WO2022206352A1 - 一种引导编辑工具、融合rna及其用途 - Google Patents

一种引导编辑工具、融合rna及其用途 Download PDF

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WO2022206352A1
WO2022206352A1 PCT/CN2022/080595 CN2022080595W WO2022206352A1 WO 2022206352 A1 WO2022206352 A1 WO 2022206352A1 CN 2022080595 W CN2022080595 W CN 2022080595W WO 2022206352 A1 WO2022206352 A1 WO 2022206352A1
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seq
cells
sequence
amino acid
fusion protein
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刘尧
仰光
李广磊
黄行许
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上海科技大学
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Definitions

  • the invention belongs to the field of biotechnology, and relates to a guide editing tool, fusion RNA and uses thereof.
  • the CRISPR/Cas9 system has been widely used in genetic manipulation [Cong, L, et.al, Science (New York, NY) 339:819-823; Shen, B, et.al, Cell Res 23:720-723. ], and won the 2020 Nobel Prize in Chemistry for its enormous influence.
  • the base editing technology (Base Editing, BE) based on the CRISPR/Cas9 system can perform single-base level manipulation of the genome [Gaudelli, NM, et.al., Nature 551:464-471; Komor, AC, et.al .,Nature 533:420-424.], compared with the traditional way of using the Homology Directed Repair (HDR) pathway after Cas9 cleavage, the efficiency is significantly improved, and it has been verified in plants, animals and human embryos.
  • HDR Homology Directed Repair
  • PE is essentially the extension of ssDNA for point mutation.
  • the basic principle is to form a fusion protein between Moroni mouse leukemia virus reverse transcriptase M-MLV and H840A mutant Cas9n, and to extend the 3' end of commonly used sgRNA.
  • the PE gRNA (pegRNA) is formed, and the extended sequence contains the binding primer (Primer Binding Site, PBS) required for reverse transcriptase and the template (Reverse Transcription template, RT template) required for repair.
  • PBS Primary Binding Site
  • RT template Reverse Transcription template
  • Reverse transcriptase reverse-transcribes PBS and RT to get the repaired DNA, so that this DNA can be used for site-directed mutagenesis, so that all types of mutations can be realized, and precise sequence insertion and deletion can be performed, which greatly expands the possibilities of gene editing. Scope [Anzalone, AV, et.al, Nature 576:149-157.].
  • the present invention provides a guide editing tool, fusion RNA and uses thereof.
  • pegRNA presents a phenomenon of head-to-tail base complementary pairing in the sequence (as shown in Figure 1), which may lead to the reduction of effectively expressed pegRNA, thereby affecting the active expression of PE.
  • Adding random sequences to the 3' end of pegRNA can reduce potential head-to-tail base pairing and improve the activity of PE.
  • it will not affect the generation of off-target, which ensures the safety of PE.
  • a guide editing tool which includes:
  • a fusion protein comprising at least one gene editor and an endonuclease
  • a fusion RNA comprising a pegRNA and a recognition site for the endonuclease described in (i);
  • the fusion protein has a reverse transcription function, and can be combined with the recognition site and cut the recognition site, thereby introducing a sequence at the 3' end of the pegRNA to avoid self-cyclization of the pegRNA .
  • the fusion RNA is sequentially pegRNA, Csy4 endonuclease recognition sequence and nick sgRNA from the 5' end to the 3' end; preferably, the Csy4 endonuclease recognition sequence is The nucleotide sequence is shown in SEQ ID NO:5.
  • the fusion protein includes, for example, Csy4 endonuclease, Cas9n and viral reverse transcriptase such as Moroni mouse leukemia from N-terminus to C-terminus. Viral reverse transcriptase M-MLV.
  • the fusion protein fuses the Csy4 endonuclease to the N-terminus of the guide editor, and performs guide editing at the target site under the guidance of the fusion RNA, which can effectively improve the editing efficiency of PE.
  • amino acid sequence of the Csy4 endonuclease is shown in SEQ ID NO: 1
  • amino acid sequence of the Cas9n is shown in SEQ ID NO: 2
  • amino acid sequence of the M-MLV is shown in shown in SEQ ID NO:3.
  • the amino acid sequence of the Csy4 endonuclease can include: the amino acid sequence shown in SEQ ID NO: 1; or the amino acid having more than 80% sequence similarity with SEQ ID NO: 1 sequence, and has the function of the amino acid sequence defined by SEQ ID NO: 1.
  • the amino acid sequence specifically refers to: the amino acid sequence shown in SEQ ID NO: 1 has undergone substitution, deletion or addition of one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or one or more (specifically can be added to the N-terminal and/or C-terminal) 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, and have amino acids such as SEQ
  • the polypeptide fragment with the function of the polypeptide fragment shown in ID NO: 1, for example, can be a Csy4 endonuclease that still has the targeting activity of the Csy4 endonuclease recognition sequence after being mutated, and more specifically can be a specific target.
  • the amino acid sequence can be more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% similar to SEQ ID NO:1.
  • the Csy4 endonuclease fragment is typically derived from Pseudomonas aeruginosa.
  • the amino acid sequence of the second Cas9n fragment can include: the amino acid sequence shown in SEQ ID NO: 2; or the amino acid sequence with more than 80% sequence similarity with SEQ ID NO: 2 , and has the function of a defined amino acid sequence.
  • the amino acid sequence in the description specifically refers to: the amino acid sequence shown in SEQ ID NO: 2 has undergone substitution, deletion or addition of one or more (specifically, 1-50, 1-30, 1-20) , 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or by adding one or more (specifically, can be added to the N-terminal and/or C-terminal) is derived from 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, and has amino acids such as
  • the polypeptide fragment with the function of the polypeptide fragment shown in SEQ ID NO: 2, for example, can still have the targeting activity of Cas9n after being mutated, and more specifically can be the activity of targeting RNA under the guidance of a suitable gRNA.
  • Said amino acid sequence can be more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% similar to SEQ ID NO:2.
  • the Cas9n fragment is typically derived from Streptococc
  • the amino acid sequence of the M-MLV fragment can include: the amino acid sequence shown in SEQ ID NO:3; or the amino acid sequence with more than 80% sequence similarity with SEQ ID NO:3 , and has the function of a defined amino acid sequence.
  • the amino acid sequence in the description specifically refers to: the amino acid sequence shown in SEQ ID NO: 3 has undergone substitution, deletion or addition of one or more (specifically, 1-50, 1-30, 1-20) , 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or by adding one or more (specifically, can be added to the N-terminal and/or C-terminal) is derived from 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, and has amino acids such as
  • the polypeptide fragment with the function of the polypeptide fragment shown in SEQ ID NO: 3, for example, can be a reverse transcription activity, more specifically, under the guidance of a primer, a single-stranded RNA (ssRNA) can be reverse transcribed as a template into Function of single-stranded DNA (ssDNA).
  • ssRNA single-stranded RNA
  • Said amino acid sequence in f) may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more similarity to SEQ ID NO:3.
  • the M-MLV fragments are generally derived from mice (Mus musculus).
  • the final fusion protein sequence is shown in SEQ ID NO:4.
  • the substitution, deletion or addition can be conservative amino acid substitution.
  • the "conservative amino acid substitution” may specifically refer to the situation in which an amino acid residue is substituted by other amino acid residues with similar side chains. Families of amino acid residues with similar side chains should be known to those skilled in the art, for example, may include, but are not limited to, basic side chains (eg, lysine, arginine, histidine), acidic Side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , non-polar side chains (e.g.
  • amino acid substitutions can include, but are not limited to, the specific situations listed in the following table.
  • the numbers in Table 1 represent the similarity between two amino acids. When the number is greater than or equal to 0, it is considered conservative.
  • Amino acid substitutions Table 2 is an exemplary conservative amino acid substitution scheme.
  • the fusion protein further includes a T2A fragment and/or a BPNLS fragment.
  • the T2A fragment is located between Csy4 endonuclease and Cas9n, and its amino acid sequence is as shown in SEQ ID NO: 6, and/or, the BPNLS fragment is located at the C end, and its amino acid sequence is as shown in SEQ ID NO:7 shown.
  • the recognition sequence of the Csy4 endonuclease included in the fusion RNA is the nucleotide sequence shown in SEQ ID NO:5, or the nucleotide sequence shown in SEQ ID NO:5.
  • the nucleotide sequence has more than 95% identity and maintains the function of being recognized by Csy4 endonuclease.
  • the DNA sequence of the Csy4 endonuclease recognition sequence fragment can include: the DNA sequence shown in SEQ ID NO:5; or the sequence similar to SEQ ID NO:5 with more than 95% sequence similarity A specific DNA sequence, and has the function of a defined DNA sequence.
  • the DNA sequence in the description specifically refers to: the DNA sequence shown in SEQ ID NO: 5 is obtained by substitution, deletion or addition of one or more (1, 2, or 3) DNAs, or A DNA fragment obtained by adding one or more (specifically, 1, 2, or 3) DNAs at the 5'-end and/or 3'-end, and having DNA as shown in SEQ ID NO: 5
  • the functional DNA fragment may have the activity of being recognized by Csy4 endonuclease, more specifically the function of being recognized by Csy4 endonuclease in the presence of Csy4 endonuclease and cleaving within the recognition sequence.
  • the DNA sequence in said can have more than 95% similarity with SEQ ID NO:5.
  • the substitution, deletion or addition can be RNA substitution.
  • the "RNA substitution” may specifically refer to the case of RNA mutation without affecting the recognition function of Csy4 endonuclease.
  • the amino acid sequence of the fusion protein is shown in SEQ ID NO:4, or is 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:4 and has the function of the fusion protein shown in the amino acid sequence of SEQ ID NO:4.
  • the second aspect of the present invention provides a fusion RNA, wherein, the fusion RNA is pegRNA, Csy4 endonuclease recognition sequence and nicking sgRNA from the 5' end to the 3' end in turn.
  • the Csy4 endonuclease recognition sequence included in the fusion RNA is the nucleotide sequence shown in SEQ ID NO:5, or has 95% identity with the nucleotide sequence shown in SEQ ID NO:5 and maintains the function of being recognized by Csy4 endonuclease.
  • the third aspect of the present invention provides a fusion protein, wherein the fusion protein sequentially includes Csy4 endonuclease, Cas9n and Moroni mouse leukemia virus reverse transcriptase from the N-terminus to the C-terminus. M-MLV.
  • the amino acid sequence of the fusion protein is shown in SEQ ID NO:4, or is 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:4 and has the function of the fusion protein shown in the amino acid sequence of SEQ ID NO:4.
  • the fourth aspect of the present invention provides an isolated nucleic acid, wherein the isolated nucleic acid comprises the first polynucleotide encoding the fusion protein according to the third aspect of the present invention; and/or, The second polynucleotide of the fusion RNA according to the second aspect of the invention is transcribed.
  • the fifth aspect of the present invention provides a recombinant expression vector, which comprises the isolated nucleic acid according to the fourth aspect of the present invention.
  • the sixth aspect of the present invention provides an expression system, which contains the recombinant expression vector as described in the fifth aspect of the present invention.
  • the expression system can be a host cell, and the host cell can express the fusion protein as described above, and the fusion protein can cooperate with the fusion RNA, so that the fusion protein can be positioned in the target region, and the guidance of the target region can be realized. edit.
  • the host cell of the expression system is selected from eukaryotic cells or prokaryotic cells, preferably selected from mouse cells, human cells, more preferably selected from mouse brain neuroma cells, human embryonic kidney
  • the cells, or human cervical cancer cells, human colon cancer cells, and human osteosarcoma cells are more preferably selected from N2a cells, HEK293T cells, Hela cells, HCT116 cells or U2OS cells.
  • the fusion RNA and the fusion protein can be expressed in the same host cell or in different host cells, and the host cells can be target cells.
  • the first polynucleotide and the second polynucleotide can be located in the same recombinant expression vector or in different recombinant expression vectors, such as pCMV, pCAG or Tet-On.
  • the seventh aspect of the present invention provides a guide editing tool according to the first aspect of the present invention, a fusion RNA according to the second aspect of the present invention, a fusion protein according to the third aspect of the present invention, and the present invention.
  • the eukaryotic organism may specifically be a metazoan, which may specifically include, but is not limited to, humans, mice, and the like.
  • the uses may specifically include, but are not limited to, point mutations, fragment insertions and deletions, etc.
  • These guided edits can be applied to edit splice acceptor/donor sites to regulate RNA splicing, and can also be used to perform models (e.g., disease models, construction of cell models, animal models, etc.) or the treatment of human diseases, etc.
  • the edited object may be an embryo, a cell, or the like.
  • the gene editing is in vitro gene editing.
  • the use involves substitution, insertion or deletion of bases.
  • the eighth aspect of the present invention provides a method for preparing the guide editing tool according to the first aspect of the present invention, characterized in that it includes the following steps: using the expression system described in the sixth aspect of the present invention , and the fusion protein and fusion RNA can be obtained respectively.
  • a ninth aspect of the present invention provides a method for guided editing, characterized in that the method comprises using the guided editing tool described in the first aspect of the present invention to perform gene editing.
  • Guide editing systems in the prior art include PE, pegRNA and nicking sgRNA.
  • Those skilled in the art can select suitable pegRNAs and nicking sgRNAs targeting specific sites according to the target editing region of the gene.
  • the sequence of the pegRNA can generally be at least partially complementary to the target region, so that it can cooperate with the PE and localize it to the target region, enabling guided editing within the target region, including all types of point mutations, such as C ⁇ G -to-A ⁇ T, G ⁇ C-to-C ⁇ G, A ⁇ T-to-C ⁇ G, T ⁇ A-to-A ⁇ T.
  • the efficiency of the guide editing system is not high; however, the use of the guide editing tool (ePE) provided by the first aspect of the present invention overcomes the above-mentioned defects.
  • the experimental methods, detection methods and preparation methods disclosed in the present invention all adopt the conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field. conventional technology. These techniques have been well described in the existing literature. For details, please refer to Sambrook et al.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol.304, Chromatin( P.M. Wassarman and A.P. Wolfffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (P.B. Becker, ed.) Humana Press, Totowa, 1999 et al.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the present invention provides a new guide editing tool (ePE), which by chimeric Csy4 endonuclease on Cas9n, compared with the editor of traditional PE, Csy4 endonuclease can be left after cutting the recognition sequence
  • ePE guide editing tool
  • a residual sequence prevents the head-to-tail base pairing of the pegRNA itself, which significantly improves the efficiency of PE editing without generating off-target effects, and has a good industrialization prospect (Figure 2).
  • Figure 1 shows that pegRNAs in a traditional form of guide editing system are formed end-to-end.
  • FIG. 2 is an improved guided editing system provided by the present invention.
  • Fig. 3 shows that the substitution efficiency of bases in HEK293 cells by the guide editing system provided by the present invention is significantly higher than that of the traditional form.
  • Figure 4 shows that there is no significant difference between the off-target rate of the guided editing system provided by the present invention and the traditional form.
  • Figure 5 shows that the base substitution efficiency of the guided editing system provided by the present invention in HeLa cells is significantly higher than that of the traditional form.
  • Figure 6 shows that the base substitution efficiency of the guide editing system provided by the present invention in mouse N2a cells is significantly higher than that of the traditional form.
  • the Csy4 endonuclease sequence (SEQ ID NO: 1) was synthesized by GenScript Biotechnology Co., Ltd., and PCR amplification was carried out using the high-fidelity enzyme kit (Vazyme, P501-d2) of Nanjing Novizan Biotechnology Co., Ltd. .
  • the forward primer is SEQ ID NO: 8: ATGGACCACTACCTCGACATTC
  • the reverse primer is SEQ ID NO: 9: GAACCAGGGAACGAAACCTCC;
  • the PCR amplification product was purified and recovered by the AxyPrep PCR Clean-up kit (Axygen, AP-PCR-500G), and was ready for use.
  • PCR amplification was performed using a high-fidelity enzyme kit (Vazyme, P501-d2) from Nanjing Novizan Biotechnology Co., Ltd.
  • the forward primer is SEQ ID NO: 10 (GTCAGATCCGCTAGAGATCC GCGGCCGCTAATACGACTCACTATAGGATGGACCACTACCTCGACATT), and the reverse primer is SEQ ID NO: 11 (GACGTCACCGCATGTTAACAGACTTCCTCTGCCCTCGAACCA GGGAACGAAACCTCCTT).
  • PCR amplification was performed using a high-fidelity enzyme kit (Vazyme, P501-d2) from Nanjing Novizan Biotechnology Co., Ltd.
  • the forward primer is SEQ ID NO: 12 (TGTTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCACCAAAGAAGAAGCGGAAAGTC)
  • the reverse primer is SEQ ID NO: 13 (TGCCGGCCCATCACTTTCAC).
  • the PCR amplification product was purified and recovered by the AxyPrep PCR Clean-up kit (Axygen, AP-PCR-500G), and was ready for use.
  • the pCMV-PE2 (Addgene #132775) plasmid was digested with NotI-HF (NEB, R3189S) and SacI-HF (NEB, R3156S) to obtain a linearized sgRNA vector.
  • NotI-HF NEB, R3189S
  • SacI-HF NEB, R3156S
  • ePE Enhanced Prime Editing
  • ePE fusion RNA used to detect the targeted editing efficiency of ePE (Enhanced Prime Editing) in eukaryotic cells was site1.
  • Subsequent detection of ePE fusion RNA at 13 endogenous gene loci in N2a cells was Dnmt1, Fgf21, Ifnar1, Trem2, Rnf2, Tyr, Fgf5, Mstn, Cftr, Hoxd13, SITE3, Ar, SITE4.
  • the sequence of the recognition site of endonuclease Csy4 is shown in SEQ ID NO:5.
  • the 20nt spacer primer of pegRNA was designed according to the target site sequence, ACCG was added to the 5' end of the upstream primer, GTTTC was added to the 3' end, and CCTTGAAAC was added to the 5' end of the downstream primer.
  • the PBS sequence and RT sequence of the pegRNA and the 20nt spacer sequence of the nicked sgRNA were designed according to the targeting site sequence.
  • the PBS sequence, RT sequence, Csy4 protein recognition sequence and spacer sequence of nicked sgRNA were synthesized on the same pair of oligonucleotide primers, GTGC was added to the 5' end of the upstream primer, and AAAC was added to the 5' end of the downstream primer. All primers were synthesized and dissolved in sterile water to 100 ⁇ M.
  • the annealed scaffold sequence needs to be phosphorylated.
  • the phosphorylation treatment system is shown in Table 11 below:
  • the primers Csy4peg-bone-F (GAGAGGGTCTCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATC, SEQ ID NO:16) and Csy4peg-bone-R (CTCTCGGTCTCACGGTGTTTCGTCCTTTCCAC, SEQ ID NO:17) were used to amplify A linearized vector fragment was obtained.
  • AxyPrep DNA gel recovery kit (Axygen, AP-GX-250G) was used for gel tapping to recover the linearized vector.
  • the linearized vector was digested with BsaI (NEB, R0535S) to obtain a fused RNA vector backbone with cohesive ends.
  • the enzyme digestion system is shown in Table 12 below:
  • the annealed product is ligated to the fusion RNA vector backbone vector to construct the target-specific fusion RNA.
  • the connection system is shown in Table 13 below:
  • the ligated products were then transformed, recovered for 30 min, plated on ammonia-resistant LB agar plates, and cultured at 37°C overnight. A single clone was selected for sequencing verification, and the correctly sequenced fusion RNA was obtained.
  • the guide editing tool (ePE) of the present invention includes the fusion protein constructed in Example 1 and the fusion RNA constructed in Example 2.
  • HEK293T cells (from ATCC) were thawed and cultured in 10 cm dishes (Corning, 430167) in DMEM (HyClone, SH30243.01) containing 10% by volume fetal bovine serum (HyClone, SV30087). The culture temperature was 37°C, and the carbon dioxide concentration was 5%. After passage, when the cell density was 80%, cells were plated into 24-well plates. 24-well plates were coated with a 1:10 dilution of polylysine solution (Sigma, P4707-50ML) before use.
  • the resuspended cells were sorted by FACS (Fluorescence activated Cell Sorting), and the cells with the top 5% of GFP fluorescence intensity were collected, and at least 10,000 cells were collected for each sample.
  • FACS Fluorescence activated Cell Sorting
  • the target site fragments of each genome were amplified by PCR using a high-fidelity enzyme kit (Vazyme, p501-d2) from Nanjing Novozymes Biotechnology Co., Ltd.
  • the PCR reaction system is shown in Table 14 below:
  • the PCR amplification products were purified and recovered by the AxyPrep PCR Clean-up kit (Axygen, AP-PCR-500G), followed by Sanger sequencing and high-throughput sequencing. 10 (2 ⁇ 150PE) were sequenced with a read depth of approximately 20 million per sample. The reads were mapped to the human reference genome (hg38) by STAR software (version 2.5.1), using annotations from GENCODE version v30. After deduplication, variants were identified by GATK HaplotypeCaller (version 4.1.2) and filtered with QD (quality by depth), all variants were validated and quantified by bam-readcount with parameters -q 20-b 30.
  • a given edit should be at least 10-fold, and these edits are required to have at least 99% of reads supporting the reference allele in wild-type samples.
  • the specific results are shown in Figure 3. It can be seen from Figure 3 that ePE can significantly improve the efficiency of guided editing compared with PE (** means p ⁇ 0.05; *** means p ⁇ 0.01).
  • 30,000 5% GFP-positive cells were collected and lysed.
  • the target site fragments of each genome were amplified by PCR using a high-fidelity enzyme kit (Vazyme, p501-d2) from Nanjing Novozymes Biotechnology Co., Ltd.
  • the PCR reaction system is shown in Table 16 below:
  • the PCR amplification products were purified and recovered by the AxyPrep PCR Clean-up kit (Axygen, AP-PCR-500G) for high-throughput sequencing.
  • AxyPrep PCR Clean-up kit Axygen, AP-PCR-500G
  • the corresponding results of the sequencing situation are shown in Figure 4, and the results show that ePE does not produce additional off-targets.
  • the amount of plasmid transfected per well was 900 ng for pCMV-Csy4-NMRT plasmid and 300 ng for fusion RNA plasmid.
  • the plasmids were mixed in 50 ⁇ l of Opti-MEM (Gibco, 11058021) medium. Taking pCMV-PE2 as the positive control group, 900ng pCMV-Csy4-NMRT, 300ng pegRNA plasmid, and 100ng nicking sgRNA were added to each well.
  • the resuspended cells are sorted by FACS (Fluorescence activated Cell Sorting). Since the GFP signal is on the pegRNA plasmid or fusion RNA plasmid, we directly sort all GFP positive cells and collect at least 10,000 cells per sample. cells.
  • FACS Fluorescence activated Cell Sorting
  • the cells collected above were directly split, and the target site fragment was amplified by PCR, and the PCR primer sequence was as SEQ ID NO: 11.
  • Each genome targeting site fragment was amplified by PCR with Novozan high-fidelity enzyme kit (Vazyme, p501-d2).
  • the PCR reaction system is shown in Table 18 below:
  • PCR amplification products were purified and recovered by AxyPrep PCR Clean-up kit (Axygen, AP-PCR-500G). PCR products with different barcodes were pooled together for deep sequencing on the Illumina Hiseq X Ten (2 ⁇ 150PE) platform at the Novogene Institute of Bioinformatics in Beijing, China. Adapter pairs with paired-end reads were removed using AdapterRemoval version 2.2.2, and paired-end read alignments of 11 bp or more were merged into a single consensus read. All processed reads were then mapped to target sequences using the BWA-MEM algorithm (BWA v0.7.16). For each locus, the mutation rate was calculated using the bam read counts with parameters -q 20-b 30.
  • Indels were calculated based on reads containing at least one inserted or deleted nucleotide in the protospacer. Indel frequencies were calculated as indel-containing reads/total mapped reads. The results of the sequencing situation are shown in Figure 5 and Figure 6 . The results showed that the target-guided editing efficiency of ePE at multiple endogenous sites in Hela cell line and N2a was significantly improved compared with PE (** indicates p ⁇ 0.05; *** indicates p ⁇ 0.01).
  • the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.
  • the above-mentioned embodiments merely illustrate the principles and effects of the present invention, but are not intended to limit the present invention.
  • anyone skilled in the art can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed in the present invention should still be covered by the claims of the present invention.

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Abstract

提供了一种引导编辑工具、融合RNA及其用途。其中所述引导编辑工具包括(i)一种融合蛋白，其包括至少一个基因编辑器与一种核酸内切酶；(ii)一种融合RNA，其包括一个pegRNA与(i)中所述核酸内切酶的识别位点；其中，所述融合蛋白具有反转录功能，且可与所述识别位点结合并对其进行剪切，从而在所述pegRNA的3'端引入序列，避免pegRNA自身环化。所述引导编辑工具可以高效地实现靶位点碱基的替换、插入与缺失等应用。

Description

一种引导编辑工具、融合RNA及其用途
本申请要求申请日为2021/4/2的中国专利申请2021103616886的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于生物技术领域，涉及一种引导编辑工具、融合RNA及其用途。
背景技术
CRISPR/Cas9系统已经被广泛应用于遗传学操作[Cong,L,et.al,Science(New York,NY)339:819-823；Shen,B,et.al,Cell Res 23:720-723.]，并因其巨大影响力获得2020年诺贝尔化学奖。基于CRISPR/Cas9系统的碱基编辑技术(Base Editing，BE)可以对基因组进行单个碱基水平的操作[Gaudelli,NM,et.al.,Nature 551:464-471；Komor,AC,et.al.,Nature 533:420-424.]，相比于传统方式利用Cas9切割后的同源重组修复(Homology Directed Repair,HDR)途径，效率明显提高，已经在植物、动物和人胚胎中验证均高效且精确编辑[Zeng,Y,et.al,Mol Ther 26:2631-2637；Li,J,et.al,Cell Res 29:174-176；Zong,Y,et.al,Nat Biotechnol 35:438-440.]，并在人胚胎中实现致病突变的修复[Zeng,Y,et.al,Mol Ther 26:2631-2637]，在小鼠疾病模型中进行了基因治疗，表现出强大的基因治疗前景[Koblan,LW,et.al,Nature 589:608-614.]。
然后，由于BE存在显著的DNA及RNA脱靶现象[Grunewald,J,et.al,Nature 37:1041-1048；Jin,S,et.al,Science(New York,NY)364:292-295.]，且BE只能针对C→T和A→G的点突变，因此应用存在着明显的限制，亟待更强大的基因编辑工具。2019年底报道的引导编辑技术(Prime Editing，PE)可以针对所有突变进行突变，包括所有的点突变类型及精确的插入和删除， 因此被寄予厚望，有望替代BE成为新一代点突变的工具[Anzalone,AV,et.al,Nature 576:149-157.]。
PE本质上是ssDNA进行点突变的延伸，其基本原理是将莫罗尼小鼠白血病病毒反转录酶M-MLV与H840A突变体Cas9n形成融合蛋白，并将常用的sgRNA的3’端进行延长形成PE gRNA(pegRNA)，延长的序列包含反转录酶所需的结合引物(Primer Binding Site，PBS)和供修复所需的模板(Reverse Transcription template，RT template)。反转录酶将PBS和RT进行反转录得到修复的DNA，从而利用这段DNA进行定点突变，从而可以实现所有类型的突变，并且可以进行序列的精确插入和删除，大大拓展了基因编辑的范围[Anzalone,AV,et.al,Nature 576:149-157.]。
引导编辑技术自2019年底被报道以来，已经应用于植物和动物上[Liu,Y,et.al,Cell Discov 6:27；Lin,Q,et.al,Nat Biotechnol 38:582-585.]，证明其载体编辑的可行性。但引导编辑的效率长期以来都较低，限制了其应用，因此对于引导编辑的优化和提高是目前研究的关键。
发明内容
为解决现有技术中引导编辑工具效率低的缺陷，本发明提供一种引导编辑工具、融合RNA及其用途。
发明人经过大量探索性研究，发现pegRNA在序列上呈现出首尾碱基互补配对的现象(如图1所示)，可能导致有效表达的pegRNA减少，从而影响PE的活性表达。对pegRNA的3’端加上随机序列可以减少潜在的首尾碱基互补配对，提高PE的活性。并且在提高PE编辑效率的情况下不会影响脱靶的产生，保证了PE的安全性。
为解决上述技术问题，本发明第一方面提供一种引导编辑工具，其包括：
(i)一种融合蛋白，其包括至少一个基因编辑器与一种核酸内切酶；
(ii)一种融合RNA，其包括一个pegRNA与(i)中所述核酸内切酶的 识别位点；
其中，所述融合蛋白具有反转录功能，且可与所述识别位点结合并对所述识别位点进行剪切，从而在所述pegRNA的3’端引入序列，避免pegRNA发生自身环化。
在一较佳的具体实施例中，所述融合RNA自5’端至3’端依次为pegRNA、Csy4核酸内切酶识别序列和切口sgRNA；优选地，所述Csy4核酸内切酶识别序列的核苷酸序列如SEQ ID NO:5所示。
在一较佳的具体实施例中，所述融合蛋白自N端至C端包括例如自N端至C端依次包括Csy4核酸内切酶、Cas9n和病毒反转录酶例如莫罗尼小鼠白血病病毒反转录酶M-MLV。所述融合蛋白将Csy4核酸内切酶融合到引导编辑器的N端，在融合RNA的引导下在靶向位点做引导编辑，能够有效提高PE的编辑效率。
优选地，所述Csy4核酸内切酶的氨基酸序列如SEQ ID NO:1所示，所述Cas9n的氨基酸序列如SEQ ID NO:2所示，和/或，所述M-MLV的氨基酸序列如SEQ ID NO:3所示。
本发明所提供的融合蛋白中，所述Csy4核酸内切酶的氨基酸序列可以包括：如SEQ ID NO:1所示的氨基酸序列；或与SEQ ID NO:1具有80％以上序列相似性的氨基酸序列、且具有SEQ ID NO:1所限定的氨基酸序列的功能。具体的，所述的氨基酸序列具体指：如SEQ ID NO:1所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，且具有氨基酸如SEQ ID NO:1所示的多肽片段的功能的多肽片段，例如，可以是Csy4核酸内切酶经过突变后依然具有Csy4核酸内切酶识别序列的靶向活性，更具体可以是能够在特殊的靶向序列的引导下靶向RNA，形成截断的 独立的两部分RNA的活性。所述的氨基酸序列可与SEQ ID NO:1具有80％、85％、90％、93％、95％、97％、或99％以上的相似性。所述Csy4核酸内切酶片段通常源自铜绿假单胞菌(Pseudomonas aeruginosa)。
本发明所提供的融合蛋白中，所述第二Cas9n片段的氨基酸序列可以包括：如SEQ ID NO:2所示的氨基酸序列；或与SEQ ID NO:2具有80％以上序列相似性的氨基酸序列、且具有所限定的氨基酸序列的功能。具体的，所述中的氨基酸序列具体指：如SEQ ID NO:2所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，且具有氨基酸如SEQ ID NO:2所示的多肽片段的功能的多肽片段，例如，可以是经过突变后依然具有Cas9n的靶向活性，更具体可以是能够在合适的gRNA的引导下靶向RNA的活性。所述中的氨基酸序列可与SEQ ID NO:2具有80％、85％、90％、93％、95％、97％、或99％以上的相似性。所述Cas9n片段通常源自化脓性链球菌(Streptococcus pyogenes)。
本发明所提供的融合蛋白中，所述M-MLV片段的氨基酸序列可以包括：如SEQ ID NO:3所示的氨基酸序列；或与SEQ ID NO:3具有80％以上序列相似性的氨基酸序列、且具有所限定的氨基酸序列的功能。具体的，所述中的氨基酸序列具体指：如SEQ ID NO:3所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，且具有氨基酸如SEQ ID NO:3所示的多肽片段的功能的多肽片段，例如，可以是具有反转录的活性，更具体是可以在引物的引导下，将单链RNA(ssRNA)作为模板反转录成为单 链DNA(ssDNA)的功能。所述f)中的氨基酸序列可与SEQ ID NO:3具有80％、85％、90％、93％、95％、97％、或99％以上的相似性。所述M-MLV片段通常源自小鼠(Mus musculus)。最终的融合蛋白序列如SEQ ID NO:4所示。
本发明所提供的融合蛋白中，所述的取代、缺失或者添加可以是保守氨基酸取代。所述“保守氨基酸取代”具体可以是指氨基酸残基被其他具有相似侧链的氨基酸残基取代的情况。具有相似侧链的氨基酸残基家族对于本领域技术人员来说应该是已知的，例如，可以是包括但不限于碱性侧链(例如赖氨酸，精氨酸，组氨酸)，酸性侧链(例如天冬氨酸，谷氨酸)，不带电荷的极性侧链(例如，甘氨酸，天冬酰胺，谷氨酰胺，丝氨酸，苏氨酸，酪氨酸，半胱氨酸)，非极性侧链(例如丙氨酸，缬氨酸，亮氨酸，异亮氨酸，脯氨酸，苯丙氨酸，甲硫氨酸，色氨酸)异亮氨酸)和芳族侧链(例如酪氨酸，苯丙氨酸，色氨酸，组氨酸)等家族。保守型氨基酸取代更具体可以包括但不限于下表中所列的具体情况，表1(氨基酸相似度矩阵)中的数字表示两个氨基酸之间的相似度，当数字大于等于0时认为是保守氨基酸取代，表2为示例性的保守氨基酸取代的方案。
表1
  C G P S A T D E N Q H K R V M I L F Y W
W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17
Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10  
F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9    
L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6      
I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5        
M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6          
V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4            
R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6              
K -5 -2 -1 0 -1 0 0 0 1 1 0 5                
H -3 -2 0 -1 -1 -1 1 1 2 3 6                  
Q -5 -1 0 -1 0 -1 2 2 1 4                    
N -4 0 -1 1 0 0 2 1 2                      
E -5 0 -1 0 0 0 3 4                        
D -5 1 -1 0 0 0 4                          
T -2 0 0 1 1 3                            
A -2 1 1 1 2                              
S 0 1 1 1                                
P -3 -1 6                                  
G -3 5                                    
C 12                                      
表2
氨基酸残基 保守替代品
Alanine D-Ala,Gly,Aib,β-Ala,L-Cys,D-Cys
Arginine D-Arg,Lys,D-Lys,Orn D-Orn
Asparagine D-Asn,Asp,D-Asp,Glu,D-Glu Gln,D-Gln
Aspartic Acid D-Asp,D-Asn,Asn,Glu,D-Glu,Gln,D-Gln
Cysteine D-Cys,S-Me-Cys,Met,D-Met,Thr,D-Thr,L-Ser,D-Ser
Glutamine D-Gln,Asn,D-Asn,Glu,D-Glu,Asp,D-Asp
Glutamic Acid D-Glu,D-Asp,Asp,Asn,D-Asn,Gln,D-Gln
Glycine Ala,D-Ala,Pro,D-Pro,Aib,β-Ala
Isoleucine D-Ile,Val,D-Val,Leu,D-Leu,Met,D-Met
Leucine Val,D-Val,Met,D-Met,D-Ile,D-Leu,Ile
Lysine D-Lys,Arg,D-Arg,Orn,D-Orn
Methionine D-Met,S-Me-Cys,Ile,D-Ile,Leu,D-Leu,Val,D-Val
Phenylalanine D-Phe,Tyr,D-Tyr,His,D-His,Trp,D-Trp
Proline D-Pro
Serine D-Ser,Thr,D-Thr,allo-Thr,L-Cys,D-Cys
Threonine D-Thr,Ser,D-Ser,allo-Thr,Met,D-Met,Val,D-Val
Tyrosine D-Tyr,Phe,D-Phe,His,D-His,Trp,D-Trp
Valine D-Val,Leu,D-Leu,Ile,D-Ile,Met,D-Met
更优选地，所述融合蛋白还包括T2A片段和/或BPNLS片段。
进一步更优选地，所述T2A片段位于Csy4核酸内切酶与Cas9n之间，其氨基酸序列如SEQ ID NO:6所示，和/或，所述BPNLS片段位于C端，其氨基酸序列如SEQ ID NO:7所示。
在一更佳的具体实施例中，所述的融合RNA包含的Csy4核酸内切酶的识别序列为如SEQ ID NO:5所示的核苷酸序列，或与SEQ ID NO:5所示的核苷酸序列具有95％以上同一性、且维持被Csy4核酸内切酶识别的功能。
本发明所提供的融合RNA中，所述Csy4核酸内切酶识别序列片段的DNA序列可以包括：如SEQ ID NO:5所示的DNA序列；或与SEQ ID NO:5具有95％以上序列相似性的DNA序列、且具有所限定的DNA序列的功能。具体的，所述中的DNA序列具体指：如SEQ ID NO:5所示的DNA序列经过取代、缺失或者添加一个或多个(1个、2个、或3个)DNA而得到的，或者在5’-末端和/或3’-末端添加一个或多个(具体可以是1个、2个、或3个)DNA而得到的，且具有DNA如SEQ ID NO:5所示的DNA片段的功能的DNA片段，例如，可以是具有被Csy4核酸内切酶识别的活性，更具体是在Csy4核酸内切酶存在的情况下被其识别，并在将识别序列内部进行切割的功能。所述中的DNA序列可与SEQ ID NO:5具有95％以上的相似性。
本发明所提供的融合RNA中，所述的取代、缺失或者添加可以是RNA取代。所述“RNA取代”具体可以是指不影响Csy4核酸内切酶识别功能的情况下的RNA突变的情况。
优选地，所述融合蛋白的氨基酸序列如SEQ ID NO:4所示，或与SEQ ID NO:4的氨基酸序列具有90％、95％、96％、97％、98％、99％或以上同一 性、且具有如SEQ ID NO:4的氨基酸序列所示的融合蛋白的功能。
为解决上述技术问题，本发明第二方面提供一种融合RNA，其中，所述融合RNA自5’端至3’端依次为pegRNA、Csy4核酸内切酶识别序列和nicking sgRNA。
优选地，所述的融合RNA包含的Csy4核酸内切酶识别序列为如SEQ ID NO:5所示的核苷酸序列，或与SEQ ID NO:5所示的核苷酸序列具有95％同一性、且维持被Csy4核酸内切酶识别的功能。
为解决上述技术问题，本发明第三方面提供一种融合蛋白，其中，所述融合蛋白自N端至C端依次包括Csy4核酸内切酶、Cas9n和莫罗尼小鼠白血病病毒反转录酶M-MLV。
优选地，所述融合蛋白的氨基酸序列如SEQ ID NO:4所示，或与SEQ ID NO:4的氨基酸序列具有90％、95％、96％、97％、98％、99％或以上同一性、且具有如SEQ ID NO:4的氨基酸序列所示的融合蛋白的功能。
为解决上述技术问题，本发明第四方面提供一种分离的核酸，其中，所述分离的核酸包括编码如本发明第三方面所述的融合蛋白的第一多核苷酸；和/或，转录如本发明第二方面所述的融合RNA的第二多核苷酸。
为解决上述技术问题，本发明第五方面提供一种重组表达载体，其包括如本发明第四方面所述的分离的核酸。
为解决上述技术问题，本发明第六方面提供一种表达系统，其含有如本发明第五方面所述的重组表达载体.
所述表达系统可以是宿主细胞，所述宿主细胞可以表达如上所述的融合蛋白，所述融合蛋白可以与融合RNA相配合，从而可以将所述融合蛋白定位到目标区域，实现目标区域的引导编辑。在本发明另一具体实施例中，所述表达系统的宿主细胞选自真核细胞或原核细胞，优选选自小鼠细胞、人细胞，更优选选自小鼠脑神经瘤细胞、人胚胎肾细胞、或人宫颈癌细胞、人结 肠癌细胞、人骨肉瘤细胞，进一步更优选选自N2a细胞、HEK293T细胞、Hela细胞、HCT116细胞或U2OS细胞。所述融合RNA与所述融合蛋白可以在同一宿主细胞中表达，也可以在不同宿主细胞中表达，所述宿主细胞可以为靶细胞。
较佳地，所述表达系统中，所述第一多核苷酸与所述第二多核苷酸可位于同一重组表达载体或不同的重组表达载体中，所述重组表达载体例如为pCMV、pCAG或Tet-On。
为解决上述技术问题，本发明第七方面提供一种如本发明第一方面所述的引导编辑工具、本发明第二方面所述的融合RNA、本发明第三方面所述的融合蛋白、本发明第四方面所述的分离的核酸或如本发明第五方面所述的表达系统在真核生物基因编辑中的用途。
所述真核生物具体可以是后生动物，具体可以是包括但不限于人、小鼠等。所述用途具体可以是包括但不限于点突变、片段插入和缺失等，这些引导编辑可以应用于编辑剪接受体/供体位点来调节RNA剪接，也可以用于进行模型(例如，疾病模型、细胞模型、动物模型等)的构建或人类疾病的治疗等。在本发明一具体实施例中，被编辑的对象可以是胚胎、细胞等。在本发明另一具体实施例中，所述基因编辑为体外基因编辑。
优选地，所述用途包括碱基的替换、插入或者缺失。
为解决上述技术问题，本发明第八方面提供一种制备如本发明第一方面所述的引导编辑工具的方法，其特征在于，其包括以下步骤：利用本发明第六方面所述的表达系统，分别获得所述的融合蛋白和融合RNA即可。
为解决上述技术问题，本发明第九方面提供一种引导编辑的方法，其特征在于，所述方法包括利用如本发明第一方面所述的引导编辑工具进行基因编辑。
现有技术中的引导编辑体系，包括PE、pegRNA和nicking sgRNA。本 领域技术人员可以根据基因的目标编辑区域，选择合适的靶向特异性位点的pegRNA和nicking sgRNA。例如，所述pegRNA的序列通常可以与目标区域至少部分互补，从而可以与所述PE相配合并将其定位到目标区域，实现靶点区域内的引导编辑，包括所有类型点突变，例如C·G-to-A·T,G·C-to-C·G,A·T-to-C·G,T·A-to-A·T。但是该引导编辑体系效率不高；而使用本发明第一方面提供的引导编辑工具(ePE)克服了上述缺陷。
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中 已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring Harbor Laboratory Press，1989 and Third edition，2001；Ausubel等，CURRENT PROTOCOLS IN MOLECULAR BIOLOGY，John Wiley&Sons，New York，1987 and periodic updates；the series METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATIN STRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS IN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，Academic Press，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，Chromatin Protocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明提供了一种新的引导编辑工具(ePE)，其通过在Cas9n上嵌合Csy4核酸内切酶，相较于传统PE的编辑器，Csy4核酸内切酶可以在切割识别序列后留下一段残留的序列，防止pegRNA自身的首尾碱基互补配对，显著提高了PE编辑的效率，同时不会产生脱靶效应，具有良好的产业化前景(图2)。
附图说明
图1为传统形式的引导编辑系统中的pegRNA会形成首尾相接的形式。
图2为本发明所提供的改良型引导编辑系统。
图3为本发明提供的引导编辑系统在HEK293细胞中对碱基的替换效率显著高于传统形式。
图4为本发明提供的引导编辑系统的脱靶率和传统形式无显著差别。
图5为本发明提供的引导编辑系统在HeLa细胞中的碱基替换效率显著高于传统形式。
图6为本发明提供的引导编辑系统在小鼠的N2a细胞中的碱基替换效率显著高于传统形式。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1编辑工具中融合蛋白的构建
1、基于Csy4核酸内切酶的引导编辑工具的构建
由金斯瑞生物科技股份有限公司合成Csy4核酸内切酶序列(SEQ ID NO:1)，使用南京诺唯赞生物科技有限公司的高保真酶试剂盒(Vazyme,P501-d2)进行PCR扩增。正向引物为SEQ ID NO:8：ATGGACCACTACCTCGACATTC，反向引物为SEQ ID NO:9：GAACCAGGGAACGAAACCTCC；
扩增体系如下表3所示：
表3
水 补水至50μL
2xbuffer 25μL
dNTP 1μL
正向引物(10μM) 2μL
反向引物(10μM) 2μL
合成Csy4核酸内切酶模板 1ng
高保真酶 1μL
PCR条件如下表4所示：
表4
Figure PCTCN2022080595-appb-000001
PCR扩增产物经通过AxyPrep PCR Clean-up试剂盒(Axygen,AP-PCR-500G)纯化回收，待用。
2、包含Csy4核酸内切酶的新一代引导编辑工具pCMV-Csy4-NMRT的构建
对步骤1得到的Csy4产物进行载体构建。使用南京诺唯赞生物科技有限公司的高保真酶试剂盒(Vazyme,P501-d2)进行PCR扩增。正向引物为SEQ ID NO:10(GTCAGATCCGCTAGAGATCC GCGGCCGCTAATACGACTCACTATAGGATGGACCACTACCTCGACATT)，反向引物为SEQ ID NO:11(GACGTCACCGCATGTTAACAGACTTCCTCTGCCCTCGAACCA GGGAACGAAACCTCCTT)。
对PE2载体进行扩增。使用南京诺唯赞生物科技有限公司的高保真酶试剂盒(Vazyme,P501-d2)进行PCR扩增。正向引物为SEQ ID NO:12(TGTTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCACCAAAGAAGAAGCGGAAAGTC),反向引物为SEQ ID NO:13(TGCCGGCCCATCACTTTCAC)。
扩增体系如下表5所示：
表5
水 补水至50μL
2xbuffer 25μL
dNTP 1μL
正向引物(10μM) 2μL
反向引物(10μM) 2μL
合成Csy4核酸内切酶模板或PE2载体 1ng
高保真酶 1μL
PCR条件如下表6所示：
表6
Figure PCTCN2022080595-appb-000002
PCR扩增产物经通过AxyPrep PCR Clean-up试剂盒(Axygen,AP-PCR-500G)纯化回收，待用。
利用NotI-HF(NEB,R3189S)和SacI-HF(NEB,R3156S)对pCMV-PE2(Addgene#132775)质粒进行酶切以得到线性化sgRNA载体。酶切体系如下表7所示：
表7
水 补水至50μL
pCMV-PE2 5μg
10×cutsmart buffer 5μL
NotI-HF酶 3μL
SacI-HF 3μL
以上反应体系配置好后，置于37℃条件下反应5h，酶切产物用AxyPrep DNA凝胶回收试剂盒(Axygen,AP-GX-250G)做割胶回收得到线性化载体。取100ng线性化载体与PCR产物片段通过南京诺唯赞生物科技有限公司的 重组酶试剂盒(Vazyme,C112)进行重组，37℃孵育30分钟并转化涂板，经Sanger测序得到正确的pCMV-Csy4-NMRT载体。连接体系如下表8所示：
表8
水 补水至20μL
5xbuffer 2μL
片段1 150ng
片段2 150ng
线性化的pCMV-PE2 100ng
重组酶 1μL
实施例2编辑工具中融合RNA的构建
检测ePE(Enhanced Prime Editing)在真核细胞中靶向编辑效率所用融合RNA为site1。后续检测ePE在HEK293T细胞的6个内源基因位点融合RNA为site1、FBN1、RIT1、RNF2、ALDOB、MSH2。后续检测ePE在N2a细胞的13个内源基因位点融合RNA为Dnmt1、Fgf21、Ifnar1、Trem2、Rnf2、Tyr、Fgf5、Mstn、Cftr、Hoxd13、SITE3、Ar、SITE4。核酸内切酶Csy4识别位点的序列如SEQ ID NO:5所示。根据靶向位点序列设计pegRNA的20nt的spacer引物，上游引物的5’端添加ACCG，3’端添加GTTTC,下游引物的5’端添加CTCTGAAAC。根据靶向位点序列设计pegRNA的PBS序列和RT序列及切口sgRNA的20nt的spacer序列。将PBS序列、RT序列、Csy4蛋白识别序列和切口sgRNA的spacer序列合成在同一对寡核苷酸引物上，上游引物添加5’端添加GTGC，下游引物的5’端添加AAAC。合成所有引物，加灭菌水溶解至100μM。合成scaffold的寡核苷酸引物scaffold-F:
Figure PCTCN2022080595-appb-000003
Figure PCTCN2022080595-appb-000004
将上述合成的引物退火，退火体系如下表9所示：
表9
正向引物 4.5μL
反向引物 4.5μL
10×NEB buffer2 1μL
退火程序如下表10所示：
表10
95℃ 5min
95-85℃ -2℃/s
85-25℃ -0.1℃/s
4℃ ∞
退火后的scaffold序列需进行磷酸化处理。磷酸化处理体系如下表11所示：
表11
水 补水至25μL
scaffold退火产物 6.25μL
10×T4 DNA ligase buffer(NEB) 2.50μL
T4PNK(NEB) 0.50μL
以pGL3-U6-sgRNA-EGFP(Addgene#107721)质粒为模板，使用引物Csy4peg-bone-F(GAGAGGGTCTCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATC，SEQ ID NO:16)和Csy4peg-bone-R(CTCTCGGTCTCACGGTGTTTCGTCCTTTCCAC，SEQ ID NO:17)扩增得到线性化载体片段。用AxyPrep DNA凝胶回收试剂盒(Axygen,AP-GX-250G)做割胶回收得到线性化载体。利用BsaI(NEB,R0535S)对线性化载体进行酶切以得到带有粘性末端的融合RNA载体骨架。酶切体系如下表12所示：
表12
水 补水至30μL
线性化载体 2μg
10×cutsmart buffer 3μL
BsaI酶 1μL
将退火产物连接到融合RNA载体骨架载体上，以构建靶向特异性融合RNA。连接体系如下表13所示：
表13
水 补水至10μL
融合RNA载体骨架 30ng
退火产物1 1μL
退火产物2 1μL
磷酸化scaffold 1μL
Solution I 5μL
连接产物随后进行转化，复苏30min，涂板于氨卞抗性的LB琼脂平板，37℃培养过夜。挑选单克隆进行测序验证，得到测序正确的融合RNA。
实施例3引导编辑工具在真核细胞中的应用
本发明所述引导编辑工具(ePE)包括实施例1构建的融合蛋白以及实施例2构建的融合RNA。
1、在人HEK293T细胞中靶向编辑
在原核细胞中筛选得到功能性的ePE后，我们进一步检测了ePE在HEK293T细胞中检测靶向引导编辑效率，过程如下：
复苏HEK293T细胞(来自ATCC)，并培养在10cm培养皿(Corning,430167)中，培养基为含有10％体积比的胎牛血清(HyClone,SV30087)的DMEM(HyClone,SH30243.01)。培养温度为37℃，二氧化碳浓度为5％。传 代后当细胞密度为80％时，细胞分盘至24孔板。24孔板使用前用1:10稀释的多聚赖氨酸溶液(Sigma，P4707-50ML)包被处理。
1)接种细胞12-14h后，细胞浓度约为80％时，进行转染。每孔转染的质粒的量是pCMV-Csy4-NMRT质粒900ng，将质粒混在50μL的Opti-MEM(Gibco,11058021)培养基中。以pCMV-PE2作为阳性对照组，每孔加900ng pCMV-PE2。
2)另外，将3μL的Lipofectamine 2000转染试剂(Thermo,11668019)混入50μL的Opti-MEM培养基，静置5分钟。
3)将混有质粒的Opti-MEM加入混有Lipofectamine 2000的Opti-MEM，慢速吹打混匀，静置20分钟。
4)将上述混和静置好的转染液分别加入培养的细胞中。
5)转染6小时后用含有10％FBS的DMEM换液。
6)转染48小时后，去培基，用PBS清洗一次细胞，然后用TE(Thermo Fisher，R001100)将细胞消化下来，再用含有10％FBS的DMEM终止消化，并离心收集细胞，最后用培养基重悬。
7)重悬后的细胞进行FACS(Fluorescence activated Cell Sorting)分选，收集GFP荧光强度前5％的细胞，每个样品至少收集10,000个细胞。
取上述收集的细胞的1/6进行直接裂解，并PCR扩增靶向位点片段，PCR引物序列如SEQ ID NO:10所示。用南京诺唯赞生物科技有限公司高保真酶试剂盒(Vazyme,p501-d2)PCR扩增各基因组靶向位点片段。PCR反应体系如下表14所示：
表14
水 补加至50μL
2×buffer 25μL
dNTP 1μL
正向引物(10μM) 2μL
反向引物(10μM) 2μL
高保真酶 1μL
细胞裂解液 3-5μL
PCR程序如下表15所示：
表15
Figure PCTCN2022080595-appb-000005
PCR扩增产物经AxyPrep PCR Clean-up试剂盒(Axygen,AP-PCR-500G)纯化回收，进行Sanger测序和高通量测序，样品均使用位于中国北京的Novogene生物信息学研究所的Illumina HiSeq X 10(2×150PE)进行测序，每个样品的读取深度约为2000万。通过STAR软件(版本2.5.1)将读段映射到人参考基因组(hg38)，使用来自GENCODE v30版的注释。删除重复后，通过GATK HaplotypeCaller(版本4.1.2)识别变体，并用QD(质量按深度)过滤，所有变体均通过bam-readcount进行验证并量化，参数为-q 20-b 30。给定的编辑至少应为10倍，并且要求这些编辑至少要有99％的读数支持野生型样品中的参考等位基因。具体结果如图3所示。由图3可知，ePE相比于PE可以显著提高引导编辑的效率(**表示p<0.05；***表示p<0.01)。
2、比较PE和ePE在人细胞中的脱靶情况
收集上述5％GFP阳性细胞30,000个，进行裂解。用南京诺唯赞生物科技有限公司高保真酶试剂盒(Vazyme,p501-d2)PCR扩增各基因组靶向位点片段。PCR反应体系如下表16所示：
表16
水 补加至50μL
2×buffer 25μL
dNTP 1μL
正向引物(10μM) 2μL
反向引物(10μM) 2μL
高保真酶 1μL
细胞裂解液 3-5μL
PCR程序如下表17所示：
表17
Figure PCTCN2022080595-appb-000006
PCR扩增产物经AxyPrep PCR Clean-up试剂盒(Axygen,AP-PCR-500G)纯化回收，进行高通量测序。测序情况对应结果如图4所示，结果表明ePE不会产生额外的脱靶。
3、ePE在更多细胞系的引导编辑结果
在上述实验中已经发现ePE针对引导编辑的效率相比于PE更高，并且不影响脱靶的产生。为了进一步阐述ePE对引导编辑效率的提高，我们在人源的Hela细胞系和鼠源的N2a上也对ePE的提高作了进一步尝试，过程如下：
1)复苏Hela细胞和N2a细胞(来自ATCC)，并分别培养在10cm培养皿(Corning,430167)中，培养基为含有10％体积比的胎牛血清(HyClone,SV30087)的DMEM(HyClone,SH30243.01)。培养温度为37℃，二氧化碳浓度为5％。传代后当细胞密度为80％时，细胞分盘至24孔板。24孔板使用前用1:10稀释的多聚赖氨酸溶液(Sigma，P4707-50ML)包被处理。
2)接种细胞12-14h后，细胞浓度约为80％时，进行转染。每孔转染的质粒的量是pCMV-Csy4-NMRT质粒900ng，融合RNA的质粒300ng。将质粒混在50μl的Opti-MEM(Gibco,11058021)培养基中。以pCMV-PE2作为阳性对照组，每孔加900ng pCMV-Csy4-NMRT，p egRNA质粒300ng，nicking sgRNA 100ng，
3)另外，将3μL的Lipofectamine 2000转染试剂(Thermo,11668019)混入50μL的Opti-MEM培养基，静置5分钟。
4)将混有质粒的Opti-MEM加入混有Lipofectamine 2000的Opti-MEM，慢速吹打混匀，静置20分钟。
5)将上述混和静置好的转染液分别加入培养的细胞中。
6)转染6小时后用含有10％FBS的DMEM换液。转染48小时后，去培基，用PBS清洗一次细胞，然后用TE(Thermo Fisher，R001100)将细胞消化下来，再用含有10％FBS的DMEM终止消化，并离心收集细胞，最后用培养基重悬。
7)重悬后的细胞进行FACS(Fluorescence activated Cell Sorting)分选，由于GFP信号在pegRNA质粒或融合RNA质粒上，因此，我们直接分选所 有的GFP阳性细胞，每个样品至少收集10,00个细胞。
上述收集的细胞直接进行裂解，并PCR扩增靶向位点片段，PCR引物序列如SEQ ID NO:11。用诺唯赞高保真酶试剂盒(Vazyme,p501-d2)PCR扩增各基因组靶向位点片段。PCR反应体系如下表18所示：
表18
Figure PCTCN2022080595-appb-000007
PCR程序如下表19所示：
表19
Figure PCTCN2022080595-appb-000008
PCR扩增产物经AxyPrep PCR Clean-up试剂盒(Axygen,AP-PCR-500G)纯化回收。将具有不同条形码的PCR产物集中在一起，在中国北京的 Novogene生物信息研究所的Illumina Hiseq X Ten(2×150PE)平台上进行深度测序。使用AdapterRemoval版本2.2.2删除了配对末端读取的适配器对，并将11bp或更多碱基的配对末端读取比对合并为单个共有读取。然后使用BWA-MEM算法(BWA v0.7.16)将所有处理的读段映射到靶序列。对于每个位点，使用参数-q 20-b 30的bam读数计数来计算突变率。基于原型间隔子中至少包含1个插入或缺失的核苷酸的读数计算插入缺失。插入缺失频率计算为含插入缺失的读段数/总映射读段数。测序情况结果见附图5和附图6。结果表明ePE在Hela细胞系和N2a的多个内源位点的靶向引导编辑效率与PE相比均有明显提高(**表示p<0.05；***表示p<0.01)。
综上所述，本发明有效克服了现有技术中的种种缺点而具高度产业利用价值。上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求书所涵盖。

Claims (10)

  1. 一种引导编辑工具，其特征在于，其包括：
    (i)一种融合蛋白，其包括至少一个基因编辑器与一种核酸内切酶；
    (ii)一种融合RNA，其包括一个pegRNA与(i)中所述核酸内切酶的识别位点；
    其中，所述融合蛋白具有反转录功能，且可与所述识别位点结合并对其进行剪切，从而在所述pegRNA的3’端引入序列，避免pegRNA自身环化。
  2. 如权利要求1所述的引导编辑工具，其特征在于，所述融合RNA自5’端至3’端依次为pegRNA、Csy4核酸内切酶识别序列和切口sgRNA；优选地，所述Csy4核酸内切酶识别序列的核苷酸序列如SEQ ID NO:5所示。
  3. 如权利要求1或2所述的引导编辑工具，其特征在于，所述融合蛋白包括例如自N端至C端依次包括Csy4核酸内切酶、Cas9n和病毒反转录酶例如莫罗尼小鼠白血病病毒反转录酶M-MLV；
    优选地，所述Csy4核酸内切酶的氨基酸序列如SEQ ID NO:1所示，所述Cas9n的氨基酸序列如SEQ ID NO:2所示，和/或，所述M-MLV的氨基酸序列如SEQ ID NO:3所示；
    更优选地，所述融合蛋白还包括T2A片段和/或BPNLS片段；
    进一步更优选地，所述T2A片段位于Csy4核酸内切酶与Cas9n之间，其氨基酸序列如SEQ ID NO:6所示，和/或，所述BPNLS片段位于C端，其氨基酸序列如SEQ ID NO:7所示。
  4. 如权利要求1～3任一项所述的引导编辑工具，其特征在于，所述的融合RNA包含的Csy4核酸内切酶的识别序列为如SEQ ID NO:5所示的核苷酸序列，或与SEQ ID NO:5所示的核苷酸序列具有95％以上同一性、且维持被Csy4核酸内切酶识别的功能；
    优选地，所述融合蛋白的氨基酸序列如SEQ ID NO:4所示，或与SEQ  ID NO:4的氨基酸序列具有90％、95％、96％、97％、98％、99％或以上同一性、且具有如SEQ ID NO:4的氨基酸序列所示的融合蛋白的功能。
  5. 一种融合RNA，其特征在于，所述融合RNA自5’端至3’端依次为pegRNA、Csy4核酸内切酶识别序列和nicking sgRNA；
    优选地，所述的融合RNA包含的Csy4核酸内切酶识别序列为如SEQ ID NO:5所示的核苷酸序列，或与SEQ ID NO:5所示的核苷酸序列具有95％同一性、且维持被Csy4核酸内切酶识别的功能。
  6. 一种融合蛋白，其特征在于，其自N端至C端依次包括Csy4核酸内切酶、Cas9n和莫罗尼小鼠白血病病毒反转录酶M-MLV；
    优选地，所述融合蛋白的氨基酸序列如SEQ ID NO:4所示，或与SEQ ID NO:4的氨基酸序列具有90％、95％、96％、97％、98％、99％或以上同一性、且具有如SEQ ID NO:4的氨基酸序列所示的融合蛋白的功能。
  7. 一种分离的核酸，其特征在于，其包括编码如权利要求6所述的融合蛋白的第一多核苷酸；和/或，转录如权利要求5所述的融合RNA的第二多核苷酸。
  8. 一种重组表达载体，其特征在于，其包括如权利要求7所述的分离的核酸。
  9. 一种表达系统，其特征在于，其含有如权利要求8所述的重组表达载体；所述表达系统的宿主细胞选自真核细胞或原核细胞，优选选自小鼠细胞、人细胞，更优选选自小鼠脑神经瘤细胞、人胚胎肾细胞、或人宫颈癌细胞、人结肠癌细胞、人骨肉瘤细胞，进一步更优选选自N2a细胞、HEK293T细胞、Hela细胞、HCT116细胞或U2OS细胞；
    较佳地，所述表达系统中，所述第一多核苷酸与所述第二多核苷酸可位于同一重组表达载体或不同的重组表达载体中，所述重组表达载体例如为pCMV、pCAG或Tet-On。
  10. 如权利要求1～4任一项所述的引导编辑工具、权利要求5所述的融 合RNA、权利要求6所述的融合蛋白、权利要求7所述的分离的核酸、权利要求8所述的重组表达载体或如权利要求9所述的表达系统在真核生物基因编辑中的用途；
    优选地，所述用途包括碱基的替换、插入或者缺失。
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