncated Cas9 protein or variant thereof; and The first polynucleotide further comprises a sequence encoding the N-terminal portion of an inteptide, the N-terminal portion of which is linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The second polynucleotide further comprises a sequence encoding the C-terminal portion of an inteptide, the C-terminal portion of which is linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The N-terminal portion and the C-terminal portion of the inteptide form a full-length inteptide.

[0211] In some embodiments, the inteptide is selected from Mxe-GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma.

[0212] In some embodiments of the compositions described herein, at least one of the polynucleotides comprises a guide RNA or a sequence encoding the guide RNA. In some embodiments, the guide RNA is a pegRNA containing a reverse transcriptase template. In some embodiments, at least one of the polynucleotides comprises both pegRNA and sgRNA, the sgRNA targeting the edited strand of the gene editing system and introducing a nick on the unedited strand, for example, in the PE3 system. In some embodiments, the polynucleotide containing the guide RNA also contains a U6 promoter. In some embodiments, the first polynucleotide comprises pegRNA, the second polynucleotide comprises sgRNA, and each polynucleotide also contains a U6 promoter.

[0213] In some embodiments of the compositions described herein, at least one of the polynucleotides comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein having enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methyltransferase or a functional fragment thereof.

[0214] In some embodiments of the compositions described herein, at least one of the carriers is an AAV carrier. In some embodiments, both carriers in the composition are AAV carriers.

[0215] In some embodiments of the compositions described herein, the Cas9 protein or a variant thereof is a Cas9 nickase (nCas9). In some embodiments, among the polynucleotides described herein, the Cas9 protein or a variant thereof is Cas9n-H840A (SEQ ID NO: 23), eSp-Cas9 (SEQ ID NO: 17), HiFi-Cas9 (SEQ ID NO: 18), Lz3-Cas9 (SEQ ID NO: 19), SpCas9-NG (SEQ ID NO: 20), Sniper-Cas9 (SEQ ID NO: 21), SuperFiCas9 (SEQ ID NO: 22), or dCas9 (SEQ ID NO: 24).

[0216] In some embodiments of the compositions described herein, the Cas9 protein or a variant thereof may be replaced by other Cas proteins that have high sequence homology with the wild-type Cas9 protein near the disclosed cleavage site.

[0217] As used herein, the term "composition" includes, but is not limited to, pharmaceutical compositions. A "pharmaceutical composition" refers to the formulation of an active pharmaceutical ingredient in a pharmaceutically acceptable or physiologically acceptable solution for administration alone or in combination with one or more other therapeutic modalities to cells or animals. It should also be understood that, where necessary, the compositions of the present invention can be administered in combination with other pharmaceutical agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or other various pharmaceutically active agents. There are no practical limitations on other components that may be included in the compositions, provided that the other pharmaceutical agents do not adversely affect the composition's ability to deliver the intended therapeutic effect. The term "pharmaceuticalally acceptable" is used herein to mean those compounds, materials, compositions, and / or dosage forms that, to a reasonable medical judgment, are suitable for use in contact with tissues of humans and animals without causing excessive toxicity, irritation, allergic reactions, or other problems or complications, and have a reasonable benefit / risk ratio.

[0218] The composition may also contain pharmaceutically acceptable carriers, diluents, or excipients. As used herein, “pharmaceutically acceptable carriers, diluents, or excipients” include, but are not limited to: any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier that has been approved by the U.S. Food and Drug Administration for use in humans or domesticated animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragali gum; malt; gelatin; talc; cocoa butter; waxes; animal and vegetable fats; paraffin wax; silicone; bentonite; silicic acid; zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginate; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer; and any other compatible substances used in pharmaceutical formulations.

[0219] Liquid pharmaceutical compositions, whether solutions, suspensions, or other similar forms, may contain one or more of the following components: a sterile diluent, such as water for injection, saline solution, preferably physiological saline; Ringer's solution; isotonic sodium chloride; a fixing oil, such as synthetic monoglycerides or diglycerides that can serve as a solvent or suspension medium; polyethylene glycol; glycerol; propylene glycol or other solvents; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate; and a tonic agent, such as sodium chloride or glucose. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0220] The composition can be suitably prepared into a formulation for intravenous, intratumoral, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ocular, or other routes of administration.

[0221] cell

[0222] In one aspect, this disclosure provides cells comprising the polynucleotides described herein.

[0223] In some implementations, the cells described herein comprise a first polynucleotide and a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or a variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or a variant thereof; and wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, at the N-terminus of the truncated Cas9 protein or a variant thereof.

[0224] In some embodiments of the cell described herein, the first polynucleotide further comprises a sequence encoding an N-terminal portion of an inteptide, the N-terminal portion of which is linked to a C-terminus encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the first polynucleotide further comprises one or more guide RNAs or a sequence encoding the guide RNA. In some embodiments, at least one guide RNA comprises a reverse transcriptase template. In some embodiments, the first polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the first polynucleotide further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moroni mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0225] In some embodiments of the cells described herein, the second polynucleotide further comprises a sequence encoding a C-terminal portion of an inteptide, the C-terminal portion of which is linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the second polynucleotide further comprises one or more guide RNAs or a sequence encoding the guide RNA. In some embodiments, at least one guide RNA comprises a reverse transcriptase template. In some embodiments, the second polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the second polynucleotide further comprises a sequence encoding a protein with enzymatic activity. In some embodiments, the protein with enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moroni mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0226] In some implementations, the cells described herein comprise a first polynucleotide and a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, at the N-terminus of the truncated Cas9 protein or variant thereof; and The first polynucleotide further comprises a sequence encoding the N-terminal portion of an intima-peptide, the N-terminal portion of which is linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The second polynucleotide further comprises a sequence encoding the C-terminal portion of an intima-peptide, the C-terminal portion of which is linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The N-terminal portion and the C-terminal portion of the inteptide form a full-length inteptide.

[0227] In some embodiments, the inteptide is selected from Mxe-GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma.

[0228] In some embodiments of the cells described herein, at least one of the polynucleotides comprises a guide RNA or a sequence encoding the guide RNA. In some embodiments, the guide RNA is a pegRNA containing a reverse transcriptase template. In some embodiments, at least one of the polynucleotides comprises both pegRNA and sgRNA, the sgRNA targeting the edited strand of the gene editing system and introducing a nick on the unedited strand, for example, in the PE3 system. In some embodiments, the polynucleotide containing the guide RNA also contains a U6 promoter. In some embodiments, the first polynucleotide comprises pegRNA, the second polynucleotide comprises sgRNA, and each polynucleotide also contains a U6 promoter.

[0229] In some embodiments of the cells described herein, at least one of the polynucleotides comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein having enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methyltransferase or a functional fragment thereof.

[0230] In one aspect, this disclosure provides cells comprising the vector described herein.

[0231] In some embodiments, the cells described herein comprise a first carrier containing a first polynucleotide and a second carrier containing a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or a variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or a variant thereof; and wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, at the N-terminus of the truncated Cas9 protein or a variant thereof.

[0232] In some embodiments of the cell described herein, the first polynucleotide further comprises a sequence encoding an N-terminal portion of an inteptide, the N-terminal portion of which is linked to a C-terminus encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the first polynucleotide further comprises one or more guide RNAs or a sequence encoding the guide RNA. In some embodiments, at least one guide RNA comprises a reverse transcriptase template. In some embodiments, the first polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the first polynucleotide further comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moroni mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0233] In some embodiments of the cells described herein, the second polynucleotide further comprises a sequence encoding a C-terminal portion of an inteptide, the C-terminal portion of which is linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the second polynucleotide further comprises one or more guide RNAs or a sequence encoding the guide RNA. In some embodiments, at least one guide RNA comprises a reverse transcriptase template. In some embodiments, the second polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the second polynucleotide further comprises a sequence encoding a protein with enzymatic activity. In some embodiments, the protein with enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moroni mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0234] In some embodiments, the cells described herein comprise a first carrier containing a first polynucleotide and a second carrier containing a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or variant thereof; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site is located between the third and fourth amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or variant thereof; and wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, at the N-terminus of the truncated Cas9 protein or variant thereof; and The first polynucleotide further comprises a sequence encoding the N-terminal portion of an intima-peptide, the N-terminal portion of which is linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The second polynucleotide further comprises a sequence encoding the C-terminal portion of an intima-peptide, the C-terminal portion of which is linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The N-terminal portion and the C-terminal portion of the inteptide form a full-length inteptide.

[0235] In some embodiments, the inteptide is selected from Mxe-GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma.

[0236] In some embodiments of the cells described herein, at least one of the polynucleotides comprises a guide RNA or a sequence encoding the guide RNA. In some embodiments, the guide RNA is a pegRNA containing a reverse transcriptase template. In some embodiments, at least one of the polynucleotides comprises both pegRNA and sgRNA, the sgRNA targeting the edited strand of the gene editing system and introducing a nick on the unedited strand, for example, in the PE3 system. In some embodiments, the polynucleotide containing the guide RNA also contains a U6 promoter. In some embodiments, the first polynucleotide comprises pegRNA, the second polynucleotide comprises sgRNA, and each polynucleotide also contains a U6 promoter.

[0237] In some embodiments of the cells described herein, at least one of the polynucleotides comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein having enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methyltransferase or a functional fragment thereof.

[0238] In some embodiments of the cells described herein, at least one of the vectors is an AAV vector. In some embodiments of the compositions described herein, both of the vectors are AAV vectors.

[0239] In some embodiments of the cells described herein, the Cas9 protein or a variant thereof is a Cas9 nickase (nCas9). In some embodiments, among the polynucleotides described herein, the Cas9 protein or a variant thereof is Cas9n-H840A (SEQ ID NO: 23), eSp-Cas9 (SEQ ID NO: 17), HiFi-Cas9 (SEQ ID NO: 18), Lz3-Cas9 (SEQ ID NO: 19), SpCas9-NG (SEQ ID NO: 20), Sniper-Cas9 (SEQ ID NO: 21), SuperFiCas9 (SEQ ID NO: 22), or dCas9 (SEQ ID NO: 24).

[0240] In some embodiments of the cells described herein, the Cas9 protein or a variant thereof may be replaced by other Cas proteins that have high sequence homology with the wild-type Cas9 protein near the disclosed cleavage site.

[0241] In some embodiments, the cells are selected from, but are not limited to, stem cells, pluripotent cells, somatic cells, cardiac cells, cardiac progenitor cells, nerve cells, glial progenitor cells, endothelial cells, T cells, B cells, pancreatic islet cells, retinal pigment epithelial cells, hepatocytes, thyroid cells, skin cells, blood cells, plasma cells, platelets, kidney cells, epithelial cells, CAR-T cells, NK cells, and CAR-NK cells. In some embodiments, the cells are derived from mammals. In some embodiments, the cells are human cells.

[0242] In some implementations, the cells are primary cells. Primary cells are cells obtained directly from human or animal tissues using enzymatic or mechanical methods. After isolation, they are placed in an artificial environment, such as plastic or glass containers, and cultured using a specialized culture medium containing the nutrients and growth factors necessary to maintain cell proliferation. Primary cells can be divided into two types: adherent cells and suspension cells. Adhesive cells require attachment to grow and are therefore called anchorage-dependent cells. Adhesive cells are typically derived from organ tissues. Suspension cells do not require attachment to a surface to grow and are therefore called anchorage-independent cells. Most suspension cells are derived from the blood system, but some tissue-derived cells, such as hepatocytes or intestinal cells, can also be cultured in suspension. Although primary cells generally have a limited lifespan, they offer many advantages compared to cell lines. Primary cell culture allows researchers to study the donor, not just the cells themselves. Various factors, such as age, medical history, race, and sex, can be considered when establishing experimental models. With the development of personalized medicine, primary cells can be used to achieve this donor variability and tissue complexity, which is difficult to achieve with more systematic and homogeneous cell lines, and cell lines cannot truly reflect the diversity of living tissues.

[0243] In some embodiments, the cells are differentiated cells. Differentiated cells are cells that have undergone differentiation. These differentiated cells are mature cells that perform specific functions. Some examples of differentiated cells are epithelial cells, skin fibroblasts, endothelial cells lining the inner walls of blood vessels, smooth muscle cells, hepatocytes, nerve cells, and human cardiomyocytes. Typically, these cells possess unique morphology, metabolic activity, membrane potential, and signal responsiveness, thereby contributing to their respective functions within body tissues or organs.

[0244] Reagent test kit

[0245] In one aspect, this disclosure provides a kit comprising the polynucleotides described herein.

[0246] In some embodiments, the kit described herein comprises a first polynucleotide and a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the N-terminal portion of a full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof splitting into two parts at a splitting site, wherein the splitting site of the full-length Cas9 protein or variant thereof is located between the second and third amino acids of any of the sequences shown in SEQ ID NO: 1-10, or, the splitting site of the full-length Cas9 protein or variant thereof is located between the first and second amino acids of SEQ ID NO: 11; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof being divided into two parts at a split site, wherein, in the full-length Cas9 protein or a variant thereof, the split site is located between the second and third amino acids of any of the sequences shown in SEQ ID NO: 1-10; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof being divided into two parts at a split site, wherein, in the full-length Cas9 protein or a variant thereof, the split site is located between the third and fourth amino acids of SEQ ID NO: 11; and wherein, the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or a variant thereof.

[0247] In some embodiments of the kit described herein, the first polynucleotide further comprises a sequence encoding an N-terminal portion of an inteptide, the N-terminal portion of which is linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the first polynucleotide further comprises one or more guide RNAs or a sequence encoding the guide RNA. In some embodiments, at least one guide RNA comprises a reverse transcriptase template. In some embodiments, the first polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the first polynucleotide further comprises a sequence encoding a protein with enzymatic activity. In some embodiments, the protein with enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moroni mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0248] In some embodiments of the kit described herein, the second polynucleotide further comprises a sequence encoding a C-terminal portion of an inteptide, the C-terminal portion of which is linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the second polynucleotide further comprises one or more guide RNAs or a sequence encoding the guide RNA. In some embodiments, at least one guide RNA comprises a reverse transcriptase template. In some embodiments, the second polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the second polynucleotide further comprises a sequence encoding a protein with enzymatic activity. In some embodiments, the protein with enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0249] In some embodiments, the kit described herein comprises a first polynucleotide and a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the N-terminal portion of a full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof being divided into two parts at a splitting site, wherein, in the full-length Cas9 protein or a variant thereof, the splitting site is located between the second and third amino acids of any of the sequences shown in SEQ ID NO: 1-10, or, in the full-length Cas9 protein or a variant thereof, the splitting site is located between the first and second amino acids of the sequence shown in SEQ ID NO: 11; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof being divided into two parts at a splitting site, wherein in the full-length Cas9 protein or variant thereof, the splitting site is located between the second and third amino acids of any of the sequences shown in SEQ ID NO: 1-10; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof being divided into two parts at a splitting site, wherein in the full-length Cas9 protein or variant thereof, the splitting site is located between the third and fourth amino acids of the sequence shown in SEQ ID NO: 11; and wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or variant thereof; and Wherein, the first polynucleotide further comprises a sequence encoding the N-terminal portion of an inteptide, the N-terminal portion of which is connected to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The second polynucleotide further comprises a sequence encoding a C-terminal portion of an inteptide, the C-terminal portion of which is connected to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The N-terminal portion and the C-terminal portion of the inteptide form a full-length inteptide.

[0250] In some embodiments, the intima peptide is selected from Mxe-GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the intima peptide is Rma.

[0251] In some embodiments of the kit described herein, at least one polynucleotide comprises a guide RNA or a sequence encoding said guide RNA. In some embodiments, the guide RNA is a pegRNA containing a reverse transcriptase template. In some embodiments, at least one of said polynucleotides comprises both pegRNA and sgRNA, said sgRNA targeting the edited strand of the gene editing system and introducing a nick on the unedited strand, for example, in the PE3 system. In some embodiments, the polynucleotide containing said guide RNA further comprises a U6 promoter. In some embodiments, the first polynucleotide comprises pegRNA, the second polynucleotide comprises sgRNA, and each polynucleotide further comprises a U6 promoter.

[0252] In some embodiments of the kits described herein, at least one polynucleotide comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the enzymatically active protein is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the enzymatically active protein is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methyltransferase or a functional fragment thereof.

[0253] In one aspect, this disclosure provides a kit comprising the vector described herein.

[0254] In some embodiments, the kit described herein comprises a first carrier and a second carrier, the first carrier comprising a first polynucleotide and the second carrier comprising a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the N-terminal portion of a full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof being divided into two parts at a splitting site, wherein, in the full-length Cas9 protein or a variant thereof, the splitting site is located between the second and third amino acids of any of the sequences shown in SEQ ID NO: 1-10, or, in the full-length Cas9 protein or a variant thereof, the splitting site is located between the first and second amino acids of the sequence shown in SEQ ID NO: 11; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof being divided into two parts at a split site, wherein, in the full-length Cas9 protein or a variant thereof, the split site is located between the second and third amino acids of any of the sequences shown in SEQ ID NO: 1-10; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the C-terminal portion of the full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof being divided into two parts at a split site, wherein, in the full-length Cas9 protein or a variant thereof, the split site is located between the third and fourth amino acids of the sequence shown in SEQ ID NO: 11; and wherein, the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or a variant thereof.

[0255] In some embodiments of the kit described herein, the first polynucleotide further comprises a sequence encoding an N-terminal portion of an inteptide, the N-terminal portion of which is linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the first polynucleotide further comprises one or more guide RNAs or a sequence encoding the guide RNA. In some embodiments, at least one guide RNA comprises a reverse transcriptase template. In some embodiments, the first polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the first polynucleotide further comprises a sequence encoding a protein with enzymatic activity. In some embodiments, the protein with enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moroni mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0256] In some embodiments of the kit described herein, the second polynucleotide further comprises a sequence encoding a C-terminal portion of an inteptide, the C-terminal portion of which is linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the inteptide is Rma. In some embodiments, the second polynucleotide further comprises one or more guide RNAs or a sequence encoding the guide RNA. In some embodiments, at least one guide RNA comprises a reverse transcriptase template. In some embodiments, the second polynucleotide further comprises an RNA polymerase III promoter. In some embodiments, the RNA polymerase III promoter is a U6 promoter. In some embodiments, the second polynucleotide further comprises a sequence encoding a protein with enzymatic activity. In some embodiments, the protein with enzymatic activity is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0257] In some embodiments, the kit described herein comprises a first carrier and a second carrier, the first carrier comprising a first polynucleotide and the second carrier comprising a second polynucleotide. The first polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises the N-terminal portion of a full-length Cas9 protein or a variant thereof, the full-length Cas9 protein or a variant thereof being divided into two parts at a splitting site, wherein, in the full-length Cas9 protein or a variant thereof, the splitting site is located between the second and third amino acids of any of the sequences shown in SEQ ID NO: 1-10, or, in the full-length Cas9 protein or a variant thereof, the splitting site is located between the first and second amino acids of the sequence shown in SEQ ID NO: 11; and The second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof being divided into two parts at a splitting site, wherein in the full-length Cas9 protein or variant thereof, the splitting site is located between the second and third amino acids of any of the sequences shown in SEQ ID NO: 1-10; or, the second polynucleotide comprises a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant comprises the C-terminal portion of the full-length Cas9 protein or variant thereof, the full-length Cas9 protein or variant thereof being divided into two parts at a splitting site, wherein in the full-length Cas9 protein or variant thereof, the splitting site is located between the third and fourth amino acids of the sequence shown in SEQ ID NO: 11; and wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, located at the N-terminus of the truncated Cas9 protein or variant thereof; and Wherein, the first polynucleotide further comprises a sequence encoding the N-terminal portion of an inteptide, the N-terminal portion of which is connected to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The second polynucleotide further comprises a sequence encoding a C-terminal portion of an inteptide, the C-terminal portion of which is connected to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof; and The N-terminal portion and the C-terminal portion of the inteptide form a full-length inteptide.

[0258] In some embodiments, the intima peptide is selected from Mxe-GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma. In some embodiments, the intima peptide is Rma.

[0259] In some embodiments of the kit described herein, at least one polynucleotide comprises a guide RNA or a sequence encoding said guide RNA. In some embodiments, the guide RNA is a pegRNA containing a reverse transcriptase template. In some embodiments, at least one of said polynucleotides comprises both pegRNA and sgRNA, said sgRNA targeting the edited strand of the gene editing system and introducing a nick on the unedited strand, for example, in the PE3 system. In some embodiments, the polynucleotide containing said guide RNA further comprises a U6 promoter. In some embodiments, the first polynucleotide comprises pegRNA, the second polynucleotide comprises sgRNA, and each polynucleotide further comprises a U6 promoter.

[0260] In some embodiments of the kits described herein, at least one polynucleotide comprises a sequence encoding a protein having enzymatic activity. In some embodiments, the enzymatically active protein is a reverse transcriptase or a functional fragment thereof. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof. In some embodiments, the enzymatically active protein is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methyltransferase or a functional fragment thereof.

[0261] In some embodiments of the kit described herein, at least one vector is an AAV vector. In some embodiments, both vectors are AAV vectors.

[0262] In some embodiments of the kits described herein, the Cas9 protein or a variant thereof is the Cas9 nickase (nCas9). In some embodiments, the Cas9 protein or a variant thereof in the polynucleotides described herein is Cas9n-H840A (SEQ ID NO: 23), eSp-Cas9 (SEQ ID NO: 17), HiFi-Cas9 (SEQ ID NO: 18), Lz3-Cas9 (SEQ ID NO: 19), SpCas9-NG (SEQ ID NO: 20), Sniper-Cas9 (SEQ ID NO: 21), SuperFiCas9 (SEQ ID NO: 22), or dCas9 (SEQ ID NO: 24).

[0263] In some embodiments of the kit described herein, the Cas9 protein or a variant thereof may be replaced by other Cas proteins that have high sequence homology with the wild-type Cas9 protein near the disclosed cleavage site.

[0264] method

[0265] In one aspect, this disclosure provides a method for altering target nucleic acids in cells, the method comprising delivering the composition or kit described herein into the cells.

[0266] In one aspect, this disclosure provides a method for altering target nucleic acids in cells, the method comprising: Provide the cells with a first polynucleotide encoding the N-terminal portion of the Cas9 protein or a variant thereof. Provide the cells with a second polynucleotide encoding the remaining C-terminal portion of the Cas9 protein or a variant thereof. Guide RNA is provided to the cells, the guide RNA containing a spacer sequence complementary to the target nucleic acid. The cell is capable of RNA trans-splicing and / or inteptide-mediated protein trans-splicing, and the cell expresses the full-length Cas9 protein or a variant thereof; and in the full-length Cas9 protein or a variant thereof, the splitting site is located between the second and third amino acids of any of the sequences shown in SEQ ID NO: 1-10; or, in the full-length Cas9 protein or a variant thereof, the splitting site is located between the first and second amino acids of the sequence shown in SEQ ID NO: 11, and the first two amino acids at the N-terminus of the second polynucleotide are replaced by cysteine ​​and phenylalanine, or cysteine ​​and tryptophan.

[0267] In some embodiments of the methods described herein, the first polynucleotide further comprises a sequence encoding the N-terminal portion of an inteptide, the N-terminal portion of which is linked to the C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof, and the second polynucleotide further comprises a sequence encoding the remaining C-terminal portion of the inteptide, the remaining C-terminal portion of which is linked to the N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof. In some embodiments, the inteptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB, and Rma.

[0268] In some embodiments, the integrin is Rma, and the sequences encoding the N-terminal and C-terminal portions of Rma are shown in Table 2. In some embodiments, the integrin is Npu, and the sequences encoding the N-terminal and C-terminal portions of Npu are shown in Table 2.

[0269] Table 2

[0270] In some embodiments of the methods described herein, the method further includes providing the cells with a protein having enzymatic activity or a polynucleotide encoding the protein having enzymatic activity. In some embodiments of the methods described herein, the method further includes providing a sequence encoding the protein having enzymatic activity. In some embodiments, the protein having enzymatic activity is a reverse transcriptase. In some embodiments, the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a functional variant thereof.

[0271] In some embodiments, the protein with enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

[0272] In some embodiments, at least one of the polynucleotides further comprises guide RNA or a sequence encoding the guide RNA. In some embodiments of the methods described herein, the guide RNA further comprises a reverse transcriptase template.

[0273] In some embodiments of the methods described herein, the first polynucleotide is provided by a first carrier, and the second polynucleotide is provided by a second carrier.

[0274] In some embodiments of the methods described herein, the first vector and the second vector are viral vectors.

[0275] In some embodiments of the methods described herein, the viral vector is selected from adenovirus, lentivirus, and adeno-associated virus (AAV).

[0276] In some embodiments of the methods described herein, the Cas9 protein or a variant thereof is a Cas9 nickase (nCas9). In some embodiments, among the polynucleotides described herein, the Cas9 protein or a variant thereof is Cas9n-H840A (SEQ ID NO: 23), eSp-Cas9 (SEQ ID NO: 17), HiFi-Cas9 (SEQ ID NO: 18), Lz3-Cas9 (SEQ ID NO: 19), SpCas9-NG (SEQ ID NO: 20), Sniper-Cas9 (SEQ ID NO: 21), SuperFiCas9 (SEQ ID NO: 22), or dCas9 (SEQ ID NO: 24).

[0277] In some embodiments of the methods described herein, the Cas9 protein or a variant thereof may be replaced by other Cas proteins that have high sequence homology with the wild-type Cas9 protein near the disclosed cleavage site.

[0278] In the methods described herein, the delivery of the described components may be performed using techniques known in the art, such as, but not limited to, electroporation, gene gun particle delivery, liposome transfection, and viral transfection.

[0279] In some embodiments of the methods described herein, the target nucleic acid is altered in vitro. In some embodiments, the methods described herein are performed in vitro.

[0280] In some embodiments of the methods described herein, the target nucleic acid is altered in vivo. In some embodiments, the methods described herein are performed in vivo. In some embodiments, the component is packaged into one or more viral vectors, such as AAV vectors, and delivered to the cells.

[0281] The Split-PE system disclosed herein is particularly suitable for in vivo gene therapy and gene editing. The choice of delivery vectors for in vivo gene editing is very limited, with viral vectors such as AAV being among the most well-established and commonly used. However, the application of AAV vectors is typically limited by their cargo capacity (approximately 4.7 kb). The Split-PE system disclosed herein provides a solution to overcome this limitation of AAV vectors, enabling the wider use of AAV and other viral vectors for in vivo gene editing and gene therapy. Example 2 demonstrates that the Split-PE system performs well in AAV infection, indicating its suitability for in vivo gene editing.

[0282] Table 3

[0283] Example

[0284] Example 1: Editing endogenous sites in human cell lines via transient transfection with Split-PE plasmid

[0285] Five PE2 cleavage sites were designed: 959-960, 1023-1026, 1039-1040, 1087-1088, and 1115-1116 (Figure 1). These five cleavage sites were combined with two integrins, Rma and Npu, to obtain eight different Split-PE constructs, namely Split-PE2-Rma-959 (…). Figure 8 (SEQ ID NO: 53 and 54), Split-PE2-Rma-1039 ( Figure 9a (SEQ ID NO: 55 and 56), Split-PE2-Rma-1087 ( Figure 10 (SEQ ID NO: 61 and 62), Split-PE2-Rma-1115 ( Figure 11 (SEQ ID NO: 63 and 64), Split-PE2-Npu-1023-CF ( Figure 12 (SEQ ID NO: 65 and 66), Split-PE2-Npu-1023-CW ( Figure 13 (SEQ ID NO: 67 and 68), Split-PE2-Npu-1039-CF ( Figure 9b (SEQ ID NO: 57 and 58) and Split-PE2-Npu-1039-CW ( Figure 9c (SEQ ID NO: 59 and 60).

[0286] First, the eight Split-PE constructs were transfected into HEK293T cells via transient plasmid transfection. Editing efficiency and the corresponding indel percentage were then assessed. Split-PE-Rma-1024 ( Figure 7 (SEQ ID NO: 51 and 52) were used as control groups. It was reported to have relatively high editing efficiency. Endogenous [materials] were selected. EMX1 The gene was used as one of the editing sites. PegRNAs were designed to achieve a G:C to T:A base substitution at a specific site (Figure 2). High-throughput sequencing results showed that at the endogenous site... EMX1 At the same time, compared with Split-PE2-Rma-1024 (control), Split-PE2-Rma-1039, Split-PE2-Rma-1087, and Split-PE2-Rma-1115 showed similar or higher single-base substitution efficiencies. Figure 3 , Figure 7-13Split-PE2-Rma-1115 (5.36%) had the highest editing efficiency, which was 1.6 times that of Split-PE2-Rma-1024 (control, 3.28%).

[0287] Choose endogenous FANCF Genes were used as another editing site. PegRNAs were designed to achieve G:C to T:A base substitutions at specific sites. High-throughput sequencing results showed that at endogenous sites… FANCF At the same time, compared with Split-PE2-Rma-1024 (control), Split-PE2-Rma-1039, Split-PE2-Rma-1087, and Split-PE2-Rma-1115 showed similar or higher single-base substitution efficiencies. Figure 14a Split-PE2-Rma-1115 (3.78%) had the highest editing efficiency, which was 1.35 times that of Split-PE2-Rma-1024 (control, 2.8%).

[0288] Choose endogenous HEK3 As a third editing site, pegRNAs were designed to achieve CTT insertion at specific sites. Figure 14b High-throughput sequencing results showed that at endogenous sites... HEK3 At this point, Split-PE2-Rma-1115 (9.14%) showed the highest editing efficiency, which was 1.08 times that of Split-PE2-Rma-1024 (control, 8.4%). Figure 14b ).

[0289] The data above indicate that when transfected into cells in plasmid form, combining the new cleavage site with Rma integrins can further improve the editing efficiency of Split-PE, thereby obtaining a more efficient Split-PE system.

[0290] Example 2: Delivery of Split-PE to endogenous sites in human cell lines via dual AAV vectors

[0291] Based on the results of transient plasmid transfection assays, Split-PE-Rma-1039, Split-PE-Rma-1087, and Split-PE-Rma-1115 were selected. They were packaged into AAV2 vectors. AAV2 is a commonly used AAV vector serotype capable of efficiently infecting HEK293T cells. First, each of the above plasmid vectors was co-packaged with helper plasmids into AAV2 vectors, and then concentrated and purified to obtain a viral titer of approximately 1 × 10^13 vg / mL. The two parts of the Split-PE combination were added to HEK293T cells at a 1:1 ratio for infection, with MOIs of 5 × 10^13 for each virus. 5 Cells were harvested 7 days after viral infection, and genomic DNA was extracted from the cells. Figure 6 High-throughput sequencing results showed that at endogenous sites... EMX1 The novel Split-PE constructs delivered via dual AAV delivery achieved single-base substitution with high efficiency. Compared to Split-PE-Rma-1024 (control, approximately 3.33%), Split-PE-Rma-959 (approximately 6.37%), Split-PE-Rma-1039 (approximately 7.27%), Split-PE-Rma-1087 (approximately 5.14%), and Split-PE-Rma-1115 (approximately 19.3%) all exhibited higher single-base substitution efficiencies. Figure 4 , Figure 7-13 The results are in good agreement with those obtained from transient plasmid transfection experiments. The single base substitution efficiency of Split-PE-Rma-1115 at the EMX1 site was approximately 19.3%, which is 5.8 times that of Split-PE-Rma-1024 (control, approximately 3.33%).

[0292] These results demonstrate that the novel Split-PE system exhibits higher editing efficiency under both transient plasmid transfection and double AAV infection conditions compared to the control group. Further testing of combinations of these splitting sites with introns in other versions of PE could lead to even higher in vivo gene editing efficiency.

[0293] Example 3: Editing endogenous sites in human cell lines via transient transfection with Split-PE plasmid

[0294] Seven additional ePE3 cleavage sites were designed: 212-213, 269-270, 309-310, 367-368, 459-460, 554-555, 940-941, and 1115-1116 (Figure 1). These seven cleavage sites were combined with the intipeptide Rma to obtain seven different Split-ePE3 constructs, namely Split-ePE3-Rma-212 (…). Figure 17 (SEQ ID NO:71 and 72), Split-ePE3-Rma-269 ( Figure 18 (SEQ ID NO: 73 and 74), Split-ePE3-Rma-309 ( Figure 19 (SEQ ID NO: 75 and 76), Split-ePE3-Rma-367 ( Figure 20 (SEQ ID NO: 77 and 78), Split-ePE3-Rma-459 ( Figure 21 (SEQ ID NO: 79 and 80), Split-ePE3-Rma-554 ( Figure 22 (SEQ ID NO: 81 and 82) and Split-ePE3-Rma-940 ( Figure 23 (SEQ ID NO: 83 and 84). The 1115-1116 splitting site is used in the ePE3 system, where the pegRNA is designed as an epegRNA, and a nick gRNA is contained in Split-ePE3-Rma-1115 ( Figure 30 (SEQ ID NO: 91 and 92).

[0295] The above eight Split-ePE3 constructs were transiently transfected into HEK293T cells using plasmids. Editing efficiency and the corresponding indel percentage were assessed. Endogenous genes were selected. EMX1 As one of the editing sites, pegRNAs are designed to achieve a G:C to T:A base substitution at a specific site. Figure 15 High-throughput sequencing results showed that at endogenous sites... EMX1 At this point, Split-ePE3-Rma-367 has the highest editing efficiency, at 25.56%.

[0296] Seven additional endogenous sites were selected to compare Split-ePE3-Rma-367 ( Figure 20 ) and Split-ePE3-Rma-1115 ( Figure 30 ) and three control constructs Split-ePE3-Npu-1024 ( Figure 27(SEQ ID NO: 85 and 86), Split-ePE3-Rma-1024 ( Figure 28 (SEQ ID NO: 87 and 88) and Split-ePE3-Rma-1105 ( Figure 29 The base substitution efficiency of (SEQ ID NO: 89 and 90). (The three constructs used as controls have been reported in Levy et al., 2020, Zhi et al., 2022, and She et al., 2023, respectively). The seven endogenous editing sites are APP (Designing epigRNA to achieve G:C to A:T base substitution) CETP (Designing epigRNA to achieve C:G to T:A base substitution) VEGFA (Designing epigRNA to achieve G:C to T:A base substitution) ALDOB (Designing epigRNA to achieve G:C to A:T base substitution) SLC30A8 (Designing epigRNA to achieve G:C to A:T base substitution) MECP2 (Designing epeRNAs to achieve C:G to T:A base substitution) and FANCF (Designing epeRNA to achieve G:C to T:A base substitution) Figure 25 At endogenous sites APP , CETP and MECP2 At the sites of endogenous VEGFA, ALDOB, SLC30A8, and FANCF, Split-ePE3-Rma-367, Split-ePE3-Rma-1115, and Split-ePE3-Npu-1024 exhibited similar editing efficiencies. At the endogenous VEGFA, ALDOB, SLC30A8, and FANCF sites, one of Split-ePE3-Rma-367 or Split-ePE3-Rma-1115 showed the highest editing efficiency compared to the three control constructs.

[0297] Four endogenous sites were selected to compare Split-ePE3-Rma-367 ( Figure 20 ) and Split-ePE3-Rma-1115 ( Figure 30 ) and three control constructs Split-ePE3-Npu-1024 ( Figure 27 Split-ePE3-Rma-1024 Figure 28 ) and Split-ePE3-Rma-1105 ( Figure 29 The base insertion efficiency of ). The four endogenous editing sites are RNF2 (Designing epigRNA to insert GTA at a specific site) HEK3(Designing epigRNA to insert CTT at a specific site) ANGPTL3 (Designing epigRNA to insert C at a specific site) and TTR (Designing epeRNA to insert A at a specific site) Figure 26 Split-ePE3-Rma-1115 exhibited the highest editing efficiency at three of the four endogenous sites: RNF2, HEK3, and TTR. (At endogenous sites...) ANGPTL3 In this regard, Split-ePE3-Npu-1024 has the highest editing efficiency, while Split-ePE3-Rma-1115 ranks second in editing efficiency.

[0298] The data above indicate that when transfected into cells in plasmid form, combining the new cleavage site with the Rma integument can further improve the editing efficiency of Split-ePE3, thereby obtaining a more efficient Split-ePE3 system.

[0299] Example 4: The method used in this example

[0300] Cell culture, transfection, and genomic DNA preparation

[0301] HEK293T (ATCC CRL-3216) cells were maintained in Dulbecco modified Eagle medium (DMEM) containing 1% GlutaMax (Thermo Fisher) supplemented with 10% fetal bovine serum (Gemini FBS) at 37°C and 5% CO2. Cells were cultured until confluence was less than 90% and then seeded onto 24-well cell culture plates (Thermo Fisher).

[0302] When transfecting the Split-PE plasmid, transfection was performed when the cell confluence reached 60%-70%. 1500 ng of each of the two Split-PE plasmids was used, and transfection was performed using 3 μL of Lipofectamine 3000 (Thermo Fisher). Cells were cultured for 3 days, and then genomic DNA was extracted using the TIANamp Genomic DNA Kit (TIANGEN).

[0303] When transducing Split-PE using AAV, approximately 60,000 HEK293T cells were seeded per well in a 24-well plate, with an MOI of 10. 6The cells were infected with AAV2 under the specified conditions. Cells were cultured for 7 days, and genomic DNA was extracted using the TIANamp Genomic DNA Kit (TIANGEN). AAV2 plasmids were purchased from General Biol, and AAV2 virus was purchased from PackGene Biotech.

[0304] Amplification for deep sequencing

[0305] Site-specific primers (Table 4) were used to generate target amplicones for deep sequencing. First, PCR amplification was performed using genomic DNA as a template with a Q5® High-Fidelity 2X Master Mix (New England Biolabs) in a 50 μL volume for 35 cycles. The PCR products were purified using AMPure XP magnetic beads (Beckman Coulter), followed by 10 cycles of amplification with primers containing sequencing adapters. The PCR products were purified again using AMPure XP magnetic beads (Beckman Coulter) and quantified using Nanodrop One (Thermo Fisher).

[0306] Deep sequencing data analysis

[0307] Deep sequencing data were processed using CRISPResso2 (https: / / github.com / pinellolab / CRISPResso2). PE editing efficiency was calculated as the percentage of correctly edited reads out of the total reads. Indel efficiency was calculated as the percentage of all mutated reads out of the total reads.

[0308] Statistical analysis

[0309] Statistical analyses were performed using GraphPad Prism. Data are presented as biological replicates and mean ± standard deviation (sd), as shown in the corresponding figures. Similarly, the legends for the corresponding figures detail the sample size and the statistical tests used. For all analyses, p < 0.05 was considered statistically significant.

[0310] Table 4

[0311] References

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Claims

1. A polynucleotide comprising a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises an N-terminal portion of a full-length Cas9 protein or a variant thereof that splits into two parts at a split site, wherein the split site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or a variant thereof, or between the first and second amino acids of SEQ ID NO: 11 of the full-length Cas9 protein or a variant thereof.

2. The polynucleotide of claim 1, further comprising a sequence encoding an N-terminal portion of an integrin, wherein the N-terminal portion of the integrin is linked to a C-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof.

3. The polynucleotide according to claim 2, wherein the in-cell peptide is selected from Mxe-GyrA, Npu, Ssp-DnaB, Ssp-GyrB and Rma.

4. The polynucleotide according to claim 3, wherein the integrin is Rma.

5. The polynucleotide according to any one of claims 1-4, further comprising one or more guide RNAs or sequences encoding therethe.

6. The polynucleotide of claim 5, wherein at least one of the guide RNAs comprises a reverse transcriptase template.

7. The polynucleotide according to any one of claims 1-6, further comprising an RNA polymerase III promoter.

8. The polynucleotide according to claim 7, wherein the RNA polymerase III promoter is the U6 promoter.

9. The polynucleotide according to any one of claims 1-8, further comprising a sequence encoding a protein having enzymatic activity.

10. The polynucleotide of claim 9, wherein the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof.

11. The polynucleotide of claim 10, wherein the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof.

12. The polynucleotide of claim 9, wherein the protein having enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

13. The polynucleotide according to any one of claims 1-12, further comprising a sequence encoding a 2A peptide (P2A).

14. The polynucleotide according to any one of claims 1-13, further comprising a sequence encoding a nuclear localization signal (NLS).

15. A polynucleotide comprising a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises a C-terminal portion of a full-length Cas9 protein or a variant thereof that splits into two parts at a split site, wherein the split site is located between the second and third amino acids of any one of SEQ ID NO: 1-10 of the full-length Cas9 protein or a variant thereof.

16. A polynucleotide comprising a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or variant thereof comprises a C-terminal portion of a full-length Cas9 protein or variant thereof that splits into two parts at a split site, wherein the split site is located between the third and fourth amino acids of SEQ ID NO: 11 in the full-length Cas9 protein or variant thereof; and wherein the sequence further encodes cysteine ​​and phenylalanine, or cysteine ​​and tryptophan, at the N-terminus of the truncated Cas9 protein or variant thereof.

17. The polynucleotide of claim 15 or 16, further comprising a sequence encoding a C-terminal portion of an inteptide, wherein the C-terminal portion of the inteptide is linked to an N-terminus of a sequence encoding the truncated Cas9 protein or a variant thereof.

18. The polynucleotide of claim 17, wherein the intended peptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB and Rma.

19. The polynucleotide of claim 18, wherein the integrin is Rma.

20. The polynucleotide according to any one of claims 15-19, further comprising one or more guide RNAs or sequences encoding therethe.

21. The polynucleotide of claim 20, wherein at least one of the guide RNAs comprises a reverse transcriptase template.

22. The polynucleotide according to any one of claims 15-21, further comprising an RNA polymerase III promoter.

23. The polynucleotide of claim 22, wherein the RNA polymerase III promoter is the U6 promoter.

24. The polynucleotide according to any one of claims 15-23, further comprising a sequence encoding a protein having enzymatic activity.

25. The polynucleotide of claim 24, wherein the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof.

26. The polynucleotide of claim 25, wherein the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof.

27. The polynucleotide of claim 24, wherein the protein having enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

28. The polynucleotide according to any one of claims 15-27, further comprising a sequence encoding a 2A peptide (P2A).

29. The polynucleotide according to any one of claims 15-28, further comprising a sequence encoding a nuclear localization signal (NLS).

30. A polynucleotide comprising a sequence encoding a truncated Cas9 protein or a variant thereof, wherein the truncated Cas9 protein or a variant thereof comprises a portion of the full-length Cas9 protein or a variant thereof located between a first cleavage site and a second cleavage site, wherein the first cleavage site is upstream of the second cleavage site, wherein at least one of the two cleavage sites is selected from the site between the second and third amino acids of any one of SEQ ID NO: 1-10 and 12, and the site between the first and second amino acids of SEQ ID NO: 11; wherein when the first cleavage site is located between the first and second amino acids of SEQ ID NO: 11, the first two amino acids at the N-terminus of the truncated Cas9 protein or a variant thereof are replaced by cysteine ​​and phenylalanine, or cysteine ​​and tryptophan.

31. The polynucleotide of claim 30, wherein both splitting sites are selected from the site between the second and third amino acids of any one of SEQ ID NO: 1-10 and 12, and the site between the first and second amino acids of SEQ ID NO: 11; wherein when the first splitting site is located between the first and second amino acids of SEQ ID NO: 11, the first two amino acids at the N-terminus of the truncated Cas9 protein or a variant thereof are replaced by cysteine ​​and phenylalanine, or cysteine ​​and tryptophan.

32. A vector comprising the polynucleotide of any one of claims 1-31.

33. The vector according to claim 32, wherein the vector is a viral vector.

34. The vector according to claim 33, wherein the viral vector is selected from adenovirus, lentivirus and adeno-associated virus (AAV).

35. The carrier according to claim 34, wherein the carrier is AAV.

36. A composition comprising the polynucleotide of any one of claims 1-31.

37. The composition of claim 36, wherein the composition comprises a first polynucleotide of any one of claims 1-14 and a second polynucleotide of any one of claims 15-29.

38. The composition of claim 37, wherein the first polynucleotide is any one of claims 2-14, and the second polynucleotide is any one of claims 17-29, wherein the N-terminal portion of the inteptide in the first polynucleotide and the C-terminal portion of the inteptide in the second polynucleotide form a full-length inteptide.

39. The composition according to any one of claims 36-38, wherein at least one of the polynucleotides comprises guide RNA or a sequence encoding it.

40. The composition according to any one of claims 36-39, wherein at least one of the polynucleotides comprises a sequence encoding a protein having enzymatic activity.

41. The composition according to claim 40, wherein the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof.

42. The composition according to claim 41, wherein the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof.

43. The composition according to claim 40, wherein the protein having enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

44. A composition comprising the carrier according to any one of claims 32-35.

45. The composition of claim 44, wherein the composition comprises a first carrier and a second carrier, the first carrier comprising a first polynucleotide of any one of claims 1-14, and the second carrier comprising a second polynucleotide of any one of claims 15-29.

46. ​​The composition of claim 45, wherein the first polynucleotide is any one of claims 2-14, and the second polynucleotide is any one of claims 17-29, wherein the N-terminal portion of the inteptide in the first polynucleotide and the C-terminal portion of the inteptide in the second polynucleotide form a full-length inteptide.

47. The composition according to any one of claims 44-46, wherein at least one of the polynucleotides comprises guide RNA or a sequence encoding thereon.

48. The composition according to any one of claims 44-47, wherein at least one of the polynucleotides comprises a sequence encoding a protein having enzymatic activity.

49. The composition according to claim 48, wherein the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof.

50. The composition of claim 49, wherein the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof.

51. The composition according to claim 48, wherein the protein having enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

52. The composition according to any one of claims 44-51, wherein both carriers are AAV carriers.

53. A cell comprising the polynucleotide of any one of claims 1-31.

54. The cell of claim 53, wherein the cell comprises a first polynucleotide of any one of claims 1-14 and a second polynucleotide of any one of claims 15-29.

55. The cell according to claim 54, wherein the first polynucleotide is any one of claims 2-14, and the second polynucleotide is any one of claims 17-29, wherein the N-terminal portion of the inteptide in the first polynucleotide and the C-terminal portion of the inteptide in the second polynucleotide form a full-length inteptide.

56. The cell according to any one of claims 53-55, wherein at least one of the polynucleotides comprises guide RNA or a sequence encoding it.

57. The cell according to any one of claims 53-56, wherein at least one of the polynucleotides comprises a sequence encoding a protein having enzymatic activity.

58. The cell of claim 57, wherein the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof.

59. The cell of claim 58, wherein the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof.

60. The cell of claim 57, wherein the protein having enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

61. A cell comprising the vector according to any one of claims 32-35.

62. The cell of claim 61, wherein the cell comprises a first carrier and a second carrier, the first carrier comprising a first polynucleotide of any one of claims 1-14, and the second carrier comprising a second polynucleotide of any one of claims 15-29.

63. The cell according to claim 62, wherein the first polynucleotide is any one of claims 2-14, and the second polynucleotide is any one of claims 17-29, wherein the N-terminal portion of the inteptide in the first polynucleotide and the C-terminal portion of the inteptide in the second polynucleotide form a full-length inteptide.

64. The cell according to any one of claims 61-63, wherein at least one of the polynucleotides comprises guide RNA or a sequence encoding thereon.

65. The cell according to any one of claims 61-64, wherein at least one of the polynucleotides comprises a sequence encoding a protein having enzymatic activity.

66. The cell of claim 65, wherein the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof.

67. The cell of claim 66, wherein the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof.

68. The cell according to claim 65, wherein the protein having enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

69. The cell according to any one of claims 61-68, wherein both vectors are AAV vectors.

70. A kit comprising the polynucleotide of any one of claims 1-31.

71. The kit according to claim 70, wherein the kit comprises a first polynucleotide according to any one of claims 1-14 and a second polynucleotide according to any one of claims 15-29.

72. The kit according to claim 71, wherein the first polynucleotide is any one of claims 2-14, and the second polynucleotide is any one of claims 17-29, wherein the N-terminal portion of the inteptide in the first polynucleotide and the C-terminal portion of the inteptide in the second polynucleotide form a full-length inteptide.

73. The kit according to any one of claims 70-72, wherein at least one of the polynucleotides comprises a guide RNA or a sequence encoding therethe.

74. The kit according to any one of claims 70-73, wherein at least one of the polynucleotides comprises a sequence encoding a protein having enzymatic activity.

75. The kit according to claim 74, wherein the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof.

76. The kit according to claim 75, wherein the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof.

77. The kit according to claim 74, wherein the protein having enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

78. A kit comprising the vector according to any one of claims 32-35.

79. The kit according to claim 78, wherein the kit comprises a first carrier and a second carrier, the first carrier comprising a first polynucleotide according to any one of claims 1-14, and the second carrier comprising a second polynucleotide according to any one of claims 15-29.

80. The kit according to claim 79, wherein the first polynucleotide is any one of claims 2-14, and the second polynucleotide is any one of claims 17-29, wherein the N-terminal portion of the inteptide in the first polynucleotide and the C-terminal portion of the inteptide in the second polynucleotide form a full-length inteptide.

81. The kit according to any one of claims 78-80, wherein at least one of the polynucleotides comprises guide RNA or a sequence encoding thereon.

82. The kit according to any one of claims 78-81, wherein at least one of the polynucleotides comprises a sequence encoding a protein having enzymatic activity.

83. The kit according to claim 82, wherein the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof.

84. The kit according to claim 83, wherein the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof.

85. The kit according to claim 82, wherein the protein having enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

86. The kit according to any one of claims 78-85, wherein both vectors are AAV vectors.

87. A method for altering a target nucleic acid in a cell, comprising delivering the following substance into the cell: a. The composition according to any one of claims 36-52; or b. The kit according to any one of claims 70-86.

88. A method for altering a target nucleic acid in a cell, comprising: a. Providing the cell with a first polynucleotide encoding a truncated Cas9 protein or a variant thereof, the truncated Cas9 protein or a variant thereof comprising the N-terminal portion of a Cas9 protein or a variant thereof that splits into two parts at a cleavage site; b. Provide the cell with a second polynucleotide encoding a truncated Cas9 protein or a variant thereof, the truncated Cas9 protein or variant thereof comprising the C-terminal portion of the Cas9 protein or variant thereof from step (a); c. Provide the cell with guide RNA, the guide RNA containing a spacer sequence complementary to the target nucleic acid. The cells described herein are capable of RNA transsplicing and / or inteptide-mediated protein transsplicing, and the cells express the complete Cas9 protein or a variant thereof; and The splitting site is located between the second and third amino acids of any of SEQ ID NO: 1-10 in the full-length Cas9 protein or its variants; or the splitting site is located between the first and second amino acids of SEQ ID NO: 11 in the full-length Cas9 protein or its variants, and the first two amino acids at the N-terminus of the second polynucleotide are replaced by cysteine ​​and phenylalanine, or cysteine ​​and tryptophan.

89. The method of claim 88, wherein the first polynucleotide further comprises a sequence encoding an N-terminal portion of an inteptide, the N-terminal portion of the inteptide being linked to the C-terminus of the sequence encoding the truncated Cas9 protein or a variant thereof in (a), and wherein the second polynucleotide further comprises a sequence encoding the remaining C-terminal portion of the inteptide, the C-terminal portion of the inteptide being linked to the N-terminus of the sequence encoding the truncated Cas9 protein or a variant thereof in (b).

90. The method of claim 89, wherein the contained peptide is selected from Mxe GyrA, Npu, Ssp-DnaB, Ssp-GyrB and Rma.

91. The method of claim 90, wherein the intima peptide is Rma.

92. The method according to any one of claims 88-91, further comprising providing the cells with a protein having enzymatic activity or a polynucleotide encoding therethe.

93. The method of claim 92, wherein at least one of the polynucleotides further comprises a sequence encoding the protein having enzymatic activity.

94. The method according to any one of claims 92-93, wherein the protein having enzymatic activity is a reverse transcriptase or a functional fragment thereof.

95. The method according to any one of claims 93-94, wherein the reverse transcriptase is Moloney mouse leukemia virus reverse transcriptase (MMLV-RT) or a variant thereof.

96. The method according to any one of claims 92-93, wherein the protein having enzymatic activity is cytidine deaminase or a functional fragment thereof, adenosine deaminase or a functional fragment thereof, or a methylase or a functional fragment thereof.

97. The method according to any one of claims 88-96, wherein at least one of the polynucleotides further comprises guide RNA or a sequence encoding thereus.

98. The method of claim 97, wherein the guide RNA comprises a reverse transcriptase template.

99. The method according to any one of claims 88-98, wherein the first polynucleotide is provided by a first carrier and the second polynucleotide is provided by a second carrier.

100. The method according to claim 99, wherein the first vector and the second vector are viral vectors.

101. The method of claim 100, wherein the viral vector is selected from adenovirus, lentivirus and adeno-associated virus (AAV).

102. The method according to claim 101, wherein the viral vector is AAV.

103. The polynucleotide according to any one of claims 1-31, wherein the Cas9 protein or a variant thereof is Cas9n-H840A, eSp-Cas9, HiFi-Cas9, Lz3-Cas9, SpCas9-NG, Sniper-Cas9, SuperFiCas9, HypaCas9, Cas9HF1, evoCas9, SpCas9, eSpCas9(1.1), xCas9, SpGCas9, SpRYCas9, Cas9-NG, Cas9-VRQR, Cas9-VRER, or dCas9.

104. The method according to any one of claims 88-102, wherein the Cas9 protein or a variant thereof is Cas9n-H840A, eSp-Cas9, HiFi-Cas9, Lz3-Cas9, SpCas9-NG, Sniper-Cas9, SuperFiCas9, HypaCas9, Cas9HF1, evoCas9, SpCas9, eSpCas9(1.1), xCas9, SpGCas9, SpRYCas9, Cas9-NG, Cas9-VRQR, Cas9-VRER, or dCas9.

105. The method according to any one of claims 87-102, wherein the method is performed in vivo.

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