CN117586987A - A single base editing system and its use - Google Patents

Abstract

The invention relates to the technical field of biology, and discloses a single base editing system and application. The said method includes: 1) A nucleic acid nicking enzyme or a polynucleotide encoding the same; 2) Cytosine deaminase or a polynucleotide encoding the same; 3) An abasic site protecting polypeptide or a polynucleotide encoding the same. The invention constructs fusion protein or over-expression plasmid containing HMCES, and constructs a novel cytosine-guanine base editor CGBEpH, which can protect abasic sites (AP sites), reduce DNA double-strand break damage generated by CGBEs, and reduce the generation of harmful low-frequency products generated in the editing process.

Description

A single base editing system and its use

Technical field

The present invention relates to the field of biotechnology, and in particular to a single base editing system and its use.

Background technique

Single base editors aim to achieve single base mutations at specific sites in genomic DNA sequences without causing DNA double-strand break damage (Gaudelli et al., 2017; Komor et al., 2016; Nishida et al., 2016), is expected to become a safer and more reliable clinical gene editing treatment method. For different genome positions and different sequences, the editing effects of single base editors will be different (Anzalone et al., 2020; Porto et al., 2020), and some low-frequency harmful by-products will be produced during this process. , It is currently believed that the production of these by-products is random and non-inducible, but in clinical treatment these small amounts of by-products may be enough to cause serious adverse consequences. Since the editing by-products of single base editors account for a small proportion, their potential production mechanisms are currently unclear. It is necessary to further explore their production mechanisms to reduce their production. This will promote the use of single base editors in clinical gene editing treatments. It is of great significance to play a role (Doudna, 2020).

The single base editor contains two components: a deaminase and a Cas cleavage enzyme defective in cleavage activity, and can achieve mutation of specific bases through deaminase initiation within a specific editing window of the genome (Anzalone et al., 2020) . In addition to these two basic components, the researchers also altered the editing product by expressing other components through fusion. For example, by fusion expression of uracil glycosylase inhibitor (UGI), the conversion mutation of cytosine to thymine can be increased by maintaining uracil (U) from being excised, while reducing the frequency of base insertion or base deletion mutations. (Komor et al., 2016). The earliest researched and invented cytosine base editors and adenine base editors can generate conversion mutations from cytosine to thymine and adenine to guanine with high efficiency (Komor et al., 2016; Komor et al. , 2017; Wang et al., 2017), however, there are also many single nucleotide mutations (SNV) in clinical practice that require transversion mutations to be corrected to normal sequences, so recent studies have reported that cytosine to guanine can be achieved Transversion mutagenic single base editors (CGBEs) (Chen et al., 2021a; Koblan et al., 2021; Kurt et al., 2021; Zhao et al., 2021), by removing UGI components or further fusion Expressing uracil glycosylase (UNG) promotes the excision of uracil and generates AP sites, which is conducive to the realization of cytosine to guanine transversion mutations, but there are also reports revealing that CGBEs will produce a higher frequency of base deletions or insertional mutation (Arbab et al., 2020; Chen et al., 2021a; Koblan et al., 2021; Kurt et al., 2021). Due to the low frequency of base deletion or insertion mutations, conventional experiments are difficult to capture sufficient information for further analysis of their mechanisms (Huang et al., 2021). It is currently believed that base deletion mutations and base insertion mutations are mutations caused by the same mechanism, and base deletion mutations caused by single base editor editing are different from base deletion mutations caused by Cas9-based strand cutting base editors. The formation mechanisms are different (Allen et al., 2018; Arbab et al., 2020; Shen et al., 2018), and there is currently no research showing whether single base editors will produce large base deletion mutations or Chromosome translocation, it can be seen that people do not yet have a clear understanding of the harmfulness of single-base editor editing by-products.

The editing products of single base editors can be optimized by interfering with the DNA damage repair pathway, such as through fusion expression of the non-homologous end joining inhibitor GAM or the homologous recombination repair factor 53BP1 (Canny et al., 2018; Chu et al. ., 2015; Maruyama et al., 2015), promotes homologous recombination repair of CRISPR/Cas9 break damage, thereby improving editing efficiency and reducing base deletion mutations. However, GAM binding to DSB ends may lead to the generation of persistent DNA breaks. The further formed base deletion mutation products cannot be detected (Komor et al., 2017); researchers used the Repair-seq method to reveal that Cas9 (Hussmann et al., 2021), single base editor (Koblanet al., 2021), and the damage repair pathway after cutting double-stranded DNA by a leading base editor (Chen et al., 2021b); some studies have also shown that trans-damage DNA synthesis repair pathway (TLS) factors generate cytosine to guanine in CGBEs Play a role in transversion mutations (Koblan et al., 2021). However, there are still many unknowns about the occurrence mechanism of low-frequency products of single base editors, including base deletion mutations, base insertion mutations, abnormal base transversion mutations, etc. It is necessary to construct an optimized single base editor that reduces low-frequency by-products. Instruments are of great significance.

Contents of the invention

In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a single base editing system and uses, in order to solve the problems existing in the prior art.

In order to achieve the above object, the present invention specifically adopts the following technical solutions.

The first aspect of the present invention protects a single base editing system, which includes:

1) Nucleic acid nickase or its encoding polynucleotide;

2) Cytosine deaminase or its encoding polynucleotide;

3) Abasic site protected polypeptide or its encoding polynucleotide.

In some embodiments of the present invention, the abasic site protected polypeptide is an APEX1 competitive inhibitor or an APEX1 mutant.

Preferably, the APEX1 competitive inhibitor is selected from HMCES.

More preferably, the HMCES is derived from eukaryotes. More preferably, derived from mice.

In some specific embodiments of the present invention, the amino acid sequence of HMCES includes:

1) The amino acid sequence shown in SEQ ID NO.1; or,

2) An amino acid sequence that has at least 80% sequence similarity with SEQ ID NO. 1 and has the function of the amino acid sequence defined in 1).

In some embodiments of the invention, the nucleic acid nickase is Cas9 nickase.

In some embodiments of the present invention, the cytosine deaminase is selected from APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D/E, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4 and mutants thereof or AID and mutants thereof one or more.

In some embodiments of the present invention, when the nucleic acid nickase or cytosine deaminase is expressed in the form of a fusion protein, the fusion protein includes a cytosine deaminase fragment and a nucleic acid nickase fragment; the abasic position Point-protected polypeptides are expressed in fusion or independently.

In certain embodiments of the present invention, the fusion protein further includes a uracil glycosylated protein fragment and/or a nuclear localization fragment and/or an abasic site protected polypeptide fragment.

In certain embodiments of the present invention, the fusion protein includes a cytosine deaminase fragment and a nucleic acid nickase fragment in sequence from the N-terminus to the C-terminus.

In some more specific embodiments of the present invention, in the fusion protein, the N-terminus of the cytosine deaminase fragment is connected to a uracil glycosylated protein fragment and/or an abasic site protected polypeptide fragment.

Preferably, the fusion protein includes an abasic site protected polypeptide fragment, a cytosine deaminase fragment and a nucleic acid nickase fragment in sequence from the N-terminus to the C-terminus.

Preferably, the fusion protein includes an abasic site protected polypeptide fragment, a cytosine deaminase fragment, a nucleic acid nickase fragment and a nuclear localization fragment in sequence from the N-terminus to the C-terminus.

Preferably, the fusion protein includes an abasic site protected polypeptide fragment, a uracil glycosylated protein fragment, a cytosine deaminase fragment and a nucleic acid nickase fragment in order from the N-terminus to the C-terminus.

Preferably, the fusion protein includes an abasic site protected polypeptide fragment, a uracil glycosylated protein fragment, a cytosine deaminase fragment, a nucleic acid nickase fragment and a nuclear localization fragment in sequence from the N-terminus to the C-terminus.

In some embodiments of the present invention, the fusion protein includes an amino acid sequence including the sequence shown in SEQ ID NO. 19 or SEQ ID NO. 20 or SEQ ID NO. 21.

In some embodiments of the present invention, the single base editing system further includes sgRNA or its encoding polynucleotide.

Preferably, the sgRNA does not contain a deaminase-preferred palindromic motif.

More preferably, the palindromic motif preferred by the deaminase is TCGA.

The second aspect of the invention protects a fusion protein for single base editing, including an abasic site protecting polypeptide fragment, a cytosine deaminase fragment and a nucleic acid nickase fragment.

In some embodiments of the present invention, the abasic site protected polypeptide is an APEX1 competitive inhibitor or an APEX1 mutant.

Preferably, the APEX1 competitive inhibitor is selected from HMCES.

More preferably, the HMCES is derived from eukaryotes. More preferably, derived from mice.

In some embodiments of the invention, the amino acid sequence of HMCES includes:

1) The amino acid sequence shown in SEQ ID NO.1; or,

2) An amino acid sequence that has at least 80% sequence similarity with SEQ ID NO. 1 and has the function of the amino acid sequence defined in 1).

In some embodiments of the present invention, the fusion protein includes an abasic site protected polypeptide fragment, a cytosine deaminase fragment and a nucleic acid nickase fragment in sequence from the N-terminus to the C-terminus.

In some embodiments of the present invention, the fusion protein further includes a uracil glycosylated protein fragment and/or a nuclear localization fragment.

Preferably, the N-terminus of the cytosine deaminase fragment is connected to a uracil glycosylation protein fragment and/or a nuclear localization fragment.

In some embodiments of the present invention, the fusion protein includes an amino acid sequence including the sequence shown in SEQ ID NO. 19 or SEQ ID NO. 20 or SEQ ID NO. 21.

A third aspect of the invention protects a polynucleotide encoding a fusion protein as described above.

A fourth aspect of the invention protects a nucleic acid construct comprising a polynucleotide as described above.

The fifth aspect of the present invention protects an expression system, which contains the nucleic acid construct as described above or the polynucleotide as described above integrated into the genome.

The sixth aspect of the present invention protects the single base editing system as described above, the fusion protein as described above, the polynucleotide as described above, the nucleic acid construct as described above or the expression as described above Use of the system in base editing.

The seventh aspect of the present invention protects a gene editing method, which includes: performing gene editing through the base editing system as described above or the fusion protein as described above.

The eighth aspect of the present invention protects the use of HMCES as an abasic site protected polypeptide for gene editing or for use in a base editing system.

In some embodiments of the invention, the HMCES is used to reduce DNA double-strand break damage in single base editing.

In some embodiments of the invention, the single base editing is cytosine to guanine base editing.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention constructs a new cytosine to guanine base editor CGBEpH by overexpressing HMCES or fusion expression to protect abasic sites (AP sites), which can reduce DNA double-strand break damage generated by CGBEs and reduce the editing process. The generation of harmful low-frequency products produced in.

Description of drawings

Figure 1 is a diagram showing the types of DNA double-strand break damage generated by existing base editors in Example 1 of the present invention.

Figure 2 shows an experimental flow chart for screening DNA damage repair factors that affect base editors in Example 1 of the present invention and the mutation maps of three base editors. in

A is the experimental design flow chart for screening DNA damage repair factors that affect base editing products. First, in the HEK293T cell line stably expressing Streptococcus pyogenes Cas9 (hereinafter abbreviated as SpCas9), an sgRNA library targeting the knockout of DNA damage repair factors was expressed through lentiviral infection. After puromycin was used to screen the infected cells for 6 days, the results were obtained. DNA damage repair factor knockout cell library. The base editor constructed based on Staphylococcus aureus Cas9 (hereinafter abbreviated as SaCas9) was then transiently transferred through Lipofectamine 2000 to target the specific SaBE-Test insertion sequence and perform base editing. The sgRNA expressed in cells was amplified by PCR and the base-edited SaBE-Test insert sequence was generated. High-throughput sequencing was performed to detect the enrichment of different base editing products occurring on the SaBE-Test insert sequence and the corresponding expressed sgRNA. , and then analyze the DNA damage repair factors that affect the generation of different types of base editing products.

B is the base substitution mutation map that occurs on the sequencing sequence containing the negative control sgRNA in the base editor AID-SaBE3 high-throughput sequencing library.

C is the base substitution mutation map that occurs on the sequencing sequence containing the negative control sgRNA in the base editor AID-SaBE1n high-throughput sequencing library.

D is the base substitution mutation map that occurs on the sequencing sequence containing the negative control sgRNA in the base editor AID-SaBE1 high-throughput sequencing library.

Figure 3 shows the proportion of different types of mutations occurring on the SaBE-Test insertion sequence in Example 1 of the present invention and a map of base deletion mutations. in

A is a pie chart showing the proportion of base substitution mutations, base deletion mutations and base insertion mutations in SaBE3 editing products.

B is a pie chart showing the proportion of base substitution mutations, base deletion mutations and base insertion mutations in SaBE1n editing products.

C is a pie chart showing the proportion of base substitution mutations, base deletion mutations and base insertion mutations in SaBE1 editing products.

D is a map of 1 bp base deletion mutations generated by SaBE3, SaBE1n, and SaBE1 editing. The y-axis represents the probability of 1 bp base deletion mutations at each site, and the x-axis represents 1-50 bp of the SaBE-Test insertion sequence.

E is a map of base deletion mutations larger than 1 bp produced by SaBE3 editing. The red line represents the probability of matching the left end of the base deletion fragment at different positions, and the blue line represents the probability of matching the right end of the base deletion fragment at different positions.

F is the map of base deletion mutations larger than 1 bp produced by SaBE1n editing.

G is the map of base deletion mutations larger than 1 bp produced by SaBE1 editing.

Figure 4 shows a graph showing the enrichment results of DNA damage repair factors screening different types of base editing products in Example 1 of the present invention. in

A shows the enrichment results of genes affecting the editing efficiency of base editors in different DNA damage repair pathways.

B is an alignment diagram of enriched genes that affect the editing efficiency of SaBE1 and SaBE1n.

C is an alignment diagram of enriched genes that affect the editing efficiency of SaBE1n and SaBE3.

D is a cluster analysis heat map of genes with different base editing product types and different DNA damage repair pathways in the library screening experiment.

Figure 5 shows the results of gene enrichment analysis and gene verification of different types of base editing products in Example 1 of the present invention. in

A shows the enrichment analysis results of base excision repair pathway (BER) factors in the editing mutation group compared with the non-mutation group.

B is the enrichment analysis result of base mismatch repair pathway (MMR) factors in the editing mutation group compared with the non-mutation group.

C is the enrichment analysis result of nucleic acid excision repair pathway (NER) factors in the editing mutation group compared with the non-mutation group.

Figure 6 shows the gene enrichment analysis affecting base deletion mutations in the CRISPR/Cas9 library screening experiment in Example 1 of the present invention and the result analysis chart of base deletion mutations occurring with various base editors. in

A shows the gene enrichment analysis results of the base deletion mutation group compared with the C>T base substitution mutation group. DNA damage repair factors that promote the generation of base deletion mutations were screened out.

B is a schematic diagram of base deletion mutations produced by the base editor.

C is the analysis result of the base editing efficiency, the proportion of C>G mutations and the deletion mutation efficiency of four base editors in the HEK293T cell line at four genomic positions.

D is the base larger than 1 bp produced by the commonly used cytosine base editor BE4max, the cytosine to guanine base editor CGBE1/AXC, and the existing AID-BE1n in our laboratory on three genome sequences. Delete mutation map.

Figure 7 shows the gene enrichment analysis and verification results of the base transversion mutations C>G and C>A in Example 1 of the present invention. in

A is the gene enrichment analysis result of the base transversion mutation C>G group compared with the C>T base substitution mutation group.

B is a schematic diagram of the base editor generating C>G base transversion mutations through two pathways: NHEJ and TLS.

C is the analysis result of changes in editing efficiency, proportion of C>G mutations and deletion mutation efficiency of CGBEs after knocking out NHEJ factor XRCC4 or TLS factor REV1 in HEK293T cell line.

D is the analysis result of the change in the proportion of C>G mutations produced by base editor editing at six genomic positions in the HEK293T cell line after knocking out the NHEJ factor XRCC4 or the TLS factor REV1 compared with wild-type cells.

Figure 8 shows an analysis diagram showing the results of chromosomal translocations caused by DSBs generated by the base editor in Example 1 of the present invention. in

A is a flow chart of the optimized method Tn5-HTGTS for capturing chromosomal translocations generated by base editors.

B is a Circos diagram of genome-wide chromosomal translocation connections captured by Tn5-HTGTS in cells indicating the action of the base editor.

C is the analysis result of the number of chromosomal translocations captured per 1000 cells when nCas9 and three base editors act on four genome positions.

D is the distribution map of chromosomal translocation connections at off-target hotspots mediated by Cas9 components at the EMX1 position.

E is the chromosomal translocation distribution map of different base editors on the off-target hotspot of AID deaminase, the ChIP-seq map of H3K27Ac, and the PRO-seq map illustrating transcription.

F is the enrichment analysis of chromosomal translocations related to histone marks, open chromosomes, and transcriptional signals produced by CGBE1 at four genomic locations.

G is the analysis of the enrichment of different motifs in the sequence where chromosomal translocation connection occurs in CGBE1.

H is a schematic diagram of the chromosomal translocation generated by the cytosine to guanine base editor.

Figure 9 shows the construction method and result verification diagram of the single base editing system CGBEpH in Example 2 of the present invention. in

D is a schematic diagram of constructing the single base editing system CGBEpH.

E shows the efficiency analysis results of base deletion mutations and repetitive sequence insertion mutations greater than 1 bp produced in cells transfected with CGBE or the single base editing system CGBEpH.

F is the result of CGBE or single base editing system CGBEpH editing efficiency and the proportion of C>G in the edited product.

G is a Circos diagram showing the distribution of translocation connections in cells edited by single base editing system CGBEs.

Figure 10 shows the construction method and result verification diagram of the fusion protein for single base editing in Example 3 of the present invention. in

A is a working model diagram that relies on HMCES protection to reduce the generation of deletion mutations.

B is a schematic diagram of the plasmid structure after fusion of HMCES and different proteins.

C is an analysis chart showing the editing efficiency, the efficiency of generating base deletion mutations and the ratio of C>G after different proteins are fused to express HMCES or GFP (control).

Figure 11 shows the verification results showing that the palindromic motif is more likely to undergo base deletion mutations in Example 4 of the present invention. in

A is the base deletion mutation map of greater than 1 bp when CGBE1 acts on the editing sequence containing a palindromic motif (TCGA) or a non-palindromic motif (TCAA).

B is a statistical diagram of the difference in efficiency of deletion mutations produced by CGBE1 after acting on five pairs of palindromic/non-palindromic motif sequences.

C is a pie chart analyzing the proportion of 3040 potential CGBE correction sites in the ClinVar database that contain palindromic motifs in the editing window.

Detailed ways

After extensive research, the inventor found that a single base editor will produce a large number of DNA double-strand breaks (DSBs) during the base editing process. These double-strand break damaged ends will be removed through end resection, template fragment insertion and filling, etc. The process is further processed into small base deletion mutations, cytosine to guanine base transversion mutations, and chromosomal translocations between the base editing sites of CGBEs and off-target sequences derived from Cas9 or cytosine deaminase. At the same time, it was discovered that APEX1 is an apurinic/apyrimidinic endonuclease that cleaves the apurinic/apyrimidine site (AP site) generated during the base editor editing process, thereby generating a single-stranded nick, which is competitively inhibited by APEX1 Agents or expression of APEX1 mutants defective in enzyme activity can inhibit the activity of APEX1, thereby reducing its cleavage of the AP site. It was further discovered that for the AP sites generated during the action of the base editor, APEX1 competitive inhibitors can also be added to inhibit their cleavage activity and reduce the breakage of AP sites, thereby reducing DNA damage and various mediated consequences. Generation of by-products. It was further found that HMCES, as a competitive inhibitor of APEX1, can protect abasic sites (AP sites) and reduce DNA damage generated during the base editing process, thereby reducing various by-products mediated by DNA damage, providing a promising future Optimizing single base editors provides new ideas and promotes the development of perfect single base editors without editing by-products. In addition, the inventors found that the editing products of different cytosine base editors are determined by both the cis-acting genome sequence and the trans-acting DNA damage repair pathway. The sgRNA targeting sequence contains palindromic motifs favored by cytosine deaminase. Significantly increasing the frequency of base deletion mutations and avoiding the selection of sgRNAs with deaminase-preferred palindromic motifs can reduce the generation of DNA double-strand break damage.

The term "competitive inhibitor" in the present invention refers to a substance that is structurally similar to the substrate of the enzyme to be inhibited and can compete with the substrate for the binding site on the enzyme molecule, thereby producing reversible or irreversible changes in enzyme activity. substance.

A first aspect of the present invention provides a single base editing system, which includes:

1) Nucleic acid nickase or its encoding polynucleotide;

2) Cytosine deaminase or its encoding polynucleotide;

3) Abasic site protected polypeptide or its encoding polynucleotide.

In the single base editing system provided by the invention, the abasic site protection polypeptide is an APEX1 competitive inhibitor or an APEX1 mutant.

Preferably, the APEX1 competitive inhibitor is selected from HMCES. The HMCES is derived from eukaryotes. More preferably, derived from mice.

Preferably, the APEX1 mutant is APEX1 with enzyme activity deficiency. APEX1 mutants such as N212A, D210N, Y171F or R177A have lower AP site cleavage activity. APEX1 mutants, nucleic acid nickases and cytosine deaminase are all protected when expressed on a single fusion protein or as isolated proteins. Abasic sites (AP sites) reduce DNA damage and the production of various by-products mediated by it.

In the single base editing system provided by the present invention, the amino acid sequence of HMCES includes:

1) The amino acid sequence shown in SEQ ID NO.1; or,

2) An amino acid sequence that has at least 80% sequence similarity with SEQ ID NO. 1 and has the function of the amino acid sequence defined in 1). Specifically, the amino acid sequence in 2) specifically refers to: the amino acid sequence shown in SEQ ID No. 1 with one or more substitutions, deletions or additions (specifically, it can be 1-50, 1-30, 1- 20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or one or more ( Specifically, it can be obtained from 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, and has The amino acid is a functional polypeptide fragment of the polypeptide fragment shown in SEQ ID No. 1. More specifically, the amino acid sequence in 2) may have a similarity of 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more with SEQ ID No. 1. The nucleotide sequence encoding HMCES includes the sequence shown in SEQ ID NO.2.

MCGRTSCHLPREVLTRACAYQDRQGRRRLPQWRDPDKYCPSYNKSPQSSSPVLLSRLHFEKDADSSDRIIIPMRWGLVPSWFKESDPSKLQFNTTNCRSDTIMEKQSFKVPLGKGRRCVVLADGFYEWQRCQGTNQRQPYFIYFPQIKTEKSGGNDASDSSDNKEKVWDNWRLLTMAGIFDCWEAPGGECLYSYSIITVDSCRGLSDIHS RMPAILDGEEAVSKWLDFGEVATQEALKLIHPIDNITFHPVSPVVNNSRNNTPECLAPADLLVKKEPKANGSSQRMMQWLATKSPKKEVPDSPKKDASGLPQWSSQFLQKSPLPAKRGATSSFLDRWLKQEKEDEPMAKKPNS(SEQ ID NO.1)

ATGTGCGGGCGAACGTCCTGTCACTTGCCCAGAGAGGTTCTCACCAGGGCCTGCGCCTATCAGGATCGGCAGGGGCCGGCGGCGGCTCCCGCAGTGGAGGGACCCCGACAAGTACTGCCCCTCCTACAACAAGAGCCCGCAGTCCAGCAGCCCCGTGCTGCTCTCCAGACTGCACTTTGAGAAGGATGCAGACTCATCAGATCGGATAATTATTCCCATGCGATGGGGCTTAGTCCCATCTTGGTTCAAAGAAAGTGATCCTTCTA AGCTGCAGTTCAACACTACCAACTGTCGTAGTGATACCATAATGGAGAAGCAGTCATTCAAGGTTTCCTCTGGGGAAAGGACGGCGGTGTGTTGTTTTAGCAGATGGATTCTACGAGTGGCAGCGGTGTCAGGGAACAAACCAGAGGCAACCATACTTCATCTATTTTCCTCAAATCAAGACAGAGAAGTCAGGTGGGAACGATGCTTCAGACAGCTCTGACAACAAGGAAAAGGTCTGGGACAACTGGAGGCTGCTGACAATGGCA GGGATCTTTGACTGCTGGGAAGCGCCAGGGGGAGAGTGCCTGTATTCCTACAGCATCATCACTGTGGATTCCTGCAGAGGTTTGAGTGACATCCACAGCAGGATGCCTGCCATACTAGATGGAGAAGAAGCAGTCTCCAAATGGCTCGACTTTGGTGAGGTCGCCACTCAGGAAGCTCTGAAGCTAATCCACCCCATAGACAATATCACCTTCCATCCAGTTTCTCCAGTGGTGAACAATTCCCGAAACAACACTCCGGAGTGTC TGGCGCCTGCTGACTTGCTGGTTAAGAAGGAGCCCAAGGCAAATGGCAGCAGTCAAAGGATGATGCAGTGGCTGGCTACAAAGTCACCCAAAAAGGAAGTCCCTGACTCACCCAAAAAGGATGCATCAGGTCTACCCCAGTGGTCCAGCCAGTTTCTCCAGAAGAGCCCATTGCCTGCTAAAAGAGGTGCTACCAGCAGCTTCCTGGATCGATGGCTGAAGCAGGAGAAGGAGGATGAGCCCATGGCCAAGAAGCCTAACAGC( SEQ ID NO.2)

In the single base editing system provided by the invention, the nucleic acid nickase is Cas9 nickase. The nucleic acid nickase is Cas9 with complete or partial loss of cleavage activity, such as dCas9 or nCas9. Cas9 nickase cleaves the target strand of DNA without cutting the non-target strand, avoiding direct DNA double-strand breaks. For example, the first Cas9 nickase fragment can still have the targeting activity of Cas9 nickase after being combined with the second Cas9 nickase fragment. More specifically, it can be the activity of targeting DNA under the guidance of a suitable sgRNA. The Cas9 nickase is selected from one or more of nSpCas9, nSaCas9, nScCas9 and nXCas9. In a specific embodiment of the invention, the Cas9 nickase is selected from nSpCas9. The sequence of nSpCas9 is shown in SEQ ID No. 3.

In the single base editing system provided by the present invention, the cytosine deaminase is selected from APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D/E, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4 or cytosine deaminase (AID ) one or more. In certain embodiments, the cytosine deaminase is selected from APOBEC1 and mutants thereof. The APOBEC1 mutants include YE1, YE2, EE, YEE, R33A and R33A/R34A. In a specific embodiment, it is APOBEC1 or APOBEC1 mutant (R33A) or AID. The amino acid sequence of the APOBEC1 mutant (R33A) is shown in SEQ ID NO.4, the amino acid sequence of APOBEC1 is shown in SEQ ID NO.5, and the amino acid sequence of the AID is shown in SEQ ID NO.6.

In the single base editing system provided by the present invention, when the nucleic acid nickase or cytosine deaminase is expressed in the form of a fusion protein, the fusion protein includes a cytosine deaminase fragment and a nucleic acid nickase fragment; Abasic site protected polypeptides are fused or expressed independently.

In the single base editing system provided by the present invention, the fusion protein further includes a uracil glycosylated protein fragment and/or a nuclear localization (NLS) fragment and/or an abasic site protected polypeptide fragment. The amino acid sequence of the nuclear localization (NLS) fragment is shown in SEQ ID NO.7, and the amino acid sequence of the uracil glycosylated protein fragment is shown in SEQ ID NO.8.

In the single base editing system provided by the invention, the fusion protein includes a cytosine deaminase fragment and a nucleic acid nickase fragment in sequence from the N-terminus to the C-terminus.

In the single base editing system provided by the present invention, in the fusion protein, the N-terminus of the cytosine deaminase fragment is connected to a uracil glycosylated protein fragment and/or an abasic site protected polypeptide fragment.

Preferably, the fusion protein includes an abasic site protected polypeptide fragment, a cytosine deaminase fragment and a nucleic acid nickase fragment in sequence from the N-terminus to the C-terminus.

Preferably, the fusion protein includes an abasic site protected polypeptide fragment, a cytosine deaminase fragment, a nucleic acid nickase fragment and a nuclear localization fragment in sequence from the N-terminus to the C-terminus.

Preferably, the fusion protein includes an abasic site protected polypeptide fragment, a uracil glycosylated protein fragment, a cytosine deaminase fragment and a nucleic acid nickase fragment in order from the N-terminus to the C-terminus.

Preferably, the fusion protein includes an abasic site protected polypeptide fragment, a uracil glycosylated protein fragment, a cytosine deaminase fragment, a nucleic acid nickase fragment and a nuclear localization fragment in sequence from the N-terminus to the C-terminus.

In the single base editing system provided by the present invention, the fusion protein includes an amino acid sequence including the sequence shown in SEQ ID NO. 19 or SEQ ID NO. 20 or SEQ ID NO. 21.

In the single base editing system provided by the present invention, the single base editing system further includes sgRNA or its encoding polynucleotide. Preferably, the sgRNA does not contain a deaminase-preferred palindromic motif. More preferably, the palindromic motif preferred by the deaminase is TCGA. The present invention found that the target site sequence containing the palindromic motif TCGA in the targeted editing region produces a higher frequency of base deletion mutations during the editing process of the base editor, resulting in base substitution of the target base editing product. Mutation frequency is low.

A second aspect of the present invention provides a fusion protein for single base editing, including an abasic site protection polypeptide fragment, a cytosine deaminase fragment and a nucleic acid nickase fragment.

In the fusion protein for single base editing provided by the present invention, the abasic site protection polypeptide fragment is an APEX1 competitive inhibitor and/or an APEX1 mutant; preferably, the APEX1 competitive inhibitor is selected from HMCES . APEX1 mutants such as N212A, D210N, Y171F or R177A have lower AP site cleavage activity. APEX1 mutants, nucleic acid nickases and cytosine deaminase are all protected when expressed on a single fusion protein or as isolated proteins. Abasic sites (AP sites) reduce DNA damage and the production of various by-products mediated by it.

Preferably, the HMCES is derived from eukaryotes, preferably from mice.

In the fusion protein for single base editing provided by the present invention, the amino acid sequence of HMCES includes:

1) The amino acid sequence shown in SEQ ID NO.1; or,

2) An amino acid sequence that has at least 80% sequence similarity with SEQ ID NO. 1 and has the function of the amino acid sequence defined in 1). Specifically, the amino acid sequence in 2) specifically refers to: the amino acid sequence shown in SEQ ID No. 1 with one or more substitutions, deletions or additions (specifically, it can be 1-50, 1-30, 1- 20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or one or more ( Specifically, it can be obtained from 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, and has The amino acid is a functional polypeptide fragment of the polypeptide fragment shown in SEQ ID No. 1. More specifically, the amino acid sequence in 2) may have a similarity of 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more with SEQ ID No. 1. The nucleotide sequence encoding HMCES includes the sequence shown in SEQ ID NO.2.

In the fusion protein for single base editing provided by the present invention, the fusion protein includes an abasic site protection polypeptide fragment, a cytosine deaminase fragment and a nucleic acid nickase fragment in order from the N-terminus to the C-terminus.

In the fusion protein for single base editing provided by the present invention, the nucleic acid nickase is Cas9 nickase. The Cas9 nickase is selected from one or more of nSpCas9, nSaCas9, nScCas9 and nXCas9. In a specific embodiment of the invention, the Cas9 nickase is selected from nSpCas9. The amino acid sequence of nSpCas9 is shown in SEQ ID NO.3.

DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKS RRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLF DDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD(SEQ ID NO.3)

In the fusion protein for single base editing provided by the invention, the cytosine deaminase is selected from APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3D/E, APOBEC3G, APOBEC3H, APOBEC4 and mutants thereof or AID and mutants thereof of one or more. In certain embodiments, the cytosine deaminase is selected from APOBEC1 and mutants thereof. The APOBEC1 mutants include YE1, YE2, EE, YEE, R33A and R33A/R34A. In a specific embodiment, it is APOBEC1 or APOBEC1 mutant (R33A) or AID. The amino acid sequence of the APOBEC1 mutant (R33A) is shown in SEQ ID NO.4, the amino acid sequence of APOBEC1 is shown in SEQ ID NO.5, and the amino acid sequence of the AID is shown in SEQ ID NO.6.

SSETGPVAVDPTLRRRIEPHEVFFDPRELAKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLP PHILWATGLK(SEQ ID NO.4)

SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQR LPPHILWATGLK(SEQ ID NO.5)

MDPATFTYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLAEAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILQ(SEQ ID NO.6)

In the fusion protein for single base editing provided by the present invention, the fusion protein further includes a uracil glycosylation protein fragment and/or a nuclear localization (NLS) fragment.

Preferably, the N-terminus of the cytosine deaminase fragment is connected to a uracil glycosylation protein fragment and/or a nuclear localization (NLS) fragment.

Preferably, the sequence of the nuclear localization (NLS) fragment is shown in SEQ ID NO.7. In the present invention, the nuclear localization fragment connected to the C-terminal is conducive to higher efficiency editing, but the nuclear localization fragment (NLS) connected to the N-terminal cannot reduce the base deletion mutations produced by CGBE, because the N-terminal short peptide affects the HMCES active.

PKKKRKV (SEQ ID NO. 7).

Preferably, the uracil glycosylated protein fragment is selected from eUNG. The amino acid sequence of eUNG is shown in SEQ ID NO.8. The uracil glycosylated protein fragment is located between the nucleic acid nickase fragment and the abasic site protected polypeptide fragment.

ANELTWHDVLAEEKQQPYFLNTLQTVASERQSGVTIYPPQKDVFNAFRFTELGDVKVVILGQDPYHGPGQAHGLAFSVRPGIAIPPSLLNMYKELENTIPGFTRPNHGYLESWARQGVLLLNTVLTVRAGQAHSHASLGWETFTDKVISLINQHREGVVFLLWGSHAQKKGAIIDKQRHHVLKAPPHPSPLSAHRGFFGCNHFVLANQWLEQR GETPIDWMPVLPAES(SEQ ID NO.8)

In the fusion protein for single base editing provided by the present invention, some or all of the abasic site protection polypeptide fragments, cytosine deaminase fragments or nucleic acid nickase fragments have connecting peptides between them.

SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO.9) (between Hmces and Ung)

ESGGSGGSGGS(SEQ ID NO.10)(between Ung and APOBEC1(R33A))

SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO.11) (between APOBEC1 (R33A) and nCas9)

SGGSKRTADGSEFE(SEQ ID NO.12)(between nCas9 and NLS)

SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO.13) (between Hmces and APOBEC1)

SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO.14) (between APOBEC1 and nCas9)

SGGSKRTADGSEFE(SEQ ID NO.15)(nCas and NLS)

SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO.16) (between Hmces and AID)

SGSETPGTSESATPES(SEQ ID NO.17)(between AID and nCas9)

SGGS(SEQ ID NO.18)(nCas and NLS)

In the fusion protein for single base editing provided by the present invention, the fusion protein includes an amino acid sequence including the sequence shown in SEQ ID NO. 19 or SEQ ID NO. 20 or SEQ ID NO. 21.

Hmces-linker peptide-eUng-linker peptide-APOBEC1(R33A)-linker peptide-nCas9-linker peptide-NLS

MCGRTSCHLPREVLTRACAYQDRQGRRRLPQWRDPDKYCPSYNKSPQSSSPVLLSRLHFEKDADSSDRIIIPMRWGLVPSWFKESDPSKLQFNTTNCRSDTIMEKQSFKVPLGKGRRCVVLADGFYEWQRCQGTNQRQPYFIYFPQIKTEKSGGNDASDSSDNKEKVWDNWRLLTMAGIFDCWEAPGGECLYSYSIITVDSCRGLSDIHS RMPAILDGEEAVSKWLDFGEVATQEALKLIHPIDNITFHPVSPVVNNSRNNTPECLAPADLLVKKEPKANGSSQRMMQWLATKSPKKEVPDSPKKDASGLPQWSSQFLQKSPLPAKRGATSSFLDRWLKQEKEDEPMAKKPNSSGGSSGGSSGSETPGTSESATPESSGGSSGGSANELTWHDVLAEEKQQPYFLNTLQTVASERQSGVTIYPPQKDVFNAFR FTELGDVKVVILGQDPYHGPGQAHGLAFSVRPGIAIPPSLLNMYKELENTIPGFTRPNHGYLESWARQGVLLLNTVLTVRAGQAHSHASLGWETFTDKVISLINQHREGVVFLLWGSHAQKKGAIIDKQRHHVLKAPHPSPLSAHRGFFGCNHFVLANQWLEQRGETPIDWMPVLPAESESGGSGGSGGSSSETGPVAVDPTLRRRIEPHEFEVFFDPRELAKETC LLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGGSSGGSSGSETPGTSESATPESSGGSSGG SDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRR LENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFY PFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDD KVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSID NKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIR KRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSKRTADGSEFEPKKKRKV(SEQ ID NO.19)

Hmces-linker peptide-APOBEC1-linker peptide-nCas9-linker peptide-NLS

MCGRTSCHLPREVLTRACAYQDRQGRRRLPQWRDPDKYCPSYNKSPQSSSPVLLSRLHFEKDADSSDRIIIPMRWGLVPSWFKESDPSKLQFNTTNCRSDTIMEKQSFKVPLGKGRRCVVLADGFYEWQRCQGTNQRQPYFIYFPQIKTEKSGGNDASDSSDNKEKVWDNWRLLTMAGIFDCWEAPGGECLYSYSIITVDSCRGLSDIHS RMPAILDGEEAVSKWLDFGEVATQEALKLIHPIDNITFHPVSPVVNNSRNNTPECLAPADLLVKKEPKANGSSQRMMQWLATKSPKKEVPDSPKKDASGLPQWSSQFLQKSPLPAKRGATSSFLDRWLKQEKEDEPMAKKPNSSGGSSGGSSGSETPGTSESATPESSGGSSGGSSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQ NTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVIT DEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSR KLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSE EVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSKRTADGSEFEPKKKRKV(SEQ IDNO.20)

Hmces-linking peptide-AID-linking peptide-nCas9-linking peptide-NLS

MCGRTSCHLPREVLTRACAYQDRQGRRRLPQWRDPDKYCPSYNKSPQSSSPVLLSRLHFEKDADSSDRIIIPMRWGLVPSWFKESDPSKLQFNTTNCRSDTIMEKQSFKVPLGKGRRCVVLADGFYEWQRCQGTNQRQPYFIYFPQIKTEKSGGNDASDSSDNKEKVWDNWRLLTMAGIFDCWEAPGGECLYSYSIITVDSCRGLSDIHS RMPAILDGEEAVSKWLDFGEVATQEALKLIHPIDNITFHPVSPVVNNSRNNTPECLAPADLLVKKEPKANGSSQRMMQWLATKSPKKEVPDSPKKDASGLPQWSSQFLQKSPLPAKRGATSSFLDRWLKQEKEDEPMAKKPNSSGGSSGGSSGSETPGTSESATPESSGGSSGGSMDPATFTYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCH VELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLAEAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILQSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQE IFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADILLLFLAAKNLSDASDI LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIAN LAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVA KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATHQSITGLYETRIDLSQLGGDS GGSPKKKRKV(SEQ ID NO.21)

A third aspect of the invention provides a polynucleotide encoding a fusion protein as described above.

A fourth aspect of the invention provides a nucleic acid construct comprising a polynucleotide as described above.

A fifth aspect of the present invention provides an expression system, which contains the nucleic acid construct as described above or the polynucleotide as described above integrated into the genome. The expression system can be E. coli or the like. In a preferred embodiment of the present invention, the expression system is DH5α.

The sixth aspect of the present invention provides the single base editing system as described above, the fusion protein as described above, the polynucleotide as described above, the nucleic acid construct as described above or the expression as described above Use of the system in base editing. The use may be, for example, base conversion, gene inactivation, etc. The single base editing system, fusion protein, polynucleotide, nucleic acid construct or expression system provided by the invention can prevent the generation of abasic sites (AP sites) during the base editing process, thereby reducing the base editing process. The DNA damage produced in the process can then reduce the production of various by-products mediated by DNA damage, thereby achieving efficient base editing.

A seventh aspect of the present invention provides a gene editing method, including: performing gene editing through the base editing system as described above or the fusion protein as described above.

An eighth aspect of the present invention provides the use of HMCES as an abasic site protection polypeptide for gene editing or for use in a base editing system.

In the uses provided by the present invention, the HMCES is used to reduce DNA double-strand break damage in single base editing.

In the uses provided by the invention, the single base editing is cytosine (C) to guanine (G) base editing.

The implementation of the present invention is described below with specific embodiments. Those familiar with this technology can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for describing specific specific embodiments, It is not intended to limit the scope of the present invention. Test methods without specifying specific conditions in the following examples usually follow conventional conditions or conditions recommended by each manufacturer.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, those skilled in the art can also use methods, equipment, and materials described in the embodiments of the present invention based on their understanding of the prior art and the description of the present invention. Any methods, equipment and materials similar or equivalent to those in the prior art may be used to implement the present invention.

In the following examples of this application, the pcDNA3 plasmid can be found in Document 1 (Citation: An W, Zhang Z, Zeng L, Yang Y, Zhu X, Wu J (2015) Cyclin Y Is Involved in the Regulation of Adipogenesis and Lipid Production.PLoS ONE 10(7):e0132721. https://doi.org/10.1371/journal.pone.0132721).

The PX33R plasmid is based on PX330 (Addgene plasmid#42230). After being digested by Sbf I and Kpn I endonucleases, it is ligated by T4 ligase to obtain the PX33R vector without Cas9 component.

Example 1 Research on editing products produced by base editors and related loss repair mechanisms

In this example, in order to analyze the editing products produced by different DNA damage types of CBE and their related DNA damage repair mechanisms, an orthogonal screening base editing system combining SpCas9 and SaCas9-based SaBE was constructed, and then the base editor was examined. During the base editing process, relevant DNA damage repair mechanisms produce different editing products. Includes the following:

1.1 Construction of base editor library

The currently widely used cytosine base editor (CBE) will theoretically form three types of DNA damage gaps, as shown in Figure 1. Commonly used CBEs (Huang et al., 2021), such as AID-SaBE3, will The non-targeted strand editing of sgRNA produces uracil (U), nCas9 with partial cleavage activity generates a nick in the targeted strand cleavage, and the fusion-expressed UGI component can inhibit the excision of uracil; CGBEs (Kurt et al., 2021; Zhao et al., 2021), such as AID-SaBE1n, which produces damage gaps similar to CBEs, but such tools do not limit or even promote the resection of U; dCBEs (Hess et al., 2017; Komor et al., 2016), such as AID-SaBE1, produces U only in non-targeted strand editing.

An orthogonal screening system combining SpCas9 and SaCas9-based SaBE, using AID mutants as cytidine deaminase components, constructed three base editors, including CBE (AID-SaBE3), CGBE (AID-SaBE1n) and dCBE(AID-SaBE1).

1) First, SpCas9 targeted cleavage of 513 DNA damage repair factors to obtain cell libraries deficient in different factors. At the same time, the specific SaBE-Test sequence located downstream of the sgRNA stem-loop structure was stably expressed. See A in Figure 2 for details.

2) Then target and edit the SaBE-Test sequence by transfecting SaBEs, and simultaneously amplify the SpCas9 sgRNA and the edited SaBE-Test sequence through the same amplicon, so that the sgRNA expression of the DNA damage repair factor can be compared with different bases The editing products correspond one to one, see A in Figure 2 for details.

The obtained cell libraries were transfected with the three base editors respectively, and each base editor had two repeated experiments.

1.2 Result analysis

1.2.1 Cytosine single base editor editing products are generated by different DNA damage-mediated

A total of 100 million cells edited by three base editors were deeply sequenced with an average of 96.5 million read sequences, achieving approximately 200-fold coverage of all sgRNA libraries (5985 sgRNAs) with low-frequency products (1%). .

Sequences containing negative control sgRNA (approximately 6 million per tool) were extracted from nearly 100 million sequencing sequences as wild-type controls, and the base substitution mutation profile of each SaBE was drawn, including the frequency of base substitution mutations, Map of location, and mutation type (C to T/G/A).

According to different DNA damage patterns, the three Sa-CBEs produced different base substitution mutation patterns. The results are shown in Figure 2.

As can be seen from Panel B in Figure 2, CBE (AID-SaBE3) mainly produces C>T base conversion mutations within the editing window.

As can be seen from Panel C in Figure 2, CGBE (AID-SaBE1n) increases the frequency of C>A/G base transversion mutations within the editing window.

As can be seen from Figure 2, D, dCBE (AID-SaBE1) has a higher frequency of base substitution mutations within the editing window and even outside the sgRNA spacer sequence, and has a higher proportion of C>A/G base transversion mutations. .

In summary, the base substitution mutations of AID-based cytosine SaBEs are consistent with expectations.

1.2.2 In-depth analysis of base transversion mutations and deletion mutations in low-frequency base editing products

From the results of 1.2.1, it can be seen that CGBE (AID-SaBE1n) and dCBE (AID-SaBE1) will produce more base transversion mutations. The profile of base substitution mutations in the SaBE-Test sequence was further analyzed.

In the SaBE-Test sequence, the patterns of base transversion mutations C>G and C>A are different. Judging from the five main editing sites, C>G tends to occur more frequently at specific sites. , such as positions close to PAM within the editing window (AID-SaBE1n and AID-SaBE3), or positions outside the sgRNA spacer on the targeting strand (AID-SaBE1), while the position where C>A is generated does not show any The preference indicates that C>G and C>A may be produced through different DNA damage repair pathways.

Deep sequencing showed that there were a large number of base deletions and insertion mutations in the CBE editing products. The results are shown in Figure 3.

It can be seen from A in Figure 3 that the CBE (AID-SaBE3) editing product contains approximately 2.7% small fragment deletion mutations.

As shown in Figure 3B, the proportion of deleted mutations in the edited product of CGBE (AID-SaBE1n) increased to 50.6%.

As can be seen from C in Figure 3, the proportion of deleted mutations in the edited product of dCBE (AID-SaBE1) is 20.1%.

Further, the results of the analysis of 1-bp and >1-bp deletion mutations were analyzed.

As can be seen from D in Figure 3, 1-bp deletion mutations often occur on targeted edited bases, and the frequency of base deletion mutations produced by dCBE (AID-SaBE1) in regions other than the sgRNA spacer is relatively high.

As can be seen from E to G in Figure 3, they are the results of analysis of deletion mutations >1 bp. By counting the start and stop sites of deletion mutations that occur, the base deletion mutation map shows that the start site often corresponds to the editing site, while the stop site corresponds to part of the C on the target strand.

It can be seen from F and G in Figure 3 that for deletion mutations >1 bp, the nCas9 cleavage enzyme activity in CGBE (AID-SaBE1n) or CBE (AID-SaBE3) will shift the termination site of the deletion mutation toward the PAM sequence. .

In summary, deep sequencing was used to analyze the base deletion mutation frequencies and patterns of three different cytosine base editors, revealing that the specific mechanisms of base deletion mutations produced by different types of base editors may be different. We then further investigated its mechanism.

1.2.3 Overview of DNA damage repair pathways mediated by cytosine base editors

The deep sequencing data first needs to go through a series of quality control (QC) steps, such as the coverage of sgRNA and the overall distribution of different sgRNAs. Then, the SaBE-Test original sequence is used as the reference sequence, and the sequenced sequence is further divided into Mutation group, C>T group, C>G group, C>A group, base deletion mutation group and base insertion mutation group.

In order to analyze the DNA damage repair pathways related to base editing products, we first screened the enriched genes by comparing the base editing mutation group and the non-mutation group, and analyzed their functions using the information from the GO database.

1) Statistics on the correlation between different DNA damage repair pathways, such as base excision repair pathway (BER), mismatch repair pathway (MMR), and nucleotide excision repair pathway (NER), and cytosine base editors. Specifically, the enriched genes that affect base editing efficiency were statistically analyzed. After GO enrichment analysis, the significant p-value of enrichment in different pathways was obtained. The results are shown in Figure 4.

As can be seen from A in Figure 4, different DNA damage repair pathways, including base excision repair pathway (BER), mismatch repair pathway (MMR) and nucleotide excision repair pathway (NER), are all involved in cytosine base editing. play different roles. The base excision repair pathway (BER) mainly plays an inhibitory role in cytosine base editing.

2) Correlation analysis was performed on the genes enriched during the base editing process of three cytosine SaBEs. The results are shown in Figure 4.

As can be seen from B in Figure 4, a correlation analysis of the genes enriched during the base editing process of the three cytosine SaBEs revealed that the genes enriched by CGBE and dCBE have a high correlation (r=0.66).

It can be seen from C in Figure 4 that the correlation between genes enriched by CGBE and CBE is weak (r=0.39).

Based on B and C in Figure 4, it can be seen that removing uracil (CGBE/dCBE) or retaining uracil (CBE) is the key factor leading to different enrichment results of DNA damage repair factors.

As shown in Figure 4B, NER factors include XPA, USP7 and ERCC4, which are enriched in the base editing mutation group.

3) The enrichment of genes encoding glycosylases and their downstream factors was statistically analyzed. The results are shown in Figure 4.

As can be seen from B and C in Figure 4, it is enriched in the non-mutation group, but it is worth noting that SMUG1 is a special factor that promotes CBE (AID-SaBE3) editing to generate C>T base conversion mutations, which may be caused by the recent SMUG1 has been reported to play a role in inducing mutations (Taglialatela et al., 2021).

4) We further selected 58 genes that were significantly enriched in the abasic mutation group for cluster analysis. The results are shown in Figure 4.

As can be seen from D in Figure 4, cluster analysis of the editing products shows that DNA double-strand break repair (DSBR) and non-homologous end joining (NHEJ) can be clearly seen based on the results of base insertion/deletion mutations. Aggregation of factors, and base insertion/deletion mutations are significantly reduced in cells lacking these factors.

5) In the CRISPR-Cas9 library screening experiment, the gene enrichment of different DNA damage repair pathway genes was compared between the C>T group and the non-mutation group. The results are shown in Figure 5.

A in Figure 5 shows the enrichment analysis results of base excision repair pathway (BER) factors after comparing the editing mutation group and the non-mutation group.

As shown in Figure 5B, the mismatch repair pathway (MMR) plays a weak role in base editing, which is consistent with the role of MMR in diverse AID-deficient mouse B lymphocytes (Rada et al., 2004) or The important role played by the lead base editor (Primediting) (Chen et al., 2021b) is different, which may be due to the defective MMR of the HEK293T cell line itself (Trojan et al., 2002), resulting in the inability of MMR factors to were screened out from the invented system.

It can be seen from Figure 5, C, that the nucleotide excision repair pathway (NER) promotes the cytosine base editing process.

Base transversion mutations and base conversion mutations clustered separately, indicating that there are different DNA damage repair pathways for base conversion and transversion. From a genetic perspective, we also found that genes with similar functions tend to cluster together. For example, 53BP1-dependent DSBR factors show obvious clustering.

Overall, the library screening overview of cytosine base editor editing results provides a rich resource for analyzing their related DNA damage repair mechanisms.

1.2.4 Base editor mediates DSB to generate small base deletion mutations

Ultra-deep sequencing of DNA damage repair gene library screening related to cytosine base editing results can provide in-depth analysis of the molecular mechanism of low-frequency small fragment base deletion mutations during the cytosine base editing process.

1) By comparing the gene enrichment of the base deletion mutation group and the C>T base substitution group, the results are shown in Figure 6.

It can be seen from A in Figure 6 that BER, DSBR and NHEJ factors can promote the generation of base deletion mutations.

It can be seen from Figure 6, B, that BER factors UNG and APEX1 mediate the conversion of the deamination product of the base editor into DSB, thereby activating the DSBR and end joining pathways. These small fragment deletion mutations may be the products of Artemis-dependent end excision repair ( Lobrich and Jeggo, 2017).

2) In order to analyze the base deletion mutation patterns of different cytosine base editors at different endogenous genomic sites, SpCas9-based cytosine base editors were selected, including BE4max (CBE) (Koblan et al., 2018) and three CGBEs, CGBE1 (Kurt et al., 2021), AXC (Koblan et al., 2021) and AID-BE1n, a total of 4 endogenous genome targeting sites were selected for testing. The results are shown in Figure 6.

As can be seen from C in Figure 6, the cytosine base editor BE4max has a higher editing efficiency, while the cytosine to guanine base editors CGBE1, AXC and AID-BE1n produce more C>G base transversions mutations, and has a higher frequency of base deletion mutations than BE4max.

3) By detecting the base deletion mutation patterns of different base editors at endogenous genomic sites in the HEK392T cell line, analyze the frequency of deletion mutations occurring at different positions.

It can be seen from D in Figure 6 that among these cytosine base editors, the deaminase components of BE4max (with UGI), CGBE1 (with eUNG), and AXC (with UdgX) are APOBEC1-derived mutants, and The deaminase component of AID-BE1n is an AID-derived mutant.

Furthermore, four commonly used base editing sites were detected, and nearly 1 million sequencing sequences of 1 million base-edited cells were analyzed for each site targeted by each editing tool. The results are shown in Figure 6.

As can be seen from D in Figure 6, both CGBE1 and AID-BE1n produce higher frequency base deletion mutations at these three endogenous genome editing sites. The start or end site of the base deletion mutation >1 bp corresponds to the base editing site or Cas9 cleavage site.

Taken together, it can be seen that DNA damage produced by CGBEs is more easily processed into DSBs and base deletion mutations.

The above jointly prove that CGBE will produce higher levels of DNA double-strand breaks (DSBs) at the targeted editing site, which deserves our attention, and these DSBs can be processed into small fragment base deletion mutations through the DNA end joining pathway. .

1.2.5 DSB end joining and translesion repair contribute to the generation of C>G base transversion mutations

C>G base transversion mutation is the target product of CGBE. In order to study the molecular mechanism involved in C>G base transversion mutations, the enriched genes of the two groups were analyzed by comparing the C>G and C>T groups. The results are shown in Figure 7.

1) From A in Figure 7, it can be seen through screening that there are three types of genes that play a role in the generation of C>G base transversions. The first type is represented by UNG. This is consistent with the recently developed CGBE which increases C by introducing UNG. >The theory of G transversion mutation is consistent (Koblan et al., 2021; Kurt et al., 2021; Zhao et al., 2021).

2) From A in Figure 7, it can be seen that the second type of factors that promote C>G base transversion are translesion repair pathway (TLS)-related factors, including the three genes RFWD3, RAD18 and DTL encoding PCNA ubiquitin ligase, and The gene REV1 encoding TLS polymerase.

3) The third category involved in the C>G base transversion process is the genes encoding two endonucleases and the genes of the NHEJ pathway.

It can be seen from A in Figure 7 that the genes encoding two endonucleases are APEX1 and XPG. APEX1 is responsible for cutting the AP site in the BER pathway, thereby generating a single-stranded nick (Robson and Hickson, 1991; Seki et al., 1991), while XPG acts as a NER pathway factor and is a structurally specific endonucleic acid cleavage enzyme responsible for cutting the 3' end of the bubble-structured double-stranded DNA (O'Donovan et al., 1994).

As can be seen from Figure 7B, APEX1 and/or XPG can convert the non-blunt DNA damage generated by the base editor into DSB, and the damaged end is filled with bases to generate a C>G base transversion mutation.

As can be seen from A in Figure 7 , the enrichment of the three core NHEJ factors XRCC4, XLF, and LIG4 indicates that they may play a promoting role in C>G base transversion mutations.

To test this model, C>G base editing was performed in cells deficient in NHEJ factors.

As can be seen from Figure 7, C, in the HEK293T cell line deficient in NHEJ factor XRCC4 or TLS factor REV1, the proportion of C>G mutations is reduced, and the frequency of accompanying base deletion mutations is also reduced. It can be seen that, in addition to the previously observed TLS pathway-mediated C>G transversion mutations (Koblan et al., 2021), a new NHEJ-dependent C>G mutation generation pathway has been discovered, and this process includes DSB Generation and end-joining processes.

In order to detect the impact of different DNA targeted editing sequences on C>G transversion mutations, four tools were tested on wild-type, XRCC4 (NHEJ factor) knockout and REV1 (TLS factor) knockout HEK293T cell lines. Mutation status to 10 base sites of 6 sgRNAs (Figure 7D).

As can be seen from D in Figure 7, the results show that TLS affects the C>G transversion frequency of all six tested sgRNAs, while NHEJ only significantly promotes the C>G transversion mutation frequency at a specific site (FANCF). .

In summary, it is demonstrated that the cis-base editing target DNA sequence and the trans-DNA damage repair factors jointly regulate the base editing results of CGBE.

1.2.6 CGBEs can cause chromatic translocation

To examine whether DSBs generated by cytosine base editors mediate the formation of chromosomal translocations, an existing high-throughput genome-wide translocation sequencing (HTGTS) method was adapted to capture rare chromosomal translocation events more efficiently. .

The newly designed Tn5-HTGTS method combines the Tn5 translocase labeling method and the chromosomal translocation cloning method. The Tn5-HTGTS method is detailed in Figure 8, A, and can detect the entire genome derived from the single base editor targeted editing site. Translocation map.

In order to verify the chromosomal translocation caused by the cytosine base editor in the endogenous genome, Tn5-HTGTS experiments were performed on cell samples transfected with the SpCas9-based cytosine base editor and 4 endogenous gene-targeting sgRNAs. .

As can be seen from Figure 8, B, compared with BE4max (purchased from Addgene #112093) (circos diagram on the left in B), CGBE including CGBE1 and AID-BE1n will produce more chromosomal translocations.

As can be seen from Figure 8 C, an average of 3 chromosomal translocations per 1000 input cells can be detected in the CGBE1 sample, while an average of 8 chromosomal translocations can be detected per 1000 input cells in the AID-BE1n sample.

It can be seen from D in Figure 8 that at the Cas-OT site, AID-BE1n produces a greater number and a wider range of chromosomal translocations than CGBE1, indicating that the chromosomal translocations produced by different deaminase components of the base editor With distinct characteristics (Liue et al., 2018), AID-BE1n produces chromosomal translocations at AID-OT sites defined by chromatin features including transcription and super-enhancers (Meng et al., 2014; Qian et al., 2014) has aggregation phenomenon.

It can be seen from E in Figure 8 that CGBE1 containing the APOBEC1 component does not have this aggregation phenomenon.

To date, we have not detected any APOBEC1 deaminase-preferred off-target sites for CGBE1.

As can be seen from F in Figure 8, the position comparison results of PRO-seq sequencing show that 7.4% and 8.8% of the chromosomal translocations generated by CGBE1 and AID-BE1n respectively fall in the transcribed region. Other sequencing results Analysis also showed that these translocation junction sites corresponded to peak sites of active transcription, chromosome openness, and activity (Figure 8F), indicating that these chromosomal translocations are likely to have adverse effects.

In order to further determine whether a small number of chromosomal translocations originate from deaminase off-target sites, we analyzed the enrichment of short fragments near the junction of chromosomal translocations.

As can be seen from G in Figure 8 , the TC motif (APOBEC1 preferential motif) was found to be enriched in CGBE1 samples.

It can be seen from G in Figure 8 that sequences with deaminase preferential targeting and palindromic motifs are more likely to undergo chromosomal translocation. For example, compared with TCH (H: non-G), CGBE1-preferred TCG is near the occurrence of chromosomal translocation. The sequence content is relatively high. In the palindrome motif, deamination occurs simultaneously on both positive and negative DNA strands, and DSBs are more likely to form after both strands are simultaneously responded to by downstream DNA damage repair factors.

In summary, from the schematic diagram summarized by H in Figure 8, it can be seen that CGBE will produce chromosomal translocations between the target editing site and the off-target preference site. The latter includes Cas-OT and deaminase-OT, while deamination Sequences characterized by palindromic motifs that are preferred by the enzyme have a higher probability of double-strand breaks.

Example 2

In this example, a recombinant vector overexpressing Hmces is constructed, and then combined with a vector containing nucleic acid nickase and cytosine deaminase to form a single base editing system and perform base editing. Specifically: clone the Hmces gene sequence into the pcDNA3 vector to obtain the recombinant vector pcDNA3-Hmces, and then simultaneously transfect the base editor and the recombinant vector pcDNA3-Hmces in a 24-well plate to construct a single base editing system CGBE-plus. -HMCES (referred to as CGBEpH). Includes the following steps:

2.1. Construction of recombinant vector pcDNA3-Hmces

2.1.1 Design and synthesize primers

Beijing Qingke Biotechnology Co., Ltd. was entrusted to synthesize primers for adding homology arms to the Hmces sequence. The amino acid sequence of Hmces includes the sequence shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. The forward and reverse primers are as follows:

Forward and reverse primers (Q-CMV-F and Hmces-EcoRI-R) used to amplify the first sequence of mouse Hmces:

Q-CMV-F:AGGCGTGTACGGTGGGAGGT(SEQ ID NO.22)

Hmces-EcoRI-R: CAGGACGTTCCGCCCGCACATggtggcgaattccagcacactggcgg (SEQ IDNO.23)

Forward and reverse primers (Hmces-F and Hmces-XhoI-R) used to amplify the second sequence of mouse Hmces:

Hmces-F: ATGTGCGGGCGAACGTCCTG (SEQ ID NO.24)

Hmces-XhoI-R:tccttgtagtcctcgagGCTGTTAGGCTTCTTGGCCA (SEQ ID NO. 25)

2.1.2 Obtain Hmces sequence with homology arms

Use ddH ₂ O to dissolve the forward and reverse primers in step 2.1.1 in this example to 100M and dilute to 10M.

Add the forward and reverse primers to the following reaction system. The reaction system is shown in Table 1. Perform the PCR reaction to obtain the PCR product.

Table 1

substance Adding amount In this example, the diluted forward and reverse primers 1μL each Template(cDNA) 1μL (about 500ng) HF buffer(5×) 5μL Phusion (F530, Thermo Fisher) 0.25μL dNTPs 0.5μL ddH ₂ O to 25μL

PCR reaction procedure:

The PCR product fragment was purified by gel and purified according to the instructions of the universal DNA purification and recovery kit (TIANGEN) to obtain Hmces containing homology arms.

2.1.3 Restriction digestion of plasmid pcDNA3

Use EcoRI and XhoI restriction endonucleases to digest plasmid pcDNA3. The digestion reaction system is as shown in Table 2 to obtain the digestion product.

Table 2

substance Adding amount NEB rCutSmart buffer(10×) 5μL EcoRI 1.5μL ikB 1.5μL Plasmid pcDNA3 5μL (approximately 5μg) ddH ₂ O to 50μL

Reaction conditions: 37°C water bath for 3 hours.

Gel recovery and purification of the enzyme digestion product fragment was carried out according to the instructions of the universal DNA purification and recovery kit (TIANGEN) to obtain the digested plasmid pcDNA3.

2.1.4 Ligase digested plasmid and target fragment

Connect the digested plasmid pcDNA3 obtained in step 2.1.3 in this example and the DNA fragment with homology arms obtained in step 2.1.2 in this example to obtain a ligation product. The ligation system is shown in Table 3.

table 3

substance Adding amount 2×Hieff Clone Enzyme Premix(YEASEN) 5μL The digested pcDNA3 plasmid obtained in step 2.1.3 in this example 2μL Hmces containing homology arms obtained in step 2.1.2 in this example 3μL ddH ₂ O to 10μL

Reaction conditions: 50℃, 30min.

2.1.5 Transformation of ligation products

Use DH5α competent to transform the ligation product obtained in step 2.1.4 in this example, and culture it on an LB plate containing ampicillin antibiotic (Amp, 100 mg/L) overnight at 37°C.

2.1.6 Pick single clones for sequencing

Pick a single colony from the ampicillin antibiotic LB plate in step 2.1.5 in this example, transfer it to LB (Amp, 100 mg/L) liquid medium and culture it overnight. Plasmid extraction was performed according to the instructions of plasmid miniprep kit (TIANGEN). The extracted plasmid was sent to the Shanghai Sequencing Department of Beijing Qingke Biotechnology Co., Ltd. for sequencing.

2.1.7 The plasmid was successfully sequenced and extracted.

The successfully sequenced plasmid was re-transformed with DH5α competent bacteria and cultured on an LB plate containing Amp (100 mg/L) overnight at 37°C. Pick a single colony and inoculate it into 300ml LB (Amp, 100mg/L) liquid medium and culture it at 37°C overnight. Collect the bacteria and extract the plasmid according to the instructions of the plasmid extraction kit (MACHEREY-NAGEL) to obtain the recombinant vector pcDNA3-Hmces.

2.2. Base editor plasmid preparation

Order plasmid CGBE1 from Addgene, containing eUNG, APOBEC1 and nCas9 expression elements.

At the same time, the plasmid PCMV-BE3 (purchased from Addgene #73020) was modified, the UGI component was removed, and the APOBEC1 component was replaced with AID, containing AID and nCas9 expression elements, to construct the plasmid AID -BE1n.

For the construction of vectors for expression of sgRNA, after the forward and reverse sequences of sgRNA were synthesized at Beijing Qingke Biotechnology Co., Ltd., they were heated, boiled and then cooled naturally to obtain a double-stranded nucleotide sequence in which the forward and reverse sequences were paired and combined, and then digested with BbsI The products of the PX33R plasmid are ligated (T4 ligase). After the ligation product is transformed into the DH5α competent state, it is cultured on an LB plate containing ampicillin antibiotic (Amp, 100 mg/L) overnight at 37°C. Single colonies are picked and amplified in the LB medium. Afterwards, the plasmid was extracted according to the instructions of the plasmid miniprep kit (purchased from TIANGEN), and sequenced by the Shanghai Sequencing Department of Beijing Qingke Biotechnology Co., Ltd. After correct comparison, the vector expressing sgRNA was obtained.

Plasmid CGBE1, plasmid AID-BE1n or vector expressing sgRNA were transformed into DH5α competent bacteria, and cultured on LB plates containing ampicillin antibiotic (Amp, 100 mg/L) overnight at 37°C.

Pick a single colony and inoculate it into 300ml LB (Amp, 100mg/L) liquid medium and culture it at 37°C overnight.

Collect the bacteria and extract the plasmid according to the instructions of the plasmid extraction kit (purchased from MACHEREY-NAGEL).

2.3. Use Lipofectamine 2000 for cell transfection

1) 16-24 hours before transfection, spread HEK293T cells into a 48-well plate at a density of 5.5×10 ⁴ cells/well, and culture them in a 37°C cell culture incubator containing 5% CO ₂ so that the next day Cell density is approximately 60-80%.

2) One hour before transfection, replace the medium with DMEM complete anti-antibody medium (added with 10% fetal calf serum, without penicillin and streptomycin antibodies).

3) Combine the plasmid CGBE1 and plasmid AID-BE1n (each 750ng) obtained in step 2.2 of this example with the recombinant vector pcDNA3-Hmces (500ng) and sgRNA (250ng) obtained in step 1.3 of this example (where sgRNA includes FANCF, RNF2, EMX1) were transfected into cells by Lipofectamine 2000 (invitrogen), with three replicates for each sample.

Plasmid CGBE1 and recombinant vector pcDNA3-Hmces form a single base editing system, referred to as CGBE1pH; plasmid AID-BE1n and recombinant vector pcDNA3-Hmces form a single base editing system, referred to as AID-CGBEpH. CGBE1pH and AID-CGBEpH are collectively referred to as the single base editing system CGBEpH. D in Figure 10 is a schematic diagram of the construction of the single base editing system CGBEpH in this embodiment.

sgRNAs targeting sequences for different sites:

FANCF: GGAATCCCTTCTGCAGCACC(SEQ ID NO.26)

RNF2：GTCATCTTAGTCATTACCTG(SEQ ID NO.27)

EMX1：GAGTCCGAGCAGAAGAAGAA(SEQ ID NO.28)

4) Three days after transfection, collect the cells and extract the genome.

2.4. Preparation of high-throughput sequencing libraries

For cell samples (>0.3×10 ⁶ cells) used for 48-well plate transfection, cell lysis buffer (200mM NaCl, 50mM Tris-Cl pH8.0, 5mM EDTA, 0.2% SDS, 100μg/ml proteinase K) was used. After lysis at 56°C overnight, genomic DNA was precipitated using ethanol precipitation, and then the genomic DNA was dissolved in TE buffer (10mM Tris-Cl pH 8.0, 0.1mM EDTA). Then the genomic DNA solution corresponding to 300,000 cells was used as a template in a 50 μl PCR reaction system for the first round of PCR amplification. In the first round of PCR amplification process, the amplification primers used for FANCF are FANCF-p5 and FANCF-p7, the amplification primers used for RNF2 are RNF2-p5 and RNF2-p7, and the amplification primers used for EMX1 are EMX1- p5 and EMX1-p7.

The PCR amplification enzymes we used in library construction include high-fidelity DNA polymerase Phusion (F530, ThermoFisher) and Pfu (FastPfu Fly DNAPolymerase, TransStart, AP231-01). Since the first round of PCR adds sequence tags to different samples, the first round of PCR products can then be mixed and used as templates for the second round of PCR amplification. The second round of PCR used PE-P5-Short and P7-index primers for amplification.

The final PCR product was cut according to the size of the target product through agarose gel electrophoresis, gel purified using a DNA purification kit (TIANGEN), and then the purified DNA product was quantified using a Qubit quantification kit (Life Technologies). High-throughput sequencing of amplified libraries was performed by illumina Hiseq (PE150).

High-throughput sequencing primers for amplification targeting the different sites mentioned above:

(Normal deoxyoligonucleotide) FANCF-p5:

TTCCCTACACGAGCTCTTCCGATCTggctacCATTGCAGAGAGGCGTATCA

(SEQ ID NO.29)

(Anti-deoxyoligonucleotide) FANCF-p7:

AGTTCAGACGTGTGCTCTTCCGATCTggctacGGGGTCCCAGGTGCTGAC

(SEQ ID NO.30)

(Normal deoxyoligonucleotide) RNF2-p5:

TTCCCTACACGACGCTCTTCCGATCTggctacAACGTAGGAATTTTGGTGGGACA

(SEQ ID NO.31)

(Anti-deoxyoligonucleotide) RNF2-p7:

AGTTCAGACGTGTGCTCTTCCGATCTggctacACGTCTCATATGCCCCTTGG

(SEQ ID NO.32)

(Normal deoxyoligonucleotide) EMX1-p5:

TTCCCTACACGAGCTCTTCCGATCTggctacGGGCCTCCTGAGTTTCTCAT

(SEQ ID NO.33)

(Anti-deoxyoligonucleotide)EMX1-p7:

AGTTCAGACGTGTGCTCTTCCGATCTggctacCTCGTGGGTTTTGTGGTTGC

(SEQ ID NO.34)

The first round of PCR amplification system was used for amplification to obtain the first round of PCR amplification products. The PCR amplification system is shown in Table 4.

substance Adding amount Ordodeoxyoligonucleotides/antideoxyoligonucleotides targeting FANCF or RNF2 or EMX1 2μL each Template (cell genomic DNA after transfection in step 2.3 in this example) 5μL (approximately 5μg) HF buffer(5×) 10μL Phusion (F530, Thermo Fisher) 0.5μL dNTPs 1μL ddH ₂ O to 50μL

First round PCR reaction procedure:

Second round of PCR amplification system:

Positive deoxyoligonucleotide (PE-P5-Short):

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC(SEQ ID NO.35)

Anti-deoxyoligonucleotide (P7-index):

CAAGCAGAAGACGGCATACGAGAT******GTGACTGGAGTTCAGACGTGT

substance Adding amount Positive deoxyoligonucleotide PE-P5-Short/anti-deoxyoligonucleotide P7-index 1μL each First round PCR reaction products 2μL HF buffer(5×) 5μL Phusion (F530, Thermo Fisher) 0.25μL dNTPs 0.5μL ddH ₂ O to 25μL

Second round PCR reaction procedure:

2.5. Results

Analyze the probability of base deletion mutations (see Figure 9E) and repetitive sequence insertion mutations greater than 1 bp (see Figure 9E) produced after cells are transfected with different single base editing systems, and analyze the base editing efficiency and the bases produced by editing. The proportion of C>G in substitution mutation products (see Figure 9F), and the chromosomal translocation distribution map (see Figure 9G).

As can be seen from E in Figure 9, CGBEpH can significantly reduce base deletion mutations and also significantly reduce DSB-mediated >1bp repeated insertion mutations.

It can be seen from F in Figure 9 that CGBEpH does not affect the base editing mutation efficiency and the proportion of C>G mutations.

It can be seen from Figure 9G that compared with CGBE1, CGBE1pH can effectively reduce the probability of CGBE1 chromosomal translocation.

Example 3 Fusion protein containing HMCES

In this example, HMCES, cytosine deaminase and nucleic acid nickase are fused and expressed to form a single base editing system, and then base editing is performed. Specifically: the coding sequence of the mouse Hmces gene was amplified and cloned into the BE4B plasmid, CGBE1 plasmid and AID-BE1n plasmid respectively, and the fusion proteins HMCES-BE4B, HMCES-CGBE1 and HMCES-AID-BE1n were constructed respectively, and then transfected. Cells undergo single base editing. Includes the following steps:

3.1. Construction of expression vector containing fusion protein

3.1.1 Design and synthesize primers

Beijing Qingke Biotechnology Co., Ltd. was entrusted to synthesize primers for adding homology arms to the Hmces sequence. Forward and reverse primers:

Used to construct fusion protein HMCES-BE4B:

Forward and reverse primers used to amplify the first sequence of mouse Hmces linked to BE4B:

Template: BE4B

Q-CMV-F: AGGCGTGTACGGTGGGAGGT (SEQ ID NO.36)

Hmces-T7-R: ACGTTCGCCCGCACATGGTGGCGGCTCTCCCTATAGT (SEQ ID NO.37) forward and reverse primers used to amplify the second sequence of mouse Hmces connected to BE4B:

Template:pcDNA3-Hmces

Hmces-F: ATGTGCGGGCGAACGTCCTG (SEQ ID NO.38)

XTEN3-Hmces-R: CCGCCAGAACTACCACCGCTGCTGTTAGGCTTCTTGGCCA (SEQ ID NO.39)

Forward and reverse primers used to amplify the third sequence of mouse Hmces linked to BE4B:

Template:BE4B-N-GFP

XTEN3-F: AGCGGTGGTAGTTCTGGCGG (SEQ ID NO.40)

XTEN-BamHI-R:TCCTGGTGTCTCGCTGCCAGA (SEQ ID NO.41)

Used to construct fusion protein HMCES-CGBE1:

Forward and reverse primers used to amplify the first sequence of mouse Hmces linked to CGBE1:

Template:CGBE1

Q-CMV-F: AGGCGTGTACGGTGGGAGGT (SEQ ID NO.42)

Hmces-T7-R：ACGTTCGCCCGCACATGGTGGCGGCTCTCCCTATAGT(SEQ ID NO.43)

Forward and reverse primers used to amplify the second sequence of mouse Hmces connected to CGBE1:

Template:pcDNA3-Hmces

Hmces-F: ATGTGCGGGCGAACGTCCTG (SEQ ID NO.44)

XTEN3-Hmces-R: CCGCCAGAACTACCACCGCTGCTGTTAGGCTTCTTGGCCA (SEQ ID NO.45)

Forward and reverse primers used to amplify the third sequence of mouse Hmces connected to CGBE1:

Template:CGBE1-N-GFP

XTEN3-F: AGCGGTGGTAGTTCTGGCGG (SEQ ID NO.46)

XTEN-BamHI-R:TCCTGGTGTCTCGCTGCCAGA (SEQ ID NO.47)

Used to construct fusion protein HMCES-AID-BE1n:

Forward and reverse primers used to amplify the first sequence of mouse Hmces linked to AID-BE1n:

Template:AID-BE1n

Q-CMV-F: AGGCGTGTACGGTGGGAGGT (SEQ ID NO.48)

Hmces-T7-R：ACGTTCGCCCGCACATGGTGGCGGCTCTCCCTATAGT(SEQ ID NO.49)

Forward and reverse primers used to amplify the second sequence of mouse Hmces linked to AID-BE1n:

Template:pcDNA3-Hmces

Hmces-F: ATGTGTGGGCGAACATCCTG (SEQ ID NO.50)

XTEN3-Hmces-R: ACCGCCAGAACTACCACCGCTCTGGCTGTAAGGACGCTTGG (SEQ ID NO.51)

Forward and reverse primers used to amplify the third sequence of mouse Hmces linked to AID-BE1n:

Template:BE4B-N-GFP

XTEN3-F: AGCGGTGGTAGTTCTGGCGG (SEQ ID NO.52)

XTEN3-NLS-R3:

ACTTCCACCTGAAGATCCACCAGATGATTCGGGAGTCGCGCTTTCAG(SEQ ID NO.53)

Forward and reverse primers used to amplify the fourth sequence of mouse Hmces linked to AID-BE1n:

Template:AID-BE1n

XTEN3-AIDmono-F：gtggatcttcaggtggaagtATGGACCCCGCTACCTTCAC(SEQ IDNO.54)

dCas9-AID-R:

GCGGACTCTGAGGTCCCGGGAGTCTCGCTGCCGCTCTGAAGGATGCGCCGAA(SEQ ID NO.55)

3.1.2 Obtain the target fragment sequence

The method is the same as the construction method in step 2.1.2 in Example 2.

3.1.3 Respectively digest plasmid BE4B, plasmid CGBE1 and plasmid AID-BE1n

Use NotI and BamHI to digest plasmid BE4B (constructed) to obtain APOBEC1 and nCas fragments; use NotI and BamHI to digest plasmid CGBE1 (purchased) to obtain eUNG, APOBEC1 and nCas fragments; use NotI and SmaI to digest plasmid CGBE1 (purchased); use NotI and SmaI to digest plasmid BE4B (constructed). Plasmid AID-BE1n (constructed) was digested to obtain AID and nCas fragments.

The enzyme digestion system and method were the same as step 2.1.3 in Example 2.

3.1.4 Ligase digested plasmid and target fragment

The target fragment obtained in step 3.1.2 was ligated with the digested plasmid obtained in step 3.1.3 respectively. The ligation method was the same as step 2.1.4 in Example 2 to obtain HM-BE4B, HM-CGBE1 and HM- respectively. AID-BE1n.

3.1.5 Transformation of ligation products

The method is the same as step 2.1.5 in Example 2.

3.1.6 Pick single clones for sequencing

The method is the same as step 2.1.6 in Example 2.

Framework of the fusion protein:

HM-CGBE1: pCMV-Hmces-Ung-APOBEC1(R33A)-nCas9-NLS-EGFP

HM-BE4B: pCMV-Hmces-APOBEC1-nCas9-NLS

HM-AID-BE1n: pCMV-Hmces-AIDmono-nCas9-NLS

After sequencing, the amino acid sequence of the fusion protein is as follows:

The amino acid sequence of HM-CGBE1 is shown in SEQ ID NO. 19, the amino acid sequence of HM-BE4B is shown in SEQ ID NO. 20, and the amino acid sequence of HM-AID-BE1n is shown in SEQ ID NO. 21.

3.1.7 The plasmid was successfully sequenced and extracted.

The method is the same as step 2.1.7 in Example 2.

3.2. Use Lipofectamine 2000 for cell transfection

Same as step 2.3 in Example 2.

At the same time, GFP was used instead of HMCES to obtain corresponding base editing systems, such as GFP-BE4B, GFP-CGBE1 and GFP-AID-BE1n, as controls.

B in Figure 10 shows the single base editing systems HMCES-BE4B, HMCES-CGBE1 and HMCES-AID-BE1n constructed after the BE4B plasmid, CGBE1 plasmid and AID-BE1n plasmid were respectively fused to express HMCES in this example.

3.3. Preparation of high-throughput sequencing libraries

Same as step 2.4 in Example 2.

3.4. Results

Analyze the editing efficiency of different base editors, the efficiency of generating base deletion mutations, and the proportion of C>G.

As can be seen from Figure 10 C, after fusion expression of HMCES, small fragment base deletion mutations were significantly reduced, and the frequency of base editing mutations did not change significantly. However, the proportion of C>G mutations in cells expressing HMCES-CGBE1 decreased slightly. However, the proportion of C>G mutations in cells expressing HMCES-AID-BE1n did not decrease.

Example 4 Reduce the generation of CGBE-mediated DSB by designing targeting sequences in cis

Through Example 1, it was found that the palindromic motif favored by deaminase is more likely to cause DNA double-strand breaks during the base editing process. In this example, the targeting sequence was designed in cis and trans to verify its effect on base editing. Effect. Specifically: 5 sgRNAs containing deaminase-preferred palindromic motifs (TCGA) and 5 sgRNAs without palindromic motifs (TCAA) were synthesized, and HEK293T cells stably expressing different sequences were obtained through lentivirus infection. CGBE was transfected into these cell lines, and then the proportion of base deletion mutations produced by CGBE in the cell genome was detected through high-throughput sequencing, and the preference of different sequences to produce base deletion mutations was analyzed. Includes the following steps:

4.1. Construction of plasmids containing targeting sites and sgRNA containing targeting sites

4.1.1. Target sequence synthesis

Sangon Bioengineering (Shanghai) Co., Ltd. was entrusted to synthesize 5 substrates containing the palindromic motif TCGA and 5 substrates without the palindromic motif TCAA, and synthesize the corresponding sgRNA.

The five substrates containing the palindromic motif TCGA include HEK3-TCGA-1, HEK3-TCGA-2, GFP153-TCGA-1, GFP153-TCGA-2 and SITE13-TCGA-2; the corresponding sgRNA includes HEK3-TCGA- 1-sgRNA-F/R, HEK3-TCGA-2-sgRNA-F/R, GFP153-TCGA-1-sgRNA-F/R, GFP153-TCGA-2-sgRNA-F/R and SITE13-TCGA-2- sgRNA-F/R.

The five substrates that do not contain the palindromic motif TCAA include HEK3-TCGA-C1, HEK3-TCGA-C2, GFP153-TCGA-C1, GFP153-TCGA-C2 and SITE13-TCGA-C2; the corresponding sgRNA includes HEK3-TCGA -C1-sgRNA-F/R, HEK3-TCGA-C2-sgRNA-F/R, GFP153-TCGA-C1-sgRNA-F/R, GFP153-TCGA-C2-sgRNA-F/R and SITE13-TCGA-C2 -sgRNA-F/R.

sequence:

HEK3-TCGA-1:

GTGGAAAGGACGAAATCGACAGACTGAGCACGTGATGGCTAGAAAGCTTGGCGTA(SEQ ID NO.56)

HEK3-TCGA-C1:

GTGGAAAGGACGAAATCAACAGACTGAGCACGTGATGGCTAGAAAGCTTGGCGTA(SEQ ID NO.57)

HEK3-TCGA-2:

GTGGAAAGGACGAAAGGCCTCGACTGAGCACGTGATGGCTAGAAAGCTTGGCGTA(SEQ ID NO.58)

HEK3-TCGA-C2:

GTGGAAAGGACGAAAGGCCTCAACTGAGCACGTGATGGCTAGAAAGCTTGGCGTA(SEQ ID NO.59)

GFP153-TCGA-1:

GTGGAAAGGACGAAATCGAACGGGCAGCTTGCCGGTGGCTAGAAAGCTTGGCGTA (SEQ ID NO.60)

GFP153-TCGA-C1:

GTGGAAAGGACGAAATCAAACGGGCAGCTTGCCGGTGGCTAGAAAGCTTGGCGTA(SEQ ID NO.61)

GFP153-TCGA-2:

GTGGAAAGGACGAAAGGGCTCGAGCAGCTTGCCGGTGGCTAGAAAGCTTGGCGTA (SEQ ID NO.62)

GFP153-TCGA-C2:

GTGGAAAGGACGAAAGGGCTCAAGCAGCTTGCCGGTGGCTAGAAAGCTTGGCGTA(SEQ ID NO.63)

SITE13-TCGA-2：

GTGGAAAGGACGAAATATATCGAATAGAGAATAGACTGCTGGCTAGAAAGCTTGGCGTA(SEQ IDNO.64)

SITE13-TCGA-C2:

GTGGAAAGGACGAAATATATCAAATAGAGAATAGACTGCTGGCTAGAAAGCTTGGCGTA(SEQ IDNO.65)

HEK3-TCGA-1-sgRNA-F: caccgTCGACAGACTGAGCACGTGA (SEQ ID NO.66)

HEK3-TCGA-1-sgRNA-R: aaacTCACGTGCTCAGTCTGTCGA (SEQ ID NO.67)

HEK3-TCGA-2-sgRNA-F: caccgGGCCTCGACTGAGCACGTGA (SEQ ID NO.68)

HEK3-TCGA-2-sgRNA-R: aaacTCACGTGCTCAGTCGAGGCC (SEQ ID NO.69)

GFP153-TCGA-1-sgRNA-F: caccgTCGAACGGGCAGCTTGCCGG (SEQ ID NO.70)

GFP153-TCGA-1-sgRNA-R: aaacCCGGCAAGCTGCCCGTTCGA (SEQ ID NO.71)

GFP153-TCGA-2-sgRNA-F: caccgGGGCTCGAGCAGCTTGCCGG (SEQ ID NO.72)

GFP153-TCGA-2-sgRNA-R: aaacCCGGCAAGCTGCTCGAGCCC (SEQ ID NO.73)

SITE13-TCGA-2-sgRNA-F: caccgTCGAATAGAGAATAGACTGC (SEQ ID NO.74)

SITE13-TCGA-2-sgRNA-R: aaacGCAGTCTATTCTCTATTCGA (SEQ ID NO.75)

HEK3-TCGA-C1-sgRNA-F: caccgTCAACAGACTGAGCACGTGA (SEQ ID NO.76)

HEK3-TCGA-C1-sgRNA-R: aaacTCACGTGCTCAGTCTGTTGA (SEQ ID NO.77)

HEK3-TCGA-C2-sgRNA-F: caccgGGCCTCAACTGAGCACGTGA (SEQ ID NO.78)

HEK3-TCGA-C2-sgRNA-R: aaacTCACGTGCTCAGTTGAGGCC (SEQ ID NO.79)

GFP153-TCGA-C1-sgRNA-F: caccgTCAAACGGGCAGCTTGCCGG (SEQ ID NO.80)

GFP153-TCGA-C1-sgRNA-R: aaacCCGGCAAGCTGCCCGTTTGA (SEQ ID NO.81)

GFP153-TCGA-C2-sgRNA-F: caccgGGGCTCAAGCAGCTTGCCGG (SEQ ID NO.82)

GFP153-TCGA-C2-sgRNA-R: aaacCCGGCAAGCTGCTTGAGCCC (SEQ ID NO.83)

SITE13-TCGA-C2-sgRNA-F: caccgAATAATAGAGAATAGACTGC (SEQ ID NO.84)

SITE13-TCGA-C2-sgRNA-R:aaacGCAGTCTATTCTCTATTATT(SEQ ID NO.85)

4.1.2. Obtaining target fragments

The method to obtain the target fragment containing sgRNA is as follows:

1) Dissolve the deoxyoligonucleotide to 100 μM with ddH ₂ O and dilute to 10 μM.

2) Add forward and reverse deoxyoligonucleotides to the following reaction system. The reaction system is shown in Table 1.

Table 1

substance Adding amount The positive deoxyoligonucleotide/antideoxyoligonucleotide of sgRNA in 4.1.1 in this example 10μL each Annealing buffer(5×) 5μL ddH ₂ O to 25μL

Reaction program: 95°C for 5 min, then gradually cool down to 10°C.

4.1.3. Enzyme digestion vector

Plasmid PX33R was digested with BbsI. The enzyme digestion system and method were the same as step 2.1.3 in Example 2.

4.1.4. Ligase digested vector and target fragment

Use T4 ligase to connect the sgRNA obtained in step 4.1.2 in this example to the digested plasmid PX33R obtained in step 4.1.3 in this example to obtain the ligation product px330R-sgRNA. The method is the same as step 2.1.4 in Example 2.

4.1.5. Transformation of ligation products

The method is the same as step 2.1.5 in Example 2.

4.1.6. Select single clones for sequencing

The method is the same as step 2.1.6 in Example 2.

4.1.7. Successfully sequenced plasmid is extracted.

The method is the same as step 2.1.7 in Example 2.

4.2. Construction of cell lines containing targeting sites

4.2.1 Construction of plasmids expressing targeting sites

The substrate sequences containing (or not containing) palindromic motifs were respectively amplified by PCR, and then ligated with NdeI and SmaI double-digested plenti-guide plasmid vectors. The ligation method was the same as step 2.1.4 in Example 2. After transforming the ligation product, selecting single clones for sequencing, and Sanger sequencing for correct comparison (the method is the same as above), the lentiviral vector plasmid expressing the target site sequence was obtained.

4.2.2 Virus packaging using FuGENE

1) Pass packaging plasmid pMD2.G (250ng), packaging plasmid psPAX2 (750ng), lentiGuide-sgRNA-Puro (1μg) into 6μL FuGENE (Roche #11814443001) for virus packaging.

2) Change the medium every 12-16 hours and add 5.5mL of fresh culture medium.

3) Collect the virus (filtered through a 0.45 μm membrane) 24 hours and 48 hours after changing the medium, and store it temporarily at 4°C.

4.2.3. Obtaining stably transfected cell lines

1) 16-24 hours before infection, spread HEK293T cells into a 6 cm culture dish at a density of 1×10 ⁶ cells/well, so that the cell density on the next day is approximately 80%.

2) Use 2mL virus, 1mL fresh medium and 3μL Polybrene to infect the cells.

3) Change the medium 24 hours after infection and add 4 mL of fresh culture medium.

24 hours after changing the medium, purinease was added for screening to obtain cell lines containing specific targeting sites.

4.2.4. Use Lipofectamine 2000 for cell transfection

Plasmid CGBE1/AID-BE1n and the ligation product px330R-sgRNA obtained in step 1.4 in this example were transfected using Lipofectamine 2000.

The specific steps are the same as step 2.3 in Example 2.

4.2.5. Preparation of high-throughput sequencing libraries

Same as step 2.4 of Example 2.

4.3. Results

High-throughput sequencing detects the proportion of base deletion mutations produced by CGBE1 in the cell genome, and analyzes the preference of different sequences to produce base deletion mutations. Subsequently, the pathogenic mutations in the ClinVar database that can be corrected by CGBE to achieve C>G mutations were counted (Arbab et al., 2020).

A in Figure 11 shows the base deletion mutation map of greater than 1 bp that occurs when CGBE1 acts on the editing sequence containing a palindromic motif (TCGA) or a non-palindromic motif (TCAA) in this example.

As can be seen from A in Figure 11, the target site sequence containing the palindromic motif TCGA in the targeted editing region produces a higher frequency of base deletion mutations during the editing process of the base editor, resulting in the target base editing product. The frequency of base substitution mutations is low; the frequency of base editing by-products of base editing in targeted editing regions that do not contain palindromic motifs is reduced, and the frequency of base deletion mutations is reduced, and the base editing efficiency is higher.

B in Figure 11 is a statistical diagram of the difference in efficiency of deletion mutations produced by plasmid CGBE1 in this example after acting on 5 pairs of palindromic/non-palindromic motif sequences.

As shown in Figure 11B, in the edited product of CGBE1, the target sequence containing the TCGA palindromic motif has a higher frequency of base deletion mutations.

C in Figure 11 is a pie chart showing the proportion of palindromic motifs contained in the editing window among the 3040 potential CGBE correction sites in the ClinVar database in this embodiment.

From C in Figure 11, it can be seen that nearly 10% of the pathogenic mutation sites contain the palindromic motif CG.

Based on the analysis of the synthetic palindromic targeting sequence and the chromosomal translocation of the preceding deaminase-OT site, it is recommended that the selection of sgRNAs with deaminase-preferring palindromic motifs should be avoided in the clinical application of CGBE.

The above embodiments only illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed in the present invention shall still be covered by the claims of the present invention.

Claims (25)

1. A single base editing system, characterized in that the single base editing system includes: 1) Nucleic acid nickase or its encoding polynucleotide; 2) Cytosine deaminase or its encoding polynucleotide; 3) Abasic site protected polypeptide or its encoding polynucleotide. 2. The single base editing system of claim 1, wherein the abasic site protection polypeptide is an APEX1 competitive inhibitor and/or an APEX1 mutant; preferably, the APEX1 competitive inhibitor The agent is selected from HMCES. 3. The single base editing system according to claim 2, wherein the amino acid sequence of the HMCES includes: 1) The amino acid sequence shown in SEQ ID NO.1; or, 2) An amino acid sequence that has 80% or more sequence similarity with SEQ ID NO. 1 and has the function of the amino acid sequence defined in 1). 4. The single base editing system of claim 1, wherein the nucleic acid nickase is Cas9 nickase; and/or the cytosine deaminase is selected from APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, One or more of APOBEC3C, APOBEC3D/E, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4 and their mutants or AID and their mutants. 5. The single base editing system of claim 1, wherein when the nucleic acid nickase or cytosine deaminase is expressed in the form of a fusion protein, the fusion protein includes a cytosine deaminase fragment and Nucleic acid nickase fragment; the abasic site protected polypeptide is fused or expressed independently. 6. The single base editing system according to claim 5, wherein the fusion protein further includes a uracil glycosylated protein fragment and/or a nuclear localization fragment and/or an abasic site protected polypeptide fragment. 7. The single base editing system of claim 6, wherein the fusion protein includes a cytosine deaminase fragment and a nucleic acid nickase fragment in order from the N-terminus to the C-terminus. 8. The single base editing system according to claim 7, wherein in the fusion protein, the N-terminus of the cytosine deaminase fragment is connected to a uracil glycosylated protein fragment and/or an abasic site. Point-protected peptide fragments. 9. The single base editing system of claim 7, wherein the fusion protein includes an amino acid sequence including the sequence shown in SEQ ID NO. 19, SEQ ID NO. 20, or SEQ ID NO. 21. 10. The single base editing system according to any one of claims 1 to 9, characterized in that the single base editing system further includes sgRNA or its encoding polynucleotide. 11. The single base editing system according to claim 10, characterized in that the sgRNA does not contain a deaminase-preferred palindrome motif; preferably, the deaminase-preferred palindrome motif is TCGA. . 12. A fusion protein for single base editing, characterized by comprising an abasic site-protecting polypeptide fragment, a cytosine deaminase fragment and a nucleic acid nickase fragment. 13. The fusion protein of claim 12, wherein the abasic site protected polypeptide fragment is an APEX1 competitive inhibitor or an APEX1 mutant; preferably, the APEX1 competitive inhibitor is selected from HMCES . 14. The fusion protein of claim 13, wherein the amino acid sequence of HMCES includes: 1) The amino acid sequence shown in SEQ ID NO.1; or, 2) An amino acid sequence having at least 80% sequence similarity with SEQ ID NO. 1 and having the function of the amino acid sequence defined in 1). 15. The fusion protein of claim 12, wherein the fusion protein includes an abasic site protected polypeptide fragment, a cytosine deaminase fragment and a nucleic acid nickase fragment in order from the N-terminus to the C-terminus. 16. The fusion protein of claim 12, wherein the fusion protein further includes a uracil glycosylated protein fragment and/or a nuclear localization fragment, preferably the N-terminus of the cytosine deaminase fragment. Attached are uracil glycosylated protein fragments and/or nuclear localization fragments. 17. The fusion protein of claim 12, wherein the fusion protein includes an amino acid sequence including the sequence shown in SEQ ID NO. 19, SEQ ID NO. 20, or SEQ ID NO. 21. 18. A polynucleotide, characterized in that it encodes the fusion protein according to any one of claims 12 to 17. 19. A nucleic acid construct, characterized in that the nucleic acid construct contains the polynucleotide of claim 18. 20. An expression system, characterized in that the expression system contains the nucleic acid construct according to claim 19 or the polynucleotide according to claim 18 is integrated into the genome. 21. The single base editing system of any one of claims 1 to 11, the fusion protein of any one of claims 12 to 17, the polynucleotide of claim 18, and the nucleic acid construct of claim 19 or the use of the expression system of claim 20 in base editing. 22. A gene editing method, comprising: performing gene editing through the base editing system according to any one of claims 1 to 11 or the fusion protein according to any one of claims 12 to 17. 23. The use of HMCES as a base-site protected polypeptide for gene editing or base editing systems. 24. The use of claim 23, wherein the HMCES is used to reduce DNA double-strand break damage in single base editing. 25. The use according to claim 23, wherein the single base editing is cytosine to guanine base editing.