WO2021155607A1 - Modified cytosine base editor and application thereof - Google Patents

Abstract

A modified cytosine base editor and application thereof. The targeted editing efficiency and fidelity of the modified cytosine base editor are significantly improved.

Description

Modified cytosine base editor and its application Technical field

The present invention belongs to the field of biotechnology. More specifically, the present invention relates to a modified cytosine base editor and its application.

Background technique

Base editing has been widely used for targeted base editing and has great potential in correcting disease-causing mutations.

CRISPR/Cas and base editors-mediated gene editing methods have been developed, and have brought great hopes for the treatment of genetic diseases caused by pathogenic mutations. Clinical applications based on CRISPR/Cas gene editing or base editing require comprehensive analysis of off-target effects to reduce the risk of harmful mutations. Although a variety of methods have been developed in the field to detect the off-target activity of genome-wide gene editing cells, including high-throughput whole-genome translocation sequencing (HTGTS), whole-genome, unbiased recognition of double-strand breaks (GUIDE-seq) And report cleavage efficiency in vitro by cycle sequencing (CIRCLE-seq). However, none of these methods can effectively detect single nucleotide variation (SNV). So far there is no effective method to detect SNV in this field.

In addition, when CRISPR/Cas is applied, the defect lies in the low editing efficiency of homology-mediated repair. Those in the art use a 16aa long XTEN linker (XTEN linker) to link the cytidine deaminase APOBEC1 and dCas9 together to construct the first generation base editor (BE1). In order to increase editing efficiency in vivo, in addition to linking cytidine deaminase and dCas9, the second-generation base editor (BE2) system also fuses base excision repair inhibitor UGI and dCas9 together to combine editing The efficiency is increased three times, up to about 20%.

In order to improve the efficiency of base editing, those skilled in the art replaced dCas9 with Cas9n to simulate mismatch repair, thereby constructing a third-generation base editor (BE3). BE3 creates a nick in the non-complementary DNA strand, and the cell uses the uracil (U)-containing DNA strand as a template for repair, thereby replicating this base editing. Among a variety of target genes in human cell lines, this BE3 system significantly improves the base editing efficiency, and its average indel (insertion-deletion) incidence is only 1.1%. For these tested target genes, these numbers are a huge improvement over Cas9-mediated HDR; the average HDR-mediated editing frequency is only 0.5%, and compared to previous single-base editing, more indels be observed. The persistence of CRISPR base editing in multiple cell divisions indicates that this method produces stable base editing. However, this BE3 system will also suffer from off-target editing.

However, previous studies have proved that the cytosine base editor (CBE) has DNA and RNA off-target effects. Reducing unnecessary DNA and RNA off-target effects is of great significance for scientific research and therapeutic applications.

Summary of the invention

The purpose of the present invention is to provide a modified cytosine base editor and its application.

In the first aspect of the present invention, a method for improving the efficiency or fidelity of targeted editing of a cytosine base editor is provided, which includes: modifying the cytosine deaminase in the cytosine base editor The cytosine deaminase includes APOBEC1 or its homologues, and the modification includes the amino acids corresponding to Trp(W) at position 90 and Arg(R) at position 126 of APOBEC1. Make a mutation and connect the cytosine base editor to the nuclear localization sequence.

In a preferred example, the cytosine base editor is BE3 gene editor system.

In another preferred example, the APOBEC1 homologue includes an enzyme selected from the group consisting of AID, APOBEC3G, APOBECA3A, CDA1.

In another preferred example, the mutation is a mutation of the cytosine deaminase corresponding to APOBEC1 at position 90 Trp to Tyr(Y); and/or, a mutation of Arg at position 126 to Glu(E) .

In another preferred embodiment, the N-terminal and/or C-terminal of the cytosine base editor is connected to a nuclear localization sequence. Preferably, it is at the C-terminus of UGI in the cytosine base editor or at the N-terminus of cytosine deaminase.

In another aspect of the present invention, there is provided a modified cytosine deaminase, said cytosine deaminase comprising APOBEC1 or a homologue thereof, the cytosine deaminase corresponding to APOBEC1 Trp( There are mutations in the amino acid of W) and Arg(R) at position 126.

In another preferred example, the APOBEC1 homologue includes an enzyme selected from the group consisting of AID, APOBEC3G, APOBECA3A, CDA1.

In another preferred example, the mutation is that the cytosine deaminase corresponding to APOBEC1's Trp at position 90 is mutated to Tyr(Y); and/or, the Arg at position 126 is mutated to Glu(E).

In another aspect of the present invention, a cytosine base editor is provided, which comprises the modified cytosine deaminase.

In another preferred embodiment, the cytosine base editor is also connected to the nuclear localization sequence; preferably, the N-terminal and/or C-terminal is connected to the nuclear localization sequence.

In another preferred example, the cytosine base editor and the nuclear localization sequence further include a linking sequence, such as a tag sequence (more specifically, a Flag tag).

In another aspect of the present invention, an isolated polynucleotide is provided, which encodes the modified cytosine deaminase or the cytosine base editor.

In another preferred embodiment, the cytosine base editor has the nucleotide sequence shown in SEQ ID NO: 2.

In another aspect of the present invention, a recombinant expression vector is provided, which comprises the polynucleotide.

In another aspect of the present invention, a genetically engineered host cell is provided, which contains the vector or the polynucleotide integrated in the genome.

In another preferred example, the editor is BE3 base editor.

In another aspect of the invention, the use of the cytosine base editor is provided for gene editing, reducing off-target effects, improving the efficiency of targeted editing, or improving the fidelity of targeted editing.

In another aspect of the invention, a method for gene editing is provided, which includes mediating gene editing with the cytosine base editor.

In another preferred embodiment, the nucleic acid sequence encoding the cytosine base editor and the sgRNA are co-injected into the receptor to perform gene editing.

In another preferred embodiment, the receptor includes: somatic cells or germ cells.

In another preferred embodiment, the germ cells include embryonic cells or fertilized eggs.

In another aspect of the invention, a reagent or kit for gene editing is provided, which includes the modified cytosine deaminase; or the cytosine base editor.

Other aspects of the present invention are obvious to those skilled in the art due to the disclosure herein.

Description of the drawings

Figure 1. Targeted editing efficiency of cytosine base editor (CBE) variants. a. The predicted rAPOBEC1 structure in various rAPOBEC1 variants. The mutated residues are highlighted and marked on the structure. b. Sequence alignment between hAPOBEC3G and rAPOBEC1. Amino acids, same residues; +, common substituents. The green triangles represent the residues in the hydrophobic active region of APOBEC3G, and the yellow stars represent the residues in the ssDNA binding region. c. The crystal structure of APOBEC3G. d. Target editing efficiency and indel frequency of different CBE variants. There are n=3 biological replicates in each group. Green and yellow indicate the residues in the helical structure and loop structure, respectively. The purple triangles indicate variants for which off-target detection is subsequently performed.

Figure 2. DNA and RNA off-target activity of CBEs variants. a. Comparison of the total number of off-target SNVs detected. Cre group n=2, BE3 group n=6, BE3 ^R126E group n=10, BE3 ^R132E group n=3, YE1-BE3 group n=8, FE1-BE3 group n=3. Compared with the Cre group, the P value on the graph was calculated by two-tailed Student's t test. b. The mutation type distribution of Cre, BE3 and 4 CBE variant treatment groups. c. Comparison of the total number of RNA off-target SNVs detected 36 hours after transfection. 3 repetitions per group. Compared with the GFP group, the two-tailed Student's t test was used to calculate the P value above each column. d. Distribution of mutation types in GFP, BE3 and 4 CBE variant treatment groups.

Figure 3. Activity of BE3-FNLS or BE3-hA3A variants. a. Targeting efficiency of BE3-FNLS or BE3-hA3A at each target. See Table 6 for target site related sequences and primer sequences. b. Comparison of the total number of off-target SNVs detected. Cre group n=2, BE3 group n=6, YE1-BE3 group n=5, BE3-hA3A ^Y130F group n=3, YE1-BE3-FNLS group n=3. Calculate the P value of each group compared with the Cre group. c. Comparison of the total number of RNA off-target SNVs detected 36 h after transfection. 3 repetitions per group. Calculate the P value of each group compared with the GFP group. d. Comparison of editing efficiency inside the window and editing efficiency outside the window. Edit window, bases 5-7. e. The distribution of indels of each mutant at each site. P value adopts two-tailed Student's t test.

Figure 4. Targeted editing of BE3 and BE3 variants at different target sites. a. Target editing efficiency and indels frequency of different versions of CBE variants at an additional 11 target sites. b. Comparison of targeting efficiency of CBE variants. c. Comparison of indel frequencies between CBE variants. d. On-target efficiency of each target engineered BE3 variant. e. Comparison of editing efficiency of CBE variants at each C of the target site. f. Comparison of the editing efficiency in the window and the editing efficiency outside the window of the CBE variant. Edit window: 5-7 bases. Each group n=3 biological replicates. P value adopts two-sided t test. The target site sequence and primer sequence are shown in Table 6.

Figure 5. Embryonic development rate of BE3 and BE3 variants. a Use sgRNA-D to detect the blastocyst rate of BE3 and BE3 variants. b Use other sgRNAs to detect the blastocyst rate of BE3-hA3A and BE3-FNLS. Each group n=3 biological replicates.

Figure 6. Targeted editing efficiency of CBE variants and editing efficiency of non-targeted SNVs. a. Target editing efficiency of BE3 and CBE variants in WGS data. b. Comparison of C-to-T and G-to-A conversion between CBE variant treatment group and Cre or BE3 group. P value adopts two-sided t test. *P<0.05, **P<0.01,***P<0.001.

Figure 7. Venn diagram of SNVs detected in each embryo through WGS data. a. ^SNVs identified in embryos treated with BE3 R126E. b. ^SNVs identified in embryos treated with BE3 R132E. c. SNVs identified in embryos treated with YE1-BE3. d. SNVs identified in embryos treated with FE1-BE3.

Figure 8. Non-target SNVs characteristics of CBE variants. The inventor's analysis detected the overlap between the SNVs and the extra-target sites predicted by Cas-OFFinder and CRISPOR.

Figure 9. BE3 variant RNA off-target efficiency 36 hours after transfection.

Figure 10. Detection of RNA off-target efficiency of CBE variants 72 hours after transfection. a. Comparison of the total number of RNA non-targeted SNVs detected 72 hours after transfection. GFP group n=6, BE3 group n=9, BE3 ^R126E group n=7, YE1-BE3 group n=2. By comparing with the Student's t test of the GFP group, the P value of each group was calculated. b. Distribution of mutation types in the GFP, BE3 and BE3 variant treatment groups. c. Off-target efficiency of BE3 variant RNA 72 hours after transfection.

Figure 11. Target editing efficiency and off-target of BE3-FNLS. a. Comparison of targeting efficiency of CBE variants. b. Comparison of editing efficiency of CBE variants at each C of the target site. c. SNVs found in embryos treated with BE3 ^{-hA3A Y130F and YE1-BE3-FNLS.} d. The overlap between the SNVs detected from the inventor's analysis and the off-target sites predicted by Cas-OFFinder and CRISPOR. e. Distribution of DNA non-targeted SNVs mutation types of embryos treated with BE3-hA3AY130F and YE1-BE3-FNLS. f. The distribution of RNA non-targeted SNVs mutation types of embryos treated with BE3-hA3AY130F and YE1-BE3-FNLS. g. The expression level of APOBEC1 in BE3 and BE3-FNLS. h. The RNA off-target rate of BE3 and BE3-FNLS 36 hours after transfection. Each group n=3 biological replicates. P value adopts two-sided t test.

Figure 12. Activity of BE3 and BE3 variants at indicated off-target sites. a. sgRNA-dependent off-target effects of BE3 variants. b. The editing frequency of BE3 variants at designated off-target sites. The P value was compared with the YE1-BE3-FNLS group using a two-sided t test. Compared with the YE1-BE3-FNLS group, a red star indicates an increase in the editing frequency, and a green star indicates a decrease in the editing frequency. *P<0.05, **P<0.01, **P<0.001. ^Plasmids expressing BE3, BE3 R126E, BE3 ^R132E , YE1-BE3, FE1-BE3, BE3-hA3A, BE3-hA3A ^Y130F , BE3-FNLS, YE1-BE3-FNLS and sgRNAs were transfected into HEK293T cells with liposome 3000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing to analyze the editing efficiency at the target site and the top ten predicted off-target sites of these sgRNAs. The target site sequence and primer sequence are shown in Table 4. Each cell represents the percentage of the total readings edited from C to T. Each group n=3 biological replicates.

Figure 13. Schematic diagram of YE1-BE3-FNLS plasmid.

Detailed ways

In the present invention, the inventors analyzed the DNA and RNA of multiple CBE variants by two-cell embryo injection whole genome off-target analysis (Genome-wide Off-target analysis by Two-cell embryo Injection, GOTI) and RNA-Seq sequencing After in-depth analysis of off-target effects, the cytosine base editor has been modified, and the targeted editing efficiency and fidelity of the cytosine base editor have been significantly improved.

The cytosine base editor includes cytosine deaminase. The cytosine deaminase includes APOBEC1 or a homologue thereof. The APOBEC1 homologues include enzymes that have the same or similar functions as APOBEC1, or enzymes that have substantially the same or substantially similar domains as APOBEC1, or those that come from a different species than APOBEC1 but play the same role in the respective species. Enzyme. For example, the APOBEC1 homologues include but are not limited to enzymes selected from the group consisting of AID, APOBEC3G, APOBECA3A, CDA1.

The present invention first provides a modified cytosine deaminase. The cytosine deaminase has mutations in the amino acids corresponding to Trp(W) at position 90 and Arg(R) at position 126 of APOBEC1, and the cytosine The deaminase is linked to the nuclear localization sequence. Preferably, the mutation is the mutation of Trp at position 90 of APOBEC1 to Tyr(Y) of the cytosine deaminase; and/or the mutation of Arg at position 126 to Glu(E).

In a preferred mode of the present invention, the cytosine deaminase and the nuclear localization sequence are also connected by a linking sequence, and the linking sequence may be any linking sequence that does not affect the functions of the two, for example, It is a tag sequence or some flexible linking sequence known in the art. Appropriate labels can be used in the present invention. For example, the tag can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE or Ty1.

The modified cytosine deaminase (modified enzyme) of the present invention can be a recombinant protein, a natural protein, a synthetic protein, and a recombinant protein is preferred. The protein of the present invention can be a natural purified product, or a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, higher plant, insect, and mammalian cells) using recombinant technology.

The present invention also includes fragments, derivatives and analogs of the engineered enzymes. As used herein, the terms "fragment", "derivative" and "analog" refer to a protein that substantially retains the same biological function or activity as the engineered enzyme of the present invention. The protein fragment, derivative or analogue of the present invention may be (i) a protein in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a protein with a substitution group in one or more amino acid residues, or (iii) a protein formed by fusing an additional amino acid sequence to the protein sequence (such as Leader sequence or secretory sequence or sequence used to purify the protein or proprotein sequence, or fusion protein). According to the definition herein, these fragments, derivatives and analogs belong to the scope well known to those skilled in the art. However, in the amino acid sequence of the modified enzyme and its fragments, derivatives and analogs, there must be conservative mutations described above in the present invention, which correspond to Trp(W) at position 90 and position 126 of APOBEC1. The amino acid of Arg(R) has the mutation, and the nuclear localization sequence is also connected.

In the present invention, the term "engineered enzyme" also includes (but is not limited to): several (usually 1-20, more preferably 1-10, still more preferably 1-8, 1- 5, 1-3, or 1-2) amino acid deletions, insertions and/or substitutions, and addition or deletion of one or several (usually within 20) at the C-terminus and/or N-terminus, preferably Within 10, more preferably within 5) amino acids. For example, in the field, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of engineered enzymes. However, in these variant forms, there must be the conservative mutations described above in the present invention, that is, the amino acids corresponding to Trp (W) at position 90 and Arg (R) at position 126 of APOBEC1 have the mutations, and they are also connected. The nuclear localization sequence described.

In the present invention, the term "engineered enzyme" also includes (but is not limited to): the amino acid sequence of the modified enzyme is more than 80%, preferably more than 85%, more preferably more than 90% , And more preferably 95% or more, such as 98% or more, 99% or more sequence identity of the derived protein that retains its protein activity. Similarly, in these derived proteins, there must be the conservative mutations described above in the present invention, that is, the amino acids corresponding to Trp (W) at position 90 and Arg (R) at position 126 of APOBEC1 have the mutations, and they are also connected. There is the nuclear localization sequence.

The present invention also provides a polynucleotide sequence encoding the engineered enzyme of the present invention or a conservative variant protein thereof.

The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.

The polynucleotide encoding the mature protein of the mutant includes: only the coding sequence of the mature protein; the coding sequence of the mature protein and various additional coding sequences; the coding sequence of the mature protein (and optional additional coding sequence) and non- Coding sequence.

The "polynucleotide encoding a protein" may include a polynucleotide encoding the protein, or a polynucleotide that also includes additional coding and/or non-coding sequences.

The full-length nucleotide sequence of the modified enzyme of the present invention or its fragments can usually be obtained by PCR amplification method, recombination method or artificial synthesis method. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used. The library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

Once the relevant sequence is obtained, the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences. At present, the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.

The present invention also relates to a vector containing the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the modified enzyme coding sequence, and a method for producing the protein of the present invention through recombinant technology.

Through conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce a recombinant engineered enzyme. Generally speaking, there are the following steps: (1). Use the polynucleotide encoding the modified enzyme of the present invention, or use the recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell; (2). Host cells cultured in a suitable medium; (3). Separating and purifying proteins from the medium or cells.

The present invention also provides a cytosine base editor containing the modified enzyme or its polynucleotide sequence. In a preferred mode of the present invention, the cytosine base editor is BE3 base editor. Other components of the cytosine base editor are known to those skilled in the art.

In the present invention, the modified enzyme polynucleotide sequence or the cytosine base editor polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can replicate and stabilize in the host. An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.

Methods well known to those skilled in the art can be used to construct an expression vector containing the modified enzyme polynucleotide sequence or the cytosine base editor polynucleotide sequence and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. The expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells.

A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell into a recipient cell.

The present invention also provides a method for gene editing, which includes mediating gene editing with the cytosine base editor of the present invention. In addition to using the cytosine base editor of the present invention for gene editing, other gene editing reagents can be used known in the art. For example, sgRNA can be designed in a manner known in the art.

In the present invention, the target of gene editing is not particularly limited, and it may be a somatic cell or a germ cell, and it may be an animal cell or a human cell.

Although the cytosine base editor (CBE) has broad prospects as a powerful gene editor, the off-target effects of DNA and RNA limit its application in science and medicine. In the specific embodiment of the present invention, the inventors screened more than 20 reasonably designed CBE mutants in detail, and analyzed the off-target effects of DNA and RNA using GOTI and RNA-Seq, respectively. The predicted residue mutations at the DNA binding site significantly reduced off-target effects, in some cases to levels comparable to unedited controls. The CBE variant YE1-BE3-FNLS obtained in the present invention has very low off-target efficiency and bystander editing while maintaining extremely high targeted editing efficiency. In the present invention, the inventors not only identified multiple residues that can specifically affect RNA and DNA off-target activity and narrow the base editing window, but also introduced a CBE variant with high fidelity and high editing efficiency, thereby expanding The application of these powerful tools in the laboratory and in the clinic.

In the specific embodiment of the present invention, the inventor screened dozens of rAPOBEC1 mutations based on the findings of multiple previous studies, and found that BE3 ^R132E , YE1-BE3, and FE1-BE3 mutations significantly reduced the off-target effects of DNA and RNA. Maintained their targeted editing activity. Interestingly, the inventors observed that the variants with reduced DNA/RNA off-target effects (BE3 ^R132E , YE1-BE3, FE1-BE3 and YE1-BE3-FNLS) also have reduced base editing windows. Rees et al. reported that bases located outside the active window but located in the R-loop region of ssDNA can still be edited, albeit with lower efficiency, especially if they are located in the favorable editing motif of rAPOBEC1. These may help explain these results.

Considering that both rAPOBEC1 and hAPOBEC3A are considered to have only one catalytic domain, the inventors predicted the possible impact of the mutation introduced in the DNA binding motif. However, given the known ability of BEs to edit DNA and RNA, rAPOBEC1 may adopt different binding modes to adapt to ssDNA and RNA. This highlights the necessity for base editing researchers to evaluate the off-target effects of base editing on DNA and RNA. The inventors speculate that the heterogeneity of this binding mode may help explain that some CBE variants discovered by the inventors not only retain high DNA off-target effects, but also significantly reduce RNA off-target effects (and vice versa) The phenomenon.

In particular, the inventors speculate that R132E affects the interaction of rAPOBEC1 with DNA and RNA, while R126E mainly affects its DNA binding ability, and Y130F mainly affects its RNA binding ability. Considering that the YE1-BE3-FNLS mutation contains both the R126E mutation and the substitution of tyrosine at the W90 residue in the hydrophobic region of rAPOBEC1, this residue is considered to be involved in the binding of rAPOBEC1 to ssDNA/RNA. The inventors preliminarily speculate that the W90Y mutation It helps to explain that the high fidelity of YE1-BE3-FNLS may be due to the change of rAPOBEC1-RNA interaction.

It is worth noting that the ideal variant YE1-BE3-FNLS screened by the inventors has both the highest targeted editing efficiency and the lowest level of indels and bystander editing. A previous study showed that multiple Cs in the editing window may increase the probability of indels occurring during editing. Compared with BE3-FNLS, YE1-BE3-FNLS significantly reduces the basic editing window, thereby reducing the distribution of multiple Cs, which may explain the significant decrease in indel frequency. In summary, the inventor’s work illustrates how to minimize the off-target effects of cytosine editing through bio-insight-driven engineering, so that these powerful gene editing tools can be used in research and therapeutic applications.

The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples usually follow the conventional conditions as described in J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the conditions described in the manufacturer The suggested conditions.

Materials and Methods

GOTI method

A mixture of mRNA and sgRNA from gene editing tools such as Cas9/BE3 was injected into a blastomere of a 2-cell stage embryo, which was derived from a wild-type female mouse X Ai9 male mouse. The action of Cre produces chimeric embryos, in which the injected cells are marked with tdTomato (red), a positive tdTomato indicates that editing has occurred, and a negative tdTomato indicates unedited. TdTomato positive cells and tdTomato negative cells were separated from chimeric embryos by FACS at E14.5 and used for WGS analysis respectively. Off-target SNV and indel were identified by comparing tdTomato+ cells and tdTomato- cells using three algorithms (Mutect2, Lofreq and Strelka for SNV analysis, and Mutect2, Scalpel and Strelka for indel analysis).

Animal care

Heterozygous Ai9 (full name B6.Cg-Gt(ROSA)26Sortm9(CAG-td-Tomato)Hze/J; JAX strain 007909) male mice and female C57BL/6 mice (4 weeks old) were mated for embryo collection. ICR females are used as recipients. The use and care of animals follow the guidelines of the Biomedical Research Ethics Committee of the Shanghai Institute of Biology, Chinese Academy of Sciences.

Mutant construction

The wild-type APOBEC1 protein sequence is shown in SEQ ID NO:1:

(1) Cytosine base editor 3 (BE3, rAPOBEC1-nCas9-UGI), including Apobec1 and Sp nCas9 enzymes, UGI enzymes, among which Apobec1 and Sp nCas9 enzymes, Sp nCas9 enzymes and UGI enzymes, respectively pass Two peptides, 16AA (sequence SGSETPGTSESATPES) and 4AA (sequence SGGS) are connected, and rAPOBEC1 is recombinant APOBEC1.

^{BE3 R126E} : The BE3 of the previous paragraph (1), in which the 126th position of the rAPOBEC1 sequence was changed from R to E.

^{BE3 R128E} : BE3 of (1), where the 128th bit of the rAPOBEC1 sequence was changed from R to E.

^{BE3 R132E} : BE3 of (1), in which position 132 of the rAPOBEC1 sequence was changed from R to E.

YE1-BE3: BE3 of (1), in which the 90th W mutation of the rAPOBEC1 sequence was changed to Y (W90Y), and the 126th position was changed from R to E (R126E).

FE1-BE3: BE3 of (1), in which the 90th position of the rAPOBEC1 sequence was changed to F (W90F), and the 126th position was changed from R to E (R126E).

BE3-hA3A: Use human APOBECA3A (human APOBECA3A) to replace apobec1 on BE3 to build a new BE3 editing tool

BE3-hA3A ^Y130F : mutation Y130 to F in human APOBECA3A.

BE3-FNLS: BE3 of (1), where the N-terminal of the rAPOBEC1 sequence is connected with the Flag tag and the NLS nuclear localization sequence (sequence: PKKKRKV), and there is also an NLS at the C-terminal of the base editor.

YE1-BE3-FNLS: For BE3-FNLS, the 90th position of the rAPOBEC1 sequence was changed to Y (W90Y), and the 126th position was changed from R to E (R126E). The schematic diagram is shown in Figure 13.

The sequence of YE1-BE3 after mutation is (SEQ ID NO: 223):

The mutant was inserted into the pCMV-BE3 plasmid to obtain the CBE mutant plasmid.

(Acquisition of mutant base editor mRNA and sgRNA)

Add the T7 promoter to the base editor coding region, and use primers F and R (base editor IVT F and base editor IVT R, etc. in Table 2) to pair the plasmids (YE1-BE3, BE3-FNLS, YE1-BE3-FNLS, hA3A) -BE3) Perform PCR amplification, purify the T7 base editor PCR product as a transcription template, and use mMESSAGE-mMACHINE T7-ULTRA-kit (Life Technologies) kit in vitro transcription (In Vitro Transcription, IVT). Through the PCR amplification of px330, the T7 promoter was added to the sgRNA template. The T7-sgRNA-PCR product was purified with MEGA-shortscript T7 kit (Life Technologies) as a template for IVT. Through PCR amplification, T7 promoter was added to Cre in vitro transcription template. Purify the T7 Cre-PCR product as an in vitro transcription template, and use mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies) for in vitro transcription. The Cas9 mRNA, Cre mRNA and sgRNAs were purified with MEGA-clear kit (Life Technologies), and eluted with RNase-free water.

Table 1, sgRNA sequence

Table 2. Primers

Among them, IVT is in vitro transcription, Tyr-C, targeting sgRNA with Tyr gene code C, Tyr-D, targeting sgRNA with Tyr gene code D, Tyr-F, targeting sgRNA with Tyr gene code F.

After obtaining the above-mentioned mRNA and sgRNA, the gene editing steps are: after mixing the mRNA and sgRNA, using a microinjector, inject it into a blastomere of a mouse 2-cell stage embryo.

Blastocyst rate calculation

Superovulated female C57BL/6 mice (4 weeks old) were mated with male C57BL/6 mice, and the fallopian tube fertilized embryos were collected 24 hours after injection of hCG. In M2 medium containing 5μg/ml cytochalasin B (CB), Cas9 mRNA and sgRNA were mixed and injected into the cytoplasm of the fertilized ovum with a FemtoJet micro-syringe (Eppendorf) under constant flow conditions. The injected embryos were cultured to the blastocyst stage in KSOM at 37°C and 5% CO _{2 in air.}

2-cell embryo injection, embryo culture and embryo transfer

Superovulated C57BL/6 female mice (4 weeks old) were bred with heterozygous Ai9 mice (full name B6.Cg-Gt(ROSA)26Sortm9(CAG-td-Tomato)Hze/J; JAX strain 007909) males, and The fallopian tube fertilized embryos were collected 23 hours after hCG injection. For 2 cell editing, a mixture of BE3 mRNA (50 ng/μl) or BE3 variant mRNA (50 ng/μl), sgRNA (50ng/μl) and Cre mRNA (2ng/μl) were injected into 2 cells 48h after hCG injection. -In the cytoplasm of embryonic blastomeres, inject in M2 medium containing 5 μg/ml cytochalasin B (CB) with a FemtoJet microsyringe (Eppendorf) at a constant flow rate. The injected embryos were cultured in a KSOM medium containing amino acids in an incubator at 37°C and 5% CO2 for 2 hours, and then transplanted into the fallopian tubes of ICR pseudo-pregnant female mice under the condition of 0.5 dpc.

clone

The NEBuilder-HiFi-DNA assembly master mix (New England Biolabs) was used for site-directed mutagenesis of BE3. Briefly, the inventors used a primer containing the desired point mutation to amplify a suitable vector plasmid by PCR. pCMV-BE3 variants-polyA-pCMV-mCherry-polyA is assembled by NEBuilder-HiFi DNA, and the PCR-amplified pCMV-mCherry polyA is combined with the digested pCMV-BE3 variant backbone. The PCR amplified U6-sgRNA was combined with the digested pCMV-EGFP-polyA backbone, and then assembled by NEBuilder-HiFi-DNA to obtain pCMV-EGFP-polyA-U6-sgRNA.

Cell culture, transfection and FACS

HEK293T cells were cultured in DMEM containing 10% fetal bovine serum (FBS) and 37°C humidified incubator containing 5% CO2. pCMV-BE3 (WT/BE3 variant)-polyA-pCMV-mCherry polyA and pCMV-EGFP-polyA-U6-sgRNA expression plasmids were co-transfected with liposome 3000 (ThermoFisher Scientific) according to the instructions. 36 or 72 hours after transfection, the cells were washed with phosphate buffered saline (PBS) and trypsinized with 0.05% trypsin EDTA. The cell suspension was filtered through a 40μm cell strainer, and EGFP/mCherry positive cells were separated by flow cytometry.

RNA sequencing

Collect about 500,000 cells (the first 5% EGFP/mCheery signal), and extract RNA according to standard protocols. To construct a library, mRNAs are fragmented and converted into cDNA using random hexamers or oligonucleotide (dT) primers. Connect the 5'end and 3'end of the cDNA to the adaptor respectively, and use the PCR method to enrich and amplify the correctly connected cDNA fragments. The concentration of the library was determined with a bioanalyzer. Sequencing is performed on the Illumina HiSeq platform.

Deep sequencing of DNA amplicons

After 72 hours, the transfected cells were taken, and EGFP+/mCherry+ cells were sorted by FACS. According to the instructions, use Tiangen DNA Extraction Kit (TIANGEN) to extract genomic DNA. The gene-specific primers on both sides of the target sequence (Tables 3 and 4) were used to amplify the target genomic site by PCR. ExTaq (TAKARA) was activated at 95°C for 3 minutes, followed by 34 cycles of PCR (at 95°C for 30 seconds, 62°C for 30 seconds, 72°C for 1 minute), and finally at 72°C for 5 minutes. Purify the DNA amplicons using the Universal DNA Purification Kit (TIANGEN) according to the instructions. The amplicon was connected to the adapter and sequenced on the Illumina HiSeq-Xten platform.

Table 3. Primers used for deep sequencing of target sites

On-target site On-target sequence(SEQ ID NO:) Forward primer(SEQ ID NO:) Reverse primer(SEQ ID NO:) EMX1 site 1 tgcccctccctccctggcccagg(14) ccagcttctgccgtttgtact(48) aactcgtagagtcccatgtctg(82) DNMT3B site 2 agagccccccctcaaagagaggg(15) gatggctgtttgtcttgtggc(49) tataaaccctgtgtgctgctt(83) EMX1 site 2 gagtccgagcagaagaagaaggg(16) gttccagaaccggaggacaa(50) attgcttgtccctctgtca(84) FANCF site 1 ggaatcccttctgcagcacctgg(17) tcccaggtgctgacgtaggta(51) atcatctcgcacgtggttc(85) HEK293 site 1 gaacacaaagcatagactgcggg(18) gctaactgtgacagcatgtgg(52) caccaacttacacacagtga(86) HEK293 site 2 ggcccagactgagcacgtgatgg(19) ttctgcttctccagccctggc(53) ttcatgcaggtgctgaaagcca(87) HEK293 site 3 ggcactgcggctggaggtggggg(20) cagagggtccaaagcaggat(54) tcaacccgaacggagacac(88) RNF2 site 1 gtcatcttagtcattacctgagg(21) cggaactcaaccattaagca(55) gttgccttcaaacctgctc(89) EMX1 site 3 gtattcacctgaaagtgtgcagg(22) cttgactgatatctccaggc(56) taggggaagttggaggaggga(90)c

PPP1R12C site 1 ggcactcgggggcgagaggaggg(23) gctcaaagtggtccggactc(57) ttaccatccctccctcgact(91) PDCD1 site 1 gcgtgacttccacatgagcgtgg(24) acaccaaccaccagggttt(58) cctcacgtagaaggaagag(92) PPP1R12C site 2 gactcacccaggagtgcgttagg(25) tcagtctccctttccttcc(59) ggctacctagatatcgcca(93) PPP1R12C site 3 gagctcactgaacgctggcatgg(26) ccttcattcctgcccttct(60) agcgacctgctatttccct(94) EMX1 site 4 gttagacccatgggagcagctgg(27) tgatctctcctctagaaactcg(61) gcccgtgtcattaagagagag(95) EMX1 site 5 agagcctgatgggaagactgagg(28) tgatctctcctctagaaactcg(62) gcccgtgtcattaagagagag(96) EMX1 site 6 gtagcctcagtcttcccatcagg(29) tgatctctcctctagaaactcg(63) gcccgtgtcattaagagagag(97) DNMT3B site 3 aagtcctcctactactgccctgg(30) tgacatcatcctactggggca(64) aaagagccgttccctataca(98) DNMT3B site 4 agtctccacacaggtgctgttgg(31) tgacatcatcctactggggca(65) aaagagccgttccctataca(99) DNMT3B site 5 tgtcccccatcctgccccagagg(32) tgacatcatcctactggggca(66) aaagagccgttccctataca(100) EMX1 site 7 tcacctgggccagggagggaggg(33) tgacatcatcctactggggca(67) aaagagccgttccctataca(101) FANCF site 2 gggaccccgccaccgtgcgccgg(34) tgggttctctctatagcca(68) ctcacgtcacagtatgtct(102) FANCF site 3 cgccgtctccaaggtgaaagcgg(35) tgggttctctctatagcca(69) ctcacgtcacagtatgtct(103) FANCF site 4 acgcctctctgcaatgctattgg(36) tgggttctctctatagcca(70) ctcacgtcacagtatgtct(104) HEK293 site 4 gacgccctctggaggaagcaggg(37) aattgatgaatcagtgctgg(71) tttctcttgggcaatatggggt(105) HEK293 site 5 cagctcctgcaccgggatactgg(38) aattgatgaatcagtgctgg(72) tttctcttgggcaatatggggt(106) PPP1R12C site 4 ctgacctgcattctctcccctgg(39) gtgatgatgcaggcctaca(73) caccccacttccgaattgg(107) PPP1R12C site 5 aggcccaggggagagaatgcagg(40) gtgatgatgcaggcctaca(74) caccccacttccgaattgg(108) PPP1R12C site 6 gaagccagtagagctcaaagtgg(41) gtgatgatgcaggcctaca(75) caccccacttccgaattgg(109) PPP1R12C site 7 tgccgtctctctcctgagtccgg(42) gtgatgatgcaggcctaca(76) caccccacttccgaattgg(110) CTNNB1 site 1 gctccttctctgagtggtaaagg(43) accattcttccactgattcag(77)t ctcatctaatgtctcagggaa(111) FANCF site 5 aagttcgctaatcccggaactgg(44) atttcgcggatgttccaatcag(78) gggcgcgacaaaaggcagcaaa(112) SRPK3 site 1 cgtcgccgatcttcacagggtgg(45) ttctgggctccgacgacgaggaa(79) atctcatccacagctgtctccg(113) FANCF site 6 gtaacgagctgcatccccgaggg(46) atttcgcggatgttccaatcag80 gggcgcgacaaaaggcagcaaa(114) PPP1R12C site 8 ggggctcaacatcggaagagggg(47) tttgccaccctatgctgacac(81) cagaaggagaaggaaaagggaa(115)

Table 4. Primers used for off-target effect deep sequencing

Site On-target sequence(SEQ ID NO:) Primer 1(SEQ ID NO:) Primer 2(SEQ ID NO:) EMX1 site 2-On-target GAGTCCGAGCAGAAGAAGAAGGG(116) To To EMX1 site 2-Off-target-1 GAGTTAGAGCAGAAGAAGAAAGG(117) TTTCTGAGGGCTGCTACCTG(155) GCCCCTCTAATACAATGGG(189) EMX1 site 2-Off-target-2 GAGTCTAAGCAGAAGAAGAAGAG(118) CTCAATGTGCTTCAACCCATC(156) ACAGAGCGAGACTCCGTCT(190) EMX1 site 2-Off-target-3 GAGTCCTAGCAGGAGAAGAAGAG(119) CAGACTCAGTAAAGCCTGGA(157) TAGGCTGGAGTGCAGTGGTG(191) EMX1 site 2-Off-target-4 GAGTCCGGGAAGGAGAAGAAAGG(120) TCTGCCTCTGACGACGAGCAA(158) GAGAAAGGCAAACAGGAGG(192) EMX1 site 2-Off-target-5 AAGTCCGAGGAGAGGAAGAAAGG(121) TTCATGGAGGGGCACAGAAG(159) GCCCTTCCAAACTAGAAGTT(193) EMX1 site 2-Off-target-6 GAATCCAAGCAGGAGAAGAAGGA(122) GAAACCGAATTATGGATGGG(160) CTCTTAGAAATGGCATTGGG(194) EMX1 site 2-Off-target-7 ACGTCTGAGCAGAAGAAGAATGG(123) TCGTCTTCCTGCAGAGGTTC(161) ACTCCCATCTTCCTCCCTA(195)

FANCF site 1-On-target GGAATCCCTTCTGCAGCACCTGG(124) To To FANCF site 1-Off-target-1 GGAACCCCGTCTGCAGCACCAGG(125) GTCTTAGTCGCCTTAGCACT(162) ATGTGCTCTGATTTCCGTG(196) FANCF site 1-Off-target-2 GGAGTCCCTCCTACAGCACCAGG(126) CATCCCGAACACAGTGACAG(163) AGATGGAAGAATGAGCAGG(197) FANCF site 1-Off-target-3 AGAGGCCCCTCTGCAGCACCAGG(127) AGGACTCAGGCAGGAGTTAG(164) TGCGGGGTGTGGATGATTT(198) FANCF site 1-Off-target-4 ACCATCCCTCCTGCAGCACCAGG(128) TAGAGTGGCATGCAACCTAG(165) AATGTGCTGGGTCTCTCCT(199) FANCF site 1-Off-target-5 TGAATCCCATCTCCAGCACCAGG(129) CAGAAACACTGGAGACCCTC(166) GATGAAGAAACTGAGGCACA(200) FANCF site 1-Off-target-6 GGAGTCCCTCCTACAGCACCAGG(130) CCGAACACAGTGACAGAAGG(167) GCCCAGTGAGACCAGTTTG(201) FANCF site 1-Off-target-7 GGAGTCCCTCCTGCAGCACCTGA(131) GGAAAATTGCTTGTCGCAGC(168) CCCCTCTGACGGTAATAAT(202) HEK293 site 1-On-target GAACACAAAGCATAGACTGCGGG(132) To To HEK293 site 1-Off-target-1 GAACACAATGCATAGATTGCCGG(133) CATATTTAATGCTCCCACACC(169) AGCCACATTGTAGACAATGAAGCC(203) HEK293 site 1-Off-target-2 AAACATAAAGCATAGACTGCAAA(134) CAGAATAGTGGGACTATGCC(170) TCACCCTCCTCCTCTCACT(204) HEK293 site 1-Off-target-3 TCAGGGTGAGCATAGACTGCCGG(135) AGATAGGACAGGTGAGGCCT(171) GGCAGGGATGAAAGGTGTC(205) HEK293 site 1-Off-target-4 TGAAGTGTTGCATAGACTGCAGG(136) ACCCCTCATGCAAATCCTAAC(172) TGGGTGGCTAGACTCAGAG(206) HEK293 site 1-Off-target-5 GGAGAGAGAGCATAGACTGCTGG(137) TCTGTACCTGCTGGGCATCCA(173) GAACATCACTCCCATCACG(207) HEK293 site 1-Off-target-6 CCAAACAAAACATAGACTGCTGG(138) GGGTAAGACTCTACCCAGGA(174) TTAATAGCAGTGTGGTGGG(208) HEK293 site 2-On-target GGCCCAGACTGAGCACGTGATGG(139) To To HEK293 site 2-Off-target-1 CACCCAGACTGAGCACGTGCTGG(140) GACAAGAGCATTAACTGCACC(175) CTCTTCTTCCGAGTGGTGG(209) HEK293 site 2-Off-target-2 GACACAGACTGGGCACGTGAGGG(141) GTGGAGTCAGCCTCGATTAC(176) GATTAGGGTTGCCAAGAGA(210) HEK293 site 2-Off-target-3 AGCTCAGACTGAGCAAGTGAGGG(142) TTCAGTCCAGACATCAGCCA(177) GGCGATGAGTAAGAGTGATGTG(211) HEK293 site 2-Off-target-4 AGACCAGACTGAGCAAGAGAGGG(143) actttggaaggtcgaagcggca(178) TGCATGGTTCATCTCCCCTA(212) HEK293 site 2-Off-target-5 GAGCCAGAATGAGCACGTGAGGG(144) GGAAATTGCGAGCAGAGGCT(179) CTGGGGTCTCTTTCTGCCTC(213) HEK293 site 2-Off-target-6 CAGGAAGCTGGAGCACGTGAGGG(145) CATCCCTTGTCTCTCTTAGG(180) TACACGTTCCACCCCTCCAACC(214) HEK293 site 2-Off-target-7 AAGGCTGAGGGAGCACGTGAAGG(146) AGTACAAGCTGATTACATCC(181) GGTGGAGACAGAAAATGAGG(215) HEK293 site 2-Off-target-8 GTCAGGGGAAGAGCACGTGACGG(147) ACTGCAGCCTGGCCCTAAAC(182) CTACCTCCAAGCCACCAAAC(216) HEK293 site 2-Off-target-9 GTTGTGAACTGAGCACGTGAGGG(148) CATTTCCTGTCAGATCACGG(183) TCAAATGCTCCACCCGCCTCA(217) HEK293 site 2-Off-target-10 ATATTTGCTGGAGCACGTGAAGG(149) TCTGAAGCTATGCGCTGGAG(184) TCAGAACCCCAATACCCCTC(218) HEK293 site 3-On-target GGCACTGCGGCTGGAGGTGGGGG(150) To To HEK293 site 3-Off-target-1 TGCACTGCGGCCGGAGGAGGTGG(151) TGGGCTCACTGCTCTCCAGAGT(185) AGGAAGGGTACTGGGGAGT(219) HEK293 site 3-Off-target-2 GGCTCTGCGGCTGGAGGGGGTGG(152) CAAGTGCTCCCCAATCCTGA(186) TGGTGAAGAGGATGGGGTGA(220)

HEK293 site 3-Off-target-4 GGCACTGCTACTGGGGGTGGTGG(153) CCGTTGCTTGTCAGCATCCT(187) ACTGCTCCCTCTGTTCTCAT(221) HEK293 site 3-Off-target-6 GGCACTGGGGTTGGAGGYGGGGG(154) CCATGGCAAACTCTCCACCA(188) GTCATTTCAGTGGCAGCGGA(222)

GOTI's FACS

In order to isolate mouse embryonic cells, the prepared tissue was enzymatically hydrolyzed in 5ml trypsin EDTA (0.05%) incubation solution at 37°C for 30 minutes, and 5ml DMEM medium and 10% fetal bovine serum (FBS) were added to stop the digestion. Then use a 1 ml pipette to homogenize the fetal tissue 30-40 times. The cell suspension was centrifuged for 6 min (800 rpm), and then the pellet was resuspended in DMEM medium containing 10% FBS. Finally, filter the cell suspension with a 40μm cell strainer, and separate the tdtomato ⁺ /tdtomato ^- cells with a flow cytometer. Through the second round of flow cytometry and fluorescence microscope analysis, it was found that the purity of the sample was greater than 95%.

Whole genome sequencing and RNA sequence data analysis

Genomic DNA was extracted from the cells using the DNeasy Blood and Tissue Kit (Cat. No. 69504, Qiagen) according to the instructions. Whole genome sequencing is performed by Illumina HiSeq X 10, with an average coverage rate of 50 times. BWA (v0.7.12) is used to map qualified sequencing reads to the reference genome (mm10). Then use Picard tool (v2.3.0) to sort and mark the mapped BAM files. In order to identify the new SNVs of the whole genome with high confidence, the inventors performed single nucleotide mutations, using the default parameters of Mutect2 (v3.5), Lofreq (v2.1.2) and Strelka (v2.7.1). Kind of algorithm. At the same time, Mutect2 (v3.5), Scalpel (v0.5.3) and Strelka (v2.7.1) were used to detect the whole genome de novo index, using default parameters. The overlap of the three SNVs or indel algorithms is considered a true mutation. All sequencing data are stored in the NCBI Sequence Reading File (SRA).

Use two previously reported algorithms, namely Cas offender (http://www.rgenome.net/Cas-officer/) and CRISPOR (http://CRISPOR.tefor.net/) to predict the potential off-target site of the target site . SNVs and indels are annotated with annovar (version 2016-02-01) using the RefSeq database.

RNA sequence data analysis uses FastQC (v0.11.3) and Trimmomatic (v0.36) for quality control. Qualified readings use STAR (v2.5.2b) and are mapped to the reference genome (integrated GRCh38) in the 2-way mode with default parameters. Then use the Picard tool (v2.3.0) to sort and mark the duplicates of the mapped BAM file. The optimized BAM file has been split read across joint connections, local realignment, basic recalibration, and variant calls using SplitNCigarReads, IndelRealigner, BaseRecalivator, and haplotype calling tools in GATK (v3.5) .

Structure prediction

To retrieve the amino acid sequences of rat APOBEC1 and human APOBEC3G from UniProt (https://www.UniProt.org/), use NCBI-blastp (https://blast.NCBI.nlm.nih.gov/blast.cgi? program= blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). According to the consensus sequence and secondary structure information of known structural proteins, the protein structure prediction server (PS) predicts the structure of rAPOBEC1. The crystal structure of APOBEC3G was downloaded from PDB (http://www.rcsb.org/3d-view/3IQS) and presented using PyMOL (v2.3.2).

Statistical Analysis

The present invention uses R version 3.5.1 (http://www.R-project.org/) for statistical analysis. All tests are two-sided, P<0.05 considered the difference to be significant.

Example 1. APOBEC1 mutants and their effects

The present inventors introduced various mutations into recombinant APOBEC1 (rAPOBEC1) in the hope of affecting DNA or RNA activity (Figure 1a). Specifically, variants include leucine-enriched N-terminal or C-terminal deletions or mutations of rAPOBEC1 (Del32, R33A, K34A, Del34, Del77, Del116, Del169, Del182, P190A, and P191A), and predicted rAPOBEC1 catalytic activity Site mutations (H61A, H61R, V62A, E63A, E63Q, C93S, C96S). After studying the structure of APOBEC3G, the inventor predicted that the R126 site of rAPOBEC1 (corresponding to the R320 site of APOBEC3G) interacts with the phosphate backbone of ssDNA (Figure 1b, c), and the R126E mutant can retain target editing activity. R128 and R132 are close to R126, and the inventors also introduced R128E and R132E mutations (Figure 1a-c). The inventors also studied the effect of a combination of point mutations (W90A, W90F, W90Y) at the active site of the hydrophobic domain of rAPOBEC1, and these mutations can reduce the width of the base editing window.

The inventors will use CBE mutant plasmids to transfect HEK293T cells to analyze its editing activity and off-target effects. Through detection on 10 genomic target sites, 7 mutants that can retain their targeted activity were screened from 23 mutants, including R33A, K34A, V62A, W90F+R126E, W90Y+R126E, R126E, and R132E , 4 of the mutants (W90F+R126E, W90Y+R126E, R126E, R132E) did not increase the mutation efficiency of indels (Figure 1d, Figure 4 and Table 5). In addition, these experiments show that the editing window of the W90F+R126E, W90Y+R126E, and R132E variants becomes narrower, Figure 4).

Table 5. P value of targeting efficiency and indel rate between CBE variants and BE3

Next, the inventors used GOTI to evaluate the DNA of mutants BE3 ^R126E , BE3 ^R132E , BE3 ^W90Y+R126E (YE1-BE37) and BE3 ^W90F+R126E (FE1-BE37) (Table 6) that have high DNA targeting efficiency. Off-target activity. First of all, it should be noted that the embryonic development of Ai9 mice has not been harmfully affected by any of these variants (Figure 5). The targeting efficiency of these variants was evaluated by whole-genome sequencing (Figure 6). Compared to wild-type embryos treated ^{BE3, BE3} R126E, BE3 R132E, the number of non-targeted SNVs DNA YE1-BE3 or FE1-BE3 treated embryos was significantly ^reduced: BE3 R126E treated embryos in DNA from non-targeted SNV 283±32 was reduced to 28±6, the ^untargeted SNV of DNA in embryos treated with BE3 R132E was 47±8, the untargeted SNV of DNA in embryos treated with YE1-BE3 was 12±2, and the DNA treated with FE1-BE3 The non-targeted SNV is 27±19.

Table 6. HiSeq×10 sequencing summary

Importantly, there was no significant difference in the number of SNVs between embryos injected with the four CBE variants and unedited control embryos (14 SNVs on average, close to the spontaneous mutation rate) (Figure 2a, Figure 7 and Table 7). In addition, compared with wild-type BE3, CBE variants showed significantly reduced mutations, and the SNV detected in these CBE variant groups did not overlap with the off-target sites analyzed using Cas-Offender and CRISPOR software (Figure 2b, Figures 6 and 8). These findings indicate that these CBE variants can produce fewer sgRNA-independent DNA off-targets.

Table 7. Non-target SNV from different algorithms

The inventors also used RNA-seq to evaluate the off-target effects of these variants on the transcriptome of the HEK293T cells transfected. Compared with wild-type BE3, three variants BE3 ^R132E , YE1-BE3 and FE1-BE3 showed significantly reduced RNA off-target editing 36h after transfection (Figures 2c and 2d). In contrast, ^{the RNA off-target editing of the BE3 R126E} mutant strain did not decrease at 36h after transfection, but it decreased significantly at 72h after transfection. Compared with the GFP-transfected control cell group, the ^{number of SNVs in the wild-type BE3 and BE3 R126E} groups increased significantly, but the number of SNVs in the BE3 ^R132E , YE1-BE3 or FE1-BE3 groups did not increase (Figures 2c, 2d, Figures 9 and 10). ). In summary, these results indicate that the BE3 ^R132E , YE1-BE3, and FE1-BE3 variants are high-fidelity base editors, and their DNA off-target and RNA off-target effects are significantly reduced compared with BE3.

Although the aforementioned three BE3 variants (BE3 ^R132E , YE1-BE3 and FE1-BE3 variants) can significantly reduce off-target effects, their targeted editing efficiency is not as good as BE3-hA3A (Figure 3a and Figure 11). BE3 (hA3AY130F) was converted from humanAPOBECA3A mutation Y130 to F. It can be observed that this mutation significantly reduces the number of off-target SNVs. The inventors used GOTI to analyze the off-target effects of BE3-hA3A, but found that BE3-hA3A is obviously toxic to embryos (Figure 5).

The inventors tried to further obtain a base editor with high target efficiency and high fidelity, introduced the Y130F mutation into BE3-hA3A, and found that the BE3-hA3A ^Y130F editor has high targeted editing efficiency (Figure 3a) , But it still produces a large number of DNA off-target SNVs (409±86) (Figure 3b and Figure 11).

Next, the inventors constructed a high-fidelity variant YE1-BE on the basis of the BE3-FNLS editor. A nuclear localization signal peptide was added to the C-terminus and N-terminus of the variant. The sequence was optimized for codons expressed in human cells. The codon-optimized DNA sequence is (SEQ ID NO: 2):

In addition to this new YE1-BE3-FNLS variant, the inventors tested the targeted editing efficiency of ^{BE3, YE1-BE3, BE3-hA3A, BE3-hA3A, Y130F, and BE3-FNLS on 21 targets of HEK293T cells.} And bystander editing. YE1-BE3-FNLS had the highest targeting efficiency, which was 70.7±5.2% (Figure 3d). It is worth noting that YE1-BE3-FNLS has the lowest indels level among the tested variants, at 0.8±0.2%, and the number of other bystander edits is also the lowest at 0.6±0.4% (Figure 3d-e). In addition, compared with BE3, YE1-BE3-FNLS also significantly reduced the off-target activity of DNA and RNA, reaching a level comparable to that of the unedited control group (Figure 3b-c). Considering that the GOTI method is used to analyze the sgRNA-independent off-target effects of gene editing proteins, the inventors also need to detect the sgRNA-dependent off-target effects of YE1-BE3-FNLS. Compared with cells treated with other BE3 variants, there was no significant difference in sgRNA-dependent off-target effects in cells transfected with YE1-BE3-FNLS (Figure 12). Therefore, this new base editor mutant meets the inventor's double standard, namely, high targeted editing efficiency and high fidelity.

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (22)

A method for improving the targeted editing efficiency or fidelity of a cytosine base editor includes: in the cytosine base editor, modifying the cytosine deaminase therein, the cytosine deaminase Including APOBEC1 or its homologues, the modification includes mutating the amino acid of the cytosine deaminase corresponding to Trp (W) at position 90 and Arg (R) at position 126 of APOBEC1, and the cytosine base The base editor is connected to the nuclear localization sequence. The method of claim 1, wherein the cytosine base editor is a BE3 gene editor system. The method of claim 1, wherein the APOBEC1 homologue comprises an enzyme selected from the group consisting of AID, APOBEC3G, APOBECA3A, CDA1. The method according to claim 1, wherein the mutation is a mutation of the cytosine deaminase corresponding to APOBEC1 at position 90 Trp to Tyr(Y); and/or, the mutation at position 126 of Arg Mutation to Glu(E). The method of claim 1, wherein the N-terminal and/or C-terminal of the cytosine base editor is connected to a nuclear localization sequence. A modified cytosine deaminase, said cytosine deaminase comprising APOBEC1 or its homologues, the cytosine deaminase corresponding to APOBEC1 90th Trp (W) and 126th Arg (R ) Has a mutation in the amino acid. The engineered cytosine deaminase according to claim 6, wherein the APOBEC1 homologue comprises an enzyme selected from the group consisting of AID, APOBEC3G, APOBECA3A, CDA1. The modified cytosine deaminase according to claim 6, wherein the mutation is that the Trp at position 90 of the cytosine deaminase corresponding to APOBEC1 is mutated to Tyr(Y); and/or , Arg at position 126 is mutated to Glu(E). A cytosine base editor, comprising the modified cytosine deaminase according to any one of claims 6-8. 9. The cytosine base editor of claim 9, wherein the cytosine base editor is also connected to a nuclear localization sequence; preferably, the N-terminal and/or C-terminal is connected to the nuclear localization sequence. 9. The cytosine base editor of claim 10, wherein the cytosine base editor and the nuclear localization sequence further comprise a linking sequence, such as a tag sequence (more specifically, a Flag tag). An isolated polynucleotide encoding the modified cytosine deaminase according to any one of claims 6-8 or the cytosine base editor according to any one of claims 9-11. The polynucleotide of claim 12, wherein the cytosine base editor has the nucleotide sequence shown in SEQ ID NO: 2. A recombinant expression vector comprising the polynucleotide of claim 12 or 13. A genetically engineered host cell, which contains the vector according to claim 14, or the polynucleotide according to claim 12 or 13 is integrated into the genome. The cytosine base editor of claim 14, wherein the editor is a BE3 base editor. The use of the cytosine base editor according to any one of claims 9-11 for gene editing, reducing off-target effects, improving the efficiency of targeted editing, or improving the fidelity of targeted editing. A method for gene editing, comprising mediating gene editing with the cytosine base editor according to any one of claims 9-11. The method of claim 18, wherein the nucleic acid sequence encoding the cytosine base editor and sgRNA are co-injected into the receptor, thereby performing gene editing. The method of claim 19, wherein the receptor comprises: a somatic cell or a germ cell. The method of claim 20, wherein the germ cells comprise embryonic cells or fertilized eggs. A reagent or kit for gene editing, which comprises the modified cytosine deaminase according to any one of claims 6-8; or which comprises the cytosine base according to any one of claims 9-11 Base editor.

Images