WO2021155607A1 - Éditeur de base de cytosine modifié et son application - Google Patents

Éditeur de base de cytosine modifié et son application Download PDF

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WO2021155607A1
WO2021155607A1 PCT/CN2020/074561 CN2020074561W WO2021155607A1 WO 2021155607 A1 WO2021155607 A1 WO 2021155607A1 CN 2020074561 W CN2020074561 W CN 2020074561W WO 2021155607 A1 WO2021155607 A1 WO 2021155607A1
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cytosine
base editor
target
sequence
editing
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PCT/CN2020/074561
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Chinese (zh)
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杨辉
左二伟
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辉大(上海)生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase

Definitions

  • the present invention belongs to the field of biotechnology. More specifically, the present invention relates to a modified cytosine base editor and its application.
  • 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.
  • HGGTS high-throughput whole-genome translocation sequencing
  • GUI-seq unbiased recognition of double-strand breaks
  • CIRCLE-seq report cleavage efficiency in vitro by cycle sequencing
  • none of these methods can effectively detect single nucleotide variation (SNV). So far there is no effective method to detect SNV in this field.
  • the defect lies in the low editing efficiency of homology-mediated repair.
  • XTEN linker 16aa long XTEN linker
  • 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%.
  • 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.
  • U uracil
  • this BE3 system significantly improves the base editing efficiency, and its average indel (insertion-deletion) incidence is only 1.1%.
  • CBE cytosine base editor
  • the purpose of the present invention is to provide a modified cytosine base editor and its application.
  • a method for improving the efficiency or fidelity of targeted editing of a cytosine base editor 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.
  • the cytosine base editor is BE3 gene editor system.
  • the APOBEC1 homologue includes an enzyme selected from the group consisting of AID, APOBEC3G, APOBECA3A, CDA1.
  • 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) .
  • 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.
  • a modified 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.
  • the APOBEC1 homologue includes an enzyme selected from the group consisting of AID, APOBEC3G, APOBECA3A, CDA1.
  • 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).
  • a cytosine base editor which comprises the modified cytosine deaminase.
  • 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.
  • the cytosine base editor and the nuclear localization sequence further include a linking sequence, such as a tag sequence (more specifically, a Flag tag).
  • an isolated polynucleotide which encodes the modified cytosine deaminase or the cytosine base editor.
  • the cytosine base editor has the nucleotide sequence shown in SEQ ID NO: 2.
  • a recombinant expression vector which comprises the polynucleotide.
  • a genetically engineered host cell which contains the vector or the polynucleotide integrated in the genome.
  • the editor is BE3 base editor.
  • 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.
  • a method for gene editing which includes mediating gene editing with the cytosine base editor.
  • nucleic acid sequence encoding the cytosine base editor and the sgRNA are co-injected into the receptor to perform gene editing.
  • the receptor includes: somatic cells or germ cells.
  • the germ cells include embryonic cells or fertilized eggs.
  • a reagent or kit for gene editing which includes the modified cytosine deaminase; or the cytosine base editor.
  • FIG. 1 Targeted editing efficiency of cytosine base editor (CBE) variants.
  • CBE cytosine base editor
  • 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.
  • FIG. 1 DNA and RNA off-target activity of CBEs variants.
  • 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.
  • FIG. 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.
  • c Comparison of the total number of RNA off-target SNVs detected 36 h after transfection. 3 repetitions per group.
  • FIG. 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.
  • FIG. 1 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.
  • FIG. 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.
  • FIG. 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.
  • FIG. 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.
  • 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.
  • FIG. 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 Distribution of DNA non-targeted SNVs mutation types of embryos treated with BE3-hA3AY130F and YE1-BE3-FNLS.
  • RNA non-targeted SNVs mutation types of embryos treated with BE3-hA3AY130F and YE1-BE3-FNLS g.
  • h. The RNA off-target rate of BE3 and BE3-FNLS 36 hours after transfection.
  • Each group n 3 biological replicates.
  • FIG. 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.
  • Figure 13 Schematic diagram of YE1-BE3-FNLS plasmid.
  • 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.
  • 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.
  • 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).
  • 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.
  • the tag can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ⁇ , 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.
  • fragment refers 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.
  • 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.
  • amino acids with similar or similar properties when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the polynucleotide sequence of the present invention can be used to express or produce a recombinant engineered enzyme.
  • the present invention also provides a cytosine base editor containing the modified enzyme or its polynucleotide sequence.
  • the cytosine base editor is BE3 base editor.
  • Other components of the cytosine base editor are known to those skilled in the art.
  • the modified enzyme polynucleotide sequence or the cytosine base editor polynucleotide sequence can be inserted into a recombinant expression vector.
  • 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.
  • 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.
  • a method for gene editing which includes mediating gene editing with the cytosine base editor of the present invention.
  • other gene editing reagents can be used known in the art.
  • sgRNA can be designed in a manner known in the art.
  • 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.
  • CBE cytosine base editor
  • the off-target effects of DNA and RNA limit its application in science and medicine.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • R132E affects the interaction of rAPOBEC1 with DNA and RNA
  • R126E mainly affects its DNA binding ability
  • Y130F mainly affects its RNA binding ability.
  • 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.
  • 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).
  • 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.
  • the wild-type APOBEC1 protein sequence is shown in SEQ ID NO: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.
  • 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.
  • 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)
  • primers F and R base editor IVT F and base editor IVT R, etc. in Table 2
  • 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.
  • 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.
  • 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.
  • 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.
  • HEK293T cells were cultured in DMEM containing 10% fetal bovine serum (FBS) and 37°C humidified incubator containing 5% CO2.
  • FBS fetal bovine serum
  • 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 40 ⁇ m cell strainer, and EGFP/mCherry positive cells were separated by flow cytometry.
  • PBS phosphate buffered saline
  • RNAs 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.
  • 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.
  • 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 40 ⁇ m 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%.
  • 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
  • mm10 reference genome
  • Picard tool v2.3.0
  • 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 Kind of algorithm.
  • 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) .
  • PS protein structure prediction server 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).
  • 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.
  • 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).
  • 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.
  • 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).
  • these experiments show that the editing window of the W90F+R126E, W90Y+R126E, and R132E variants becomes narrower, Figure 4).
  • 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
  • the DNA treated with FE1-BE3 The non-targeted SNV is 27 ⁇ 19.
  • RNA-seq used to evaluate the off-target effects of these variants on the transcriptome of the HEK293T cells transfected.
  • three variants BE3 R132E , YE1-BE3 and FE1-BE3 showed significantly reduced RNA off-target editing 36h after transfection ( Figures 2c and 2d).
  • 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.
  • BE3-hA3A Figure 3a and Figure 11
  • BE3 hA3AY130F
  • BE3 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 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):
  • YE1-BE3-FNLS 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).

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Abstract

L'invention concerne un éditeur de base de cytosine modifié et son application. L'efficacité d'édition ciblée et la fidélité de l'éditeur de base de cytosine modifié sont considérablement améliorées.
PCT/CN2020/074561 2020-02-07 2020-02-07 Éditeur de base de cytosine modifié et son application WO2021155607A1 (fr)

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CN107164377A (zh) * 2017-06-12 2017-09-15 王小平 基于碱基编辑的基因敲除方法及其应用
CN108513575A (zh) * 2015-10-23 2018-09-07 哈佛大学的校长及成员们 核碱基编辑器及其用途
WO2018165629A1 (fr) * 2017-03-10 2018-09-13 President And Fellows Of Harvard College Éditeur de base cytosine à guanine
CN108822217A (zh) * 2018-02-23 2018-11-16 上海科技大学 一种基因碱基编辑器
CN109321584A (zh) * 2017-12-27 2019-02-12 华东师范大学 一种简单定性/定量检测单碱基基因编辑技术工作效率的报告系统
WO2019042284A1 (fr) * 2017-09-01 2019-03-07 Shanghaitech University Protéines de fusion pour une précision améliorée dans l'édition de base
WO2019126709A1 (fr) * 2017-12-22 2019-06-27 The Broad Institute, Inc. Systèmes cas12b, procédés et compositions pour l'édition de base d'adn ciblée
WO2019126762A2 (fr) * 2017-12-22 2019-06-27 The Broad Institute, Inc. Systèmes cas12a, procédés et compositions d'édition ciblée de bases d'arn

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108513575A (zh) * 2015-10-23 2018-09-07 哈佛大学的校长及成员们 核碱基编辑器及其用途
WO2018165629A1 (fr) * 2017-03-10 2018-09-13 President And Fellows Of Harvard College Éditeur de base cytosine à guanine
CN107164377A (zh) * 2017-06-12 2017-09-15 王小平 基于碱基编辑的基因敲除方法及其应用
WO2019042284A1 (fr) * 2017-09-01 2019-03-07 Shanghaitech University Protéines de fusion pour une précision améliorée dans l'édition de base
WO2019126709A1 (fr) * 2017-12-22 2019-06-27 The Broad Institute, Inc. Systèmes cas12b, procédés et compositions pour l'édition de base d'adn ciblée
WO2019126762A2 (fr) * 2017-12-22 2019-06-27 The Broad Institute, Inc. Systèmes cas12a, procédés et compositions d'édition ciblée de bases d'arn
CN109321584A (zh) * 2017-12-27 2019-02-12 华东师范大学 一种简单定性/定量检测单碱基基因编辑技术工作效率的报告系统
CN108822217A (zh) * 2018-02-23 2018-11-16 上海科技大学 一种基因碱基编辑器

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