WO2023125814A1 - 腺嘌呤脱氨酶及其应用 - Google Patents

腺嘌呤脱氨酶及其应用 Download PDF

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WO2023125814A1
WO2023125814A1 PCT/CN2022/143408 CN2022143408W WO2023125814A1 WO 2023125814 A1 WO2023125814 A1 WO 2023125814A1 CN 2022143408 W CN2022143408 W CN 2022143408W WO 2023125814 A1 WO2023125814 A1 WO 2023125814A1
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editing
amino acid
sequence
seq
ads
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陈亮
李大力
陈曦
汝高盟
李长青
高弘毅
朱碧云
杨倞
白思佳
丁若一
刘明耀
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华东师范大学
上海邦耀生物科技有限公司
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Priority claimed from CN202111641225.1A external-priority patent/CN116410963A/zh
Priority claimed from CN202111644450.0A external-priority patent/CN116410964A/zh
Application filed by 华东师范大学, 上海邦耀生物科技有限公司 filed Critical 华东师范大学
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Definitions

  • the invention belongs to the field of gene editing, and in particular relates to an adenine deaminase and application thereof.
  • the essence of human genetic diseases is due to gene mutations. About 60% of genetic diseases are caused by single base mutations. The traditional use of homologous recombination mediated by genome editing technology to correct such genetic diseases is very inefficient (0.1%-5 %) [1,2] .
  • the single base editor derived from the CRISPR system is an emerging high-efficiency base editing technology in recent years. Due to its advantages such as no DNA double-strand breaks, no need for recombination templates, and high-efficiency editing, it has shown great promise in basic research and clinical disease treatment. application prospects.
  • Classical base editors are mainly divided into cytosine base editor CBE and adenine base editor ABE, the former is derived from Streptococcus pyogenes (Streptococcus pyogenes) spCas9n with impaired activity, rat-derived cytosine base editor Composed of ammoniaase rAPOBEC1 and uracil glycosidase inhibitors, in which Cas9 protein uses NGG as PAM to recognize and specifically bind DNA, and then under the action of deaminase and DNA repair, it finally targets upstream of NGG (position 21-23)
  • the 20bp range of the sequence realizes the replacement of C ⁇ GT ⁇ A, and the editing window is mainly located at positions 4-8 [2] .
  • the latter is the fusion of bacterial-derived TadA and spCas9.
  • ABE7.10 which can act on single-stranded DNA.
  • the editing region is mainly located at positions 4-7.
  • the average editing efficiency of A ⁇ TG ⁇ C caused by this system in human cells is about 53%, which is much higher than the efficiency of base mutation mediated by homologous recombination, and the purity of the product is as high as 99.9% % and extremely low indels (insertions and deletions) occurred [3] .
  • the technical problem to be solved by the present invention is to provide an adenine deaminase and its application in order to overcome the lack of cytosine base editors with high precision, high safety and high efficiency editing in the prior art.
  • the base editor comprising the adenine deaminase completely avoids the editing of adenine and greatly improves the editing of cytosine;
  • the base editor comprising the adenine deaminase can be accurate, efficient and safe It mediates cytosine editing in a specific way, showing high editing activity and low off-target events, so it has extremely high safety, which will greatly promote its wide application in the field of gene editing.
  • the present invention uses adenine deaminase TadA-8e (only 167 amino acids) as an entry point, and through analysis and rational design of the crystal structure of TadA-8e, identifies the "molecular switch" that induces ABE to convert into CGBE "( Figure 1), that is, completely abolishing the adenine editing ability of ABE itself, greatly increasing the editing properties of the substrate cytosine, and developing the ABE-driven ADS-BE1 (currently the smallest CGBE) after multiple rounds of screening and transformation, It can cause precise C-G to G-C base transversion editing at positions 5-7.
  • ADS-BE1 exhibits higher editing activity and lower off-target events. Therefore, it has extremely high security.
  • ADS-BE1 that transforms ABE into CGBE (C G base editor) (uracil glucosidase inhibitor)
  • C G base editor uracil glucosidase inhibitor
  • base editor uracil glucosidase inhibitor
  • the first aspect of the present invention provides a kind of adenine deaminase, the aminoacid sequence of described adenine deaminase is the mutated sequence of SEQ ID NO:2, and the position of the mutation is the 28th, the 30th One or two of the 46th and 46th digits.
  • the mutation is selected from one or more of the following:
  • amino acid residue N at position 46 is replaced with L or P.
  • the mutation site is position 46, and position 27, 29 or 48.
  • the mutation is selected from any of the following:
  • amino acid residue N at position 46 is replaced by L
  • amino acid residue P at position 29 is replaced by A
  • the second aspect of the present invention provides a cytosine base editor, the cytosine base editor includes nuclease and adenine deaminase according to the first aspect of the present invention.
  • the nuclease and the adenine deaminase are connected by a linker, and the linker can be conventional in the art, preferably such as SEQ ID NO: 3, SEQ ID NO: 9 or SEQ ID NO :10 shown.
  • the nuclease is preferably a Cas protein and variants thereof, and the Cas protein is preferably selected from spCas9 derived from Saccharomyces cerevisiae, SaCas9 derived from Staphylococcus aureus, LbCas12a derived from Lachnospiraceae bacteria, and Amidococcus It belongs to enAsCas12a of bacterial origin; the Cas protein variant is preferably selected from VQR-spCas9, VRER-spCas9, spRY, spNG, SaCas9-KKH and SaCas9-NG.
  • the nuclease is spCas9, and its amino acid sequence is preferably as shown in SEQ ID NO:4.
  • the cytosine base editor further comprises a uracil DNA glycosylase inhibitor (UGI).
  • UMI uracil DNA glycosylase inhibitor
  • the UGI is connected to the nuclease through a linker, and the copy number of the UGI is at least one, such as one, two or three.
  • the third aspect of the present invention provides a fusion protein, said fusion protein sequentially comprises a signal peptide sequence, adenine deaminase as described in the first aspect, a nuclease and a signal peptide sequence from the N-terminal to the C-terminal; Said nuclease is as described in the second aspect of the present invention.
  • the fusion protein can also be fused with other functional proteins, and the other functional proteins can improve the cytosine editing efficiency, regulate the editing window or precise editing efficiency of the fusion protein.
  • the signal peptide sequence is a nuclear localization signal sequence or a polyA signal sequence, the nuclear localization signal sequence is preferably shown in SEQ ID NO: 1; the polyA signal sequence is preferably BGH polyA.
  • the fusion protein further includes UGI as described in the second aspect of the present invention.
  • the polyA signal sequence may be a conventional polyadenylation signal sequence derived from eukaryotic cells or prokaryotic cells in the art.
  • the BGH polyA is bovine growth hormone polyadenylation signal.
  • the fusion protein sequentially comprises a signal peptide sequence, adenine deaminase, nuclease, signal peptide sequence, UGI and signal peptide sequence from the N-terminal to the C-terminal;
  • the UGI is preferably defined by an amino acid sequence such as The linker shown in SEQ ID NO: 7 is connected to the signal peptide sequence at its N-terminal.
  • UGI is at least two copies
  • UGI is preferably connected by a linker as shown in SEQ ID NO:8.
  • the length of the linker can be adjusted according to what is known in the art, so as to improve the cytosine editing efficiency of the fusion protein, regulate the editing window or precise editing efficiency.
  • the fourth aspect of the present invention provides an isolated nucleic acid encoding the adenine deaminase as described in the first aspect of the present invention, the cytosine base editor as described in the second aspect of the present invention Or the fusion protein as described in the third aspect of the present invention.
  • the fifth aspect of the present invention provides a gene expression cassette, which includes a promoter element and a gene element of interest; wherein the gene element of interest is the nucleic acid according to the fourth aspect of the present invention.
  • the promoter element may be conventional in the art, preferably selected from CMV, CAG, PGK, EF1 ⁇ , Ctsk and Lp1.
  • the sixth aspect of the present invention provides a cytosine base editing system, which includes: sgRNA and the cytosine base editor according to the second aspect of the present invention.
  • the target sequence of the sgRNA is as shown in any of SEQ ID NOs: 11-24 or SEQ ID NOs: 83-93.
  • the seventh aspect of the present invention provides a pharmaceutical composition, which comprises the adenine deaminase as described in the first aspect of the present invention, the cytosine base as described in the second aspect of the present invention
  • the editor the fusion protein according to the third aspect of the present invention or the cytosine base editing system according to the sixth aspect of the present invention, and a pharmaceutically acceptable carrier.
  • the eighth aspect of the present invention provides a base editing method, the base editing method comprising:
  • base editing occurs in the target cell; the base editing includes editing C to T and C to G.
  • the source of the target cells is an isolated cell line; preferably, the cell line is a mammalian cell line.
  • the isolated cell line is 293T cells, HELA cells, U2OS cells, NIH3T3 cells or N2A cells.
  • the base editing method may be for non-therapeutic purposes.
  • the ninth aspect of the present invention provides a method for constructing an animal model, crop breeding or manufacturing a base editing device, the method comprising using the adenine deaminase as described in the first aspect of the present invention, the adenine deaminase as described in the present invention
  • the tenth aspect of the present invention provides a pharmaceutical composition for gene therapy, the pharmaceutical composition comprising the adenine deaminase as described in the first aspect of the present invention, as described in the second aspect of the present invention
  • the cytosine base editor of the present invention the fusion protein as described in the third aspect of the present invention, the nucleic acid as described in the fourth aspect of the present invention, the gene expression cassette as described in the fifth aspect of the present invention or the gene expression cassette as described in the fifth aspect of the present invention
  • the cytosine base editing system according to the sixth aspect; the gene therapy is a gene therapy for treating diseases caused by gene mutations.
  • the eleventh aspect of the present invention provides a method for gene therapy, the method is to use the adenine deaminase as described in the first aspect of the present invention, the cytosine base as described in the second aspect of the present invention A base editor, a fusion protein as described in the third aspect of the present invention, a nucleic acid as described in the fourth aspect of the present invention, a gene expression cassette as described in the fifth aspect of the present invention, or a gene expression cassette as described in the sixth aspect of the present invention
  • the cytosine base editing system treats diseases caused by gene mutations.
  • the twelfth aspect of the present invention provides a recombinant expression vector comprising the nucleic acid according to the fourth aspect of the present invention or the gene expression cassette according to the fifth aspect of the present invention.
  • the thirteenth aspect of the present invention provides a transformant comprising the nucleic acid as described in the fourth aspect of the present invention, the gene expression cassette as described in the fifth aspect of the present invention or the gene expression cassette as described in the present invention
  • the recombinant expression vector of the twelfth aspect is provided.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the adenine deaminase of the present invention completely avoids the editing of adenine, and greatly improves the editing of cytosine, realizing the transversion editing of a single base from C-G to G-C or editing from C-G to T-A.
  • the base editor containing the adenine deaminase can mediate cytosine editing with high precision, high efficiency and safety, with reduced indels and smaller size, showing high editing activity and low off-target events, so it has extremely high High safety will greatly promote its wide application in the field of gene editing.
  • Figure 1 is a design construct (PDB: 6VPC) based on the crystal structure of Tad-8e.
  • Figure 2 is a comparison of A3, A4 and C6 base editing achieved by 30 ABE8e mutants constructed at the FANCF site 1 site on 293T.
  • Figure 3 is a comparison of C>G base editing at 12 endogenous sites on 293T by ADS-BE1, CGBE1, and APO1-nCas9-XRCC1.
  • Figure 4 is a comparison of average C>G base editing at different positions of ADS-BE1, CGBE1, and APO1-nCas9-XRCC1 (12 endogenous sites are counted).
  • Figure 5 is a comparison of indels generated by ADS-BE1, CGBE1, APO1-nCas9-XRCC1, and ABE8e (counting 12 endogenous sites).
  • Figure 6A, Figure 6B and Figure 6C are the independent off-target comparisons of ADS-BE1, CGBE1, APO1-nCas9-XRCC1, and ABE8e at ABE site 23.
  • Figure 7 is a comparison of C to G efficiency generated by ADS-BE1, CGBE1, APO1-nCas9-XRCC1, and ABE8e at the target site ABE site 23.
  • Figure 8 is the map of nSaCas9-R-loop.
  • Figure 9 is a schematic diagram showing the comparison of base editing from C to T achieved by 15 ADS-BE2 series and eADS-BE2 at the FGF6-sg4 site on 293T.
  • Figure 10 is a schematic diagram of the comparison of base editing from C to T achieved by two ADS-BE2 series and two eADS-BE2 series at Site A and FGF6-sg4 on 293T.
  • Figure 11 shows 8 endogenous sites of 4 ADS-BE2 series, 3 eADS-BE and control BE4max-PU, BE4max-YE1-PU, BE4max-YEE-PU, eA3A-PU, A3G5.13-PU on 293T Schematic diagram of point-to-point C to T base editing.
  • Figure 12 shows the average C of different positions of eADS-BE2.1, eADS-BE2.6, ADS-BE2.5 and BE4max-PU, BE4max-YE1-PU, BE4max-YEE-PU, eA3A-PU, A3G5.13-PU Schematic diagram of comparison to T base editing (8 endogenous sites counted).
  • Figure 13 is a comparison of indels generated by eADS-BE2.1, eADS-BE2.6, ADS-BE2.5 and BE4max-PU, BE4max-YE1-PU, BE4max-YEE-PU, eA3A-PU, A3G5.13-PU (Statistics of 8 endogenous sites) schematic diagram.
  • Figure 14 shows eADS-BE2.1, eADS-BE2.6, ADS-BE2.5 and BE4max-PU, BE4max-YE1-PU, BE4max-YEE-PU, eA3A-PU, A3G5.13-PU in VEGFA site 2 Schematic diagram of site-generated-independent off-target comparison.
  • Figure 15 shows eADS-BE2.1, eADS-BE2.6, ADS-BE2.5 and BE4max-PU, BE4max-YE1-PU, BE4max-YEE-PU, eA3A-PU, A3G5.13-PU in VEGFA site 2 Schematic diagram of the comparison of C to T efficiencies generated by sites.
  • amino acid sequence of bNLS used in the examples is as follows:
  • the amino acid sequence of TadA8e is as follows:
  • the amino acid sequence of the linker Linker1 is as follows:
  • linker Linker18 is: PAP.
  • the amino acid sequence of the linker Linker15 is: PAPAPAP (SEQ ID NO: 9).
  • the amino acid sequence of the linker Linker13 is as follows: PAPAP (SEQ ID NO: 10).
  • amino acid sequence of spCas9n(D10A) is as follows:
  • amino acid sequence of UGI is as follows:
  • amino acid sequence of 2 ⁇ UGI is as follows:
  • the amino acid sequence of the linker Linker0 between 2 ⁇ UGI is: SGGSGGSGGS (SEQ ID NO: 8).
  • the amino acid sequence of P2A is: MTNFSLLKQAGDVEENPGP (SEQ ID NO: 7).
  • Example 1 ABE8e single point mutations ABE8e-N46L and ABE8e-N46P can eliminate editing A to G editing and produce efficient C to G editing
  • ABE8e single-point mutation sequences were synthesized according to Table 1, using ABE8e as the vector (Addgene#138489), and then seamlessly cloned and assembled (the kit was purchased from: Vazyme ClonExpress MultiS One Step Cloning Kit, C113-01 ), the target site is to synthesize two oligos according to Table 2, plus CACC on the positive strand, and AAAC on the reverse strand, and connect to U6-sgRNA-EF1 ⁇ -GFP that has been digested with BbsI; plasmid U6-sgRNA-EF1 ⁇ -
  • the nucleotide sequence of GFP is shown in SEQ ID NO: 136, wherein, the coding sequence of the sgRNA targeting the target sequence is represented by continuous N.
  • each functional block of ABE8e is: bNLS+TadA8e+Linker1+spCas9n(D10A)+bNLS.
  • each functional block of ADS-BE1 is: bNLS+TadA8e(N46L)+Linker1+spCas9n(D10A)+bNLS.
  • amino acid sequence of bNLS in the construct is the 2-19 positions of SEQ ID NO: 1, and the first M is omitted.
  • the amino acid sequence of TadA-8e in the construct is the 2-167 positions of SEQ ID NO: 2, and the first M is omitted.
  • amino acid sequence of spCas9n (D10A) in the construct is 2-1368 of SEQ ID NO: 4, and the first M is omitted.
  • HEK293T HEK293T cells are ATCC CRL-3216 cell line
  • HEK293T cells are ATCC CRL-3216 cell line
  • the genomic DNA of the cells was extracted with Tiangen Cell Genome Extraction Kit (DP304). Then use the Hi-Tom Gene Editing Detection Kit (Novogene) operating procedure to design the corresponding identification primers (as shown in Table 3), that is, add a bridge sequence 5'-ggagtgagtacggtgtgc- at the 5' end of the forward identification primer 3' (SEQ ID NO:53), the reverse identification primer 5' end plus the bridge sequence 5'-gagttggatgctggatgg-3' (SEQ ID NO:54), that is, a round of PCR product is obtained, and then the round of PCR product is used as The template was subjected to two rounds of PCR products, and then mixed together for gel cutting, recovery and purification, and then sent to the company for sequencing (sequencing company: Suzhou Jinweizhi Biotechnology Co., Ltd.).
  • Embodiment 2 ADS-BE1 working characteristic description
  • HEK293T HEK293T cells are ATCC CRL-3216 cell line
  • HEK293T cells are ATCC CRL-3216 cell line
  • the genomic DNA of the cells was extracted with Tiangen Cell Genome Extraction Kit (DP304). Then use the Hi-Tom Gene Editing Detection Kit (Novogene) operating process to design the corresponding identification primers (as shown in Table 3), that is, add the 5' end of the forward identification primer as shown in SEQ ID NO:53
  • the bridging sequence shown, the reverse identification primer 5' end plus the bridging sequence shown in SEQ ID NO: 54 that is, a round of PCR product is obtained, and then the round of PCR product is used as a template to carry out the second round of PCR product, and then mixed in After gel cutting, recovery and purification, they were sent to the company for sequencing (sequencing company: Suzhou Jinweizhi Biotechnology Co., Ltd.).
  • ADS-BE1 also has a preference for motifs (the background sequence of target C).
  • ADS- BE1 is as efficient as traditional CGBE1 and APO-nCas9-XRCC1, but for GC and CC sequence targets, the editing efficiency of ADS-BE1 is higher than that of traditional CGBE1 and APO-nCas9-XRCC1; but for AC sequence targets, ADS-BE1 is extremely Insensitive, the editing efficiency is far lower than traditional CGBE1 and APO-nCas9-XRCC1. From the above results, it can be concluded that the extremely narrow window of C5/C6 and the motif sequence preference endow ADS-BE1 with high-precision editing performance.
  • Embodiment 3 ADS-BE1 safety evaluation
  • HEK293 HEK293T cells are ATCC CRL-3216 cell line
  • HEK293T cells are ATCC CRL-3216 cell line
  • the genomic DNA of the cells was extracted with Tiangen Cell Genome Extraction Kit (DP304). Then use the Hi-Tom Gene Editing Detection Kit (Novogene) operating process to design the corresponding identification primers (as shown in Table 3), that is, add the 5' end of the forward identification primer as shown in SEQ ID NO:53
  • the bridging sequence shown, the reverse identification primer 5' end plus the bridging sequence shown in SEQ ID NO: 54 that is, to obtain a round of PCR products, and then use the first round of PCR products as templates to perform two rounds of PCR products, and then mix them in After gel cutting, recovery and purification, they were sent to the company for sequencing (sequencing company: Suzhou Jinweizhi Biotechnology Co., Ltd.).
  • the indels produced by ADS-BE1 are far lower than the traditional CGBE series, and are on par with the original ABE8e; according to the Cas9-independent off-target evaluation (Figure 6A, Figure 6B, Figure 6C and Table 8), detection at 4 R-loop sites found that ADS-BE1 caused very low C editing (C to G, C to T, C to A), and these small off-target editing events showed that ADS-BE1 was more High safety; at the same time, the target site produces highly efficient C to G editing ( Figure 7 and Table 9).
  • Example 4 Structure-oriented design and linker transformation to obtain high-precision eADS-BE2 series and wide-window ADS-BE2.5
  • ADS-BE2-Linker18, ADS-BE2-Linker15, ADS-BE2-Linker13 and eADS-BE2 were obtained by changing the length of Linker.
  • BE4max (Addgene #112093) as a template
  • a PCR instrument Veriti 96-well gradient PCR instrument, Applied Biosystems
  • seamless clone assembly the kit is Vazyme ClonExpress MultiS One Step Cloning Kit, C113-01
  • ADS-BE2 was used as the vector for seamless cloning and assembly
  • ADS-BE2-Linker18, ADS-BE2-Linker15 , ADS-BE2-Linker13, and eADS-BE2 series are directly obtained by changing the length of the Linker through seamless cloning assembly.
  • the target site is to synthesize two oligos according to Table 11, plus CACC on the positive strand, and AAAC on the reverse strand, and connect to U6-sgRNA-EF1 ⁇ -GFP that has been digested with BbsI
  • Oligo-up is a forward primer
  • Oligo-dn is a reverse primer
  • HEK293T HEK293T cells are ATCC CRL-3216 cell line
  • HEK293T cells are ATCC CRL-3216 cell line
  • the genomic DNA of the cells was extracted with Tiangen Cell Genome Extraction Kit (DP304). Then use the Hi-Tom Gene Editing Detection Kit (Novogene) operating procedure to design the corresponding identification primers (as shown in Table 13), that is, add a bridge sequence 5'-ggagtgagtacggtgtgc- at the 5' end of the forward identification primer 3' (SEQ ID NO:53), the reverse identification primer 5' end plus the bridge sequence 5'-gagttggatgctggatgg-3' (SEQ ID NO:54), that is, a round of PCR product is obtained, and then the round of PCR product is used as The template was subjected to two rounds of PCR products, and then mixed together for gel cutting, recovery and purification, and then sent to the company for sequencing (the sequencing service provider is Suzhou Jinweizhi Biotechnology Co., Ltd.).
  • the sequencing service provider is Suzhou Jinweizhi Biotechnology Co., Ltd.
  • F is the forward identification primer
  • R is the reverse identification primer
  • Rep.1, Rep.2 and Rep.3 are in triplicate.
  • ADS-BE2 was C5, C6 and C7, and the editing efficiency of C7 was 20%.
  • the editing range of ADS-BE2.1 and ADS-BE2.6 was limited to C5 and C6, and the editing efficiency of C7 was 2.2% and 1.4%, respectively, which decreased by 8.1 and 13.3 times, respectively, and reduced bystander cytosine to a certain extent.
  • ADS-BE2.5 greatly expanded the editing range of cytosine, and the editing efficiency of C7 and C8 positions was 3.2 times and 29.4 times that of ADS-BE2, respectively.
  • Embodiment 5 Description of eADS-BE2.1, eADS-BE2.6 and wide window ADS-BE2.5 working characteristics
  • HEK293T HEK293T cells are ATCC CRL-3216 cell line
  • HEK293T cells are ATCC CRL-3216 cell line
  • HEK293T cells were digested and inoculated into 96-well plates at 2 ⁇ 10 5 cells/well.
  • the genomic DNA of the cells was extracted with Tiangen Cell Genome Extraction Kit (DP304). Then use the Hi-Tom Gene Editing Detection Kit (Novogene) operating procedure to design the corresponding identification primers (as shown in Table 12), that is, add a bridge sequence 5'-ggagtgagtacggtgtgc- at the 5' end of the forward identification primer 3' (SEQ ID NO:53), the reverse identification primer 5' end plus the bridge sequence 5'-gagttggatgctggatgg-3' (SEQ ID NO:54), that is, a round of PCR product is obtained, and then the round of PCR product is used as The template was subjected to two rounds of PCR products, and then mixed together for gel cutting, recovery and purification, and then sent to the company for sequencing (the sequencing service provider is Suzhou Jinweizhi Biotechnology Co., Ltd.).
  • the sequencing service provider is Suzhou Jinweizhi Biotechnology Co., Ltd.
  • Table 16-1 4 ADS-BE2 series, 3 eADS-BE and control BE4max-PU, BE4max-YE1-PU, BE4max-YEE-PU, eA3A-PU and A3G5.13-PU in EGFR-sg39 on 293T Source site realizes C to T base editing (unit, %)
  • Table 16-2 4 ADS-BE2 series, 3 eADS-BE and control BE4max-PU, BE4max-YE1-PU, BE4max-YEE-PU, eA3A-PU and A3G5.13-PU at the endogenous position of LAG3sg4 on 293T Points to achieve C to T base editing (unit, %)
  • Table 16-3 4 ADS-BE2 series, 3 eADS-BE and control BE4max-PU, BE4max-YE1-PU, BE4max-YEE-PU, eA3A-PU and A3G5.13-PU in HBG-sg14 on 293T Source site realizes C to T base editing (unit, %)
  • Table 16-4 4 ADS-BE2 series, 3 eADS-BE and control BE4max-PU, BE4max-YE1-PU, BE4max-YEE-PU, eA3A-PU and A3G5.13-PU in FANCF-sg17 on 293T Source site realizes C to T base editing (unit, %)
  • Table 16-5 4 ADS-BE2 series, 3 eADS-BE and control BE4max-PU, BE4max-YE1-PU, BE4max-YEE-PU, eA3A-PU and A3G5.13-PU in VEGFA site 2 on 293T Source site realizes C to T base editing (unit, %)
  • Table 16-6 4 ADS-BE2 series, 3 eADS-BE and control BE4max-PU, BE4max-YE1-PU, BE4max-YEE-PU, eA3A-PU and A3G5.13-PU endogenous at Site A on 293T Site realized C to T base editing (unit, %)
  • Table 16-7 4 ADS-BE2 series, 3 eADS-BE and control BE4max-PU, BE4max-YE1-PU, BE4max-YEE-PU, eA3A-PU and A3G5.13-PU in FGF6-sg4 on 293T Source site realizes C to T base editing (unit, %)
  • Table 16-8 4 ADS-BE2 series, 3 eADS-BE and control BE4max-PU, BE4max-YE1-PU, BE4max-YEE-PU, eA3A-PU and A3G5.13-PU endogenous to PDCD1site 2 on 293T Site realized C to T base editing (unit, %)
  • ADS-BE2-Linker18, ADS-BE2-Linker15 and ADS-BE2-Linker13 also reduced the editing efficiency of C7 to a certain extent, while eADS-BE2 with Linker deleted not only exhibited the lowest C7 editing, but also further reduced C6
  • the editing of making the editing more preferred to the single base C5.
  • the editing sites of eADS-BE2.1 and eADS-BE2.6 on the endogenous test targets Site A and FGF6-sg4 are more concentrated in C5/C6, for the site A target C5 position, eADS-BE2.1 and
  • the editing efficiency of eADS-BE2.6 was 1.7 times and 2 times of the original, respectively.
  • the editing efficiency was 1.2 times and 1.1 times of the original, while the adjacent C7 was reduced by 6.6 times and 3.6 times, respectively.
  • BE4max-PU, BE4max-YE1-PU, BE4max-YEE-PU, eA3A-PU, A3G5 .13-PU is the control
  • the target site is VEGFA site 2
  • design 6 Cas9-independent off-target detection targets (as shown in Table 11), synthesize two oligos, add CACC to the positive strand, and add AAAC to the reverse strand, Connect to the nSaCas9-R-loop vector (as shown in Figure 8) that has been digested with BbsI.
  • HEK293T HEK293T cells are ATCC CRL-3216 cell line
  • HEK293T cells are ATCC CRL-3216 cell line
  • HEK293T cells were digested and inoculated into 96-well plates at 2 ⁇ 10 5 cells/well.
  • the genomic DNA of the cells was extracted with Tiangen Cell Genome Extraction Kit (DP304). Then use the Hi-Tom Gene Editing Detection Kit (Novogene) operating procedure to design the corresponding identification primers (as shown in Table 12), that is, add a bridge sequence 5'-ggagtgagtacggtgtgc- at the 5' end of the forward identification primer 3' (SEQ ID NO:53), the 5' end of the reverse identification primer is added with a bridge sequence 5'-gagttggatgctggatgg-3' (SEQ ID NO:54), that is, a round of PCR product is obtained, and then a round of PCR product is used as The template was subjected to two rounds of PCR products, and then mixed together for gel cutting, recovery and purification, and then sent to the company for sequencing (the sequencing service provider is Suzhou Jinweizhi Biotechnology Co., Ltd.).
  • the sequencing service provider is Suzhou Jinweizhi Biotechnology Co.,
  • the average indels production efficiency of BE4max-PU, BE4max-YE1-PU, BE4max-YEE-PU, eA3A-PU and A3G5.13-PU in the control group were 6.2%, 3.4%, 1.5%, 4.5%, respectively. % and 3.9%, ADS-BE2.5, eADS-BE2.1 and eADS-BE2.6 produce indels with an average efficiency of 4.6%, 1.8% and 1.2%, and the wide window ADS-BE2.5 produces indels significantly lower than the classic The BE4max-PU; eADS-BE2.1 and eADS-BE2.6 maintain a low level of indels while ensuring high precision.
  • Gaudelli NM Komor AC, Rees HA, et al., Programmable base editing of a*t to g*c in genomic DNA without DNA cleavage. Nature, 2017, 551: 464-471.
  • Zhao D Li J, Li S, et al., Glycosylase base editors enable c-to-a and c-to-g base changes. Nat Biotechnol, 2021, 39:35-40.

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Abstract

本发明公开了一种腺嘌呤脱氨酶及其应用。所述腺嘌呤脱氨酶的氨基酸序列为SEQ ID NO:2经突变的序列，所述突变的位点为第28位、第30位、第46位和中的一位或两位。本发明的腺嘌呤脱氨酶完全避免了腺嘌呤编辑，并极大提高了对胞嘧啶的编辑，实现精准的C-G到G-C碱基颠换编辑以及单个碱基C-G到T-A编辑。包含该腺嘌呤脱氨酶的碱基编辑器可高精度、高效、安全地介导胞嘧啶编辑，indels降低、体积更小，展示出较高的编辑活性和较低的脱靶事件，因此具备极高的安全性，将极大促进其在基因编辑领域的广泛应用。

Description

腺嘌呤脱氨酶及其应用
本申请要求申请日为2021/12/29的中国专利申请2021116444500和申请日为2021/12/29的中国专利申请2021116412251的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于基因编辑领域，具体涉及一种腺嘌呤脱氨酶及其应用。
背景技术
人类遗传病发生的本质是由于基因突变，60％左右的遗传疾病由单个碱基突变引起，传统的利用基因组编辑技术介导的同源重组进行纠正这类遗传病非常低效(0.1％-5％) [1,2]。基于CRISPR系统衍生出来的单碱基编辑器是近年来新兴的高效碱基编辑技术，因不产生DNA双链断裂、无须重组模板、高效编辑等优势使其在基础研究和临床疾病治疗展示了巨大的应用前景。
经典的碱基编辑器主要分为胞嘧啶碱基编辑器CBE和腺嘌呤碱基编辑器ABE，前者由活性受损的来源于酿脓链球菌(Streptococcus pyogenes)spCas9n、大鼠来源的胞嘧啶脱氨酶rAPOBEC1和尿嘧啶糖苷酶抑制剂组成，其中Cas9蛋白以NGG作为PAM识别并特异结合DNA，紧接着在脱氨酶以及DNA修复的作用下，最终在NGG(21-23位)上游靶向序列20bp范围实现C·G-T·A的替换，编辑窗口主要位于4-8位 [2]。后者则是将细菌来源的TadA与spCas9融合，在定向进化和蛋白质工程化改造技术的辅助下，经历7轮进化最终获得可作用于单链DNA的腺嘌呤碱基编辑器ABE7.10，活性编辑区域主要位于4-7位，该系统在人类细胞中引起A·T-G·C的平均编辑效率约为53％，远高于利用同源重组介导碱基突变的效率，其产物纯度高达99.9％以及极低的indels(插入和缺失)发生 [3]。在CBE开发过程中，科学家发现尿嘧啶糖苷酶UNG影响C到G产物发生 [4]，因此，科学家在CBE的基础上再融合尿嘧啶糖苷酶UNG [5,6]/UNG变体/碱基切除修复蛋白XRCC1等修复因子 [7]，开发出CGBE系列，在DNA特定区域引起C-G到G-C的碱基颠换。然而，现有的CGBE系列由于多个融合蛋白的掺入，形成较大体积且复杂的构建体系为后期基因治疗带来阻碍，并且融合修复因子的CGBE其碱基编辑范围可覆盖3-9位，也无法兼容精准靶向和高效编辑 [7,8]。与此同时，多个课题组报道ABE除高效介导A到G的编辑外，也会引起低频的胞嘧啶编辑 [5,9-11]。然而致病的点突变事件中，往往需要精确地修正1个碱基，额外的碱基编辑(bystander突变，无法区别目标C和邻近C)反而会带来副作用，缺乏真正意义的精准，虽然YE1、YEE等胞嘧啶碱基编辑器一定程度缩窄靶向范围，但损失了靶向编辑效率，且多靶点不适用；eA3A-BE3、A3G-BE5.13等CBE依靠序列偏好性提高目标C的靶向性，但仍无法去除其他序列背景中C的编辑。
发明内容
本发明所要解决的技术问题是为了克服现有技术缺少同时具有高精度、高安全性、高效编辑的胞嘧啶碱基编辑器的不足，提供一种腺嘌呤脱氨酶及其应用。本发明包含该腺嘌呤脱氨酶的碱基编辑器完全避免了腺嘌呤编辑，并极大提高了对胞嘧啶的编辑；包含该腺嘌呤脱氨酶的碱基编辑器可精准、高效、安全地介导胞嘧啶编辑，展示出较高的编辑活性和较低的脱靶事件，因此具备极高的安全性，将极大促进其在基因编辑领域的广泛应用。
发明人基于上述难点，本发明以腺嘌呤脱氨酶TadA-8e(仅167个氨基酸)作为切入点，通过对TadA-8e晶体结构分析和理性设计，鉴定出诱导ABE转换为CGBE的“分子开关”(图1)，即完全废除ABE本身的腺嘌呤编辑能力，极大增加对底物胞嘧啶的编辑特性，经多轮筛选改造开发出ABE驱动的ADS-BE1(目前体积最小的CGBE)，可在5-7位引起精准的C-G到G-C碱基颠换编辑，相对于传统胞嘧啶脱氨酶衍生而来的CGBE系列，ADS-BE1展示出较高的编辑活性和较低的脱靶事件，因此具备极高的安全性。
进一步地，发明人意外发现在腺嘌呤脱氨酶上的特定位点进行突变可以尝试在改造ABE转换为CGBE(C·G碱基编辑器)的载体(ADS-BE1)基础上融合2×UGI(尿嘧啶糖苷酶抑制剂)，基于结构导向设计引入不同类型的突变，进而开发不依赖于胞嘧啶脱氨酶的高活性碱基编辑器(base editor)，获得高效介导C到T突变的ADS-BE2系列，发现相对传统BE4max，ADS-BE2系列中ADS-BE2.5脱靶和indels降低、体积更小，并保持高效的C到T突变的能力；在高效进行靶向编辑的ADS-BE2.1和ADS-BE2.6上，通过结构导向设计和Linker改造双策略开发的胞嘧啶编辑编辑器eADS-BE2.1和eADS-BE2.6精度更高，可在C5/C6位引起精准的C-G到T-A碱基编辑，展示出较高的编辑活性和较低的脱靶事件，具备极高的安全性。
本发明通过以下技术方案解决上述技术问题：
本发明的第一方面提供了一种腺嘌呤脱氨酶，所述腺嘌呤脱氨酶的氨基酸序列为SEQ ID NO:2经突变的序列，所述突变的位点为第28位、第30位、第46位和中的一位或两位。
在一些实施方案中，所述突变选自以下一种或多种：
(1)第28位氨基酸残基V替换为A或G；
(2)第30位氨基酸残基V替换为F；
(3)第46位氨基酸残基N替换为A、G、L或P。
在一些具体实施方案中，(3)中，第46位氨基酸残基N替换为L或P。
本发明一些实施方案中，所述突变的位点为第46位，以及第27位、第29位或第48位。
在一些具体实施方案中，所述突变选自以下任一：
(4)第46位氨基酸残基N替换为L，并且第27位氨基酸残基E替换为R；
(5)第46位氨基酸残基N替换为L，并且第29位氨基酸残基P替换为A；和
(6)第46位氨基酸残基N替换为L，并且第48位氨基酸残基A替换为M。
本发明的第二方面提供了一种胞嘧啶碱基编辑器，所述胞嘧啶碱基编辑器包括核酸酶和如本发明第一方面所述的腺嘌呤脱氨酶。
在一些实施方案中，所述核酸酶与所述腺嘌呤脱氨酶通过连接子连接，所述连接子可为本领域常规，优选如SEQ ID NO:3、SEQ ID NO:9或SEQ ID NO:10所示。
本发明中，所述核酸酶优选为Cas蛋白及其变体，所述Cas蛋白优选选自酿酒酵母来源的spCas9、金黄色葡萄球菌来源的SaCas9、毛螺菌科细菌来源的LbCas12a和酸胺球菌属细菌来源的enAsCas12a；所述Cas蛋白变体优选选自VQR-spCas9、VRER-spCas9、spRY、spNG、SaCas9-KKH和SaCas9-NG。
在一些具体实施方案中，所述核酸酶为spCas9，其氨基酸序列优选如SEQ ID NO:4所示。
在一些实施方案中，所述胞嘧啶碱基编辑器还包括尿嘧啶DNA糖基化酶抑制剂(UGI)。
在一些具体实施方案中，所述UGI通过连接子与所述核酸酶连接，所述UGI的拷贝数为至少一个，例如一个、两个或三个。
本发明的第三方面提供了一种融合蛋白，所述融合蛋白自N端至C端依次包含信号肽序列、如第一方面所述的腺嘌呤脱氨酶、核酸酶和信号肽序列；所述核酸酶如本发明的第二方面所述。
本发明中，所述融合蛋白还可融合其他功能蛋白，所述其他功能蛋白能够提高该融合蛋白的胞嘧啶编辑效率、调控编辑窗口或精准编辑效率。
在一些实施方案中，所述信号肽序列为核定位信号序列或polyA信号序列，所述核定位信号序列优选如SEQ ID NO:1所示；所述polyA信号序列优选为BGH polyA。
在一些实施方案中，所述融合蛋白还包括UGI，所述UGI如本发明的第二方面所述。
本发明中，所述polyA信号序列可为本领域常规来自真核细胞或原核细胞的多腺苷酸化信号序列。
本发明中，所述BGH polyA为牛生长激素多腺苷酸化信号。
在一些具体实施方案中，所述融合蛋白自N端至C端依次包含信号肽序列、腺嘌呤脱氨酶、核酸酶、信号肽序列、UGI和信号肽序列；所述UGI优选通过氨基酸序列如SEQ ID NO:7所示的连接子与其N端的信号肽序列连接。
当UGI为至少两个拷贝时，UGI之间优选通过如SEQ ID NO:8所示的连接子连接。
本发明中，所述连接子的长度可根据本领域公知进行调整，以利于提高该融合蛋白的胞嘧啶编辑效率、调控编辑窗口或精准编辑效率。
本发明的第四方面提供了一种分离的核酸，所述核酸编码如本发明的第一方面所述的腺嘌呤脱氨酶、如本发明的第二方面所述的胞嘧啶碱基编辑器或者如本发明的第三方面所述的融合蛋白。
本发明的第五方面提供了一种基因表达盒，所述基因表达盒包括启动子元件和目的基因元件；其中，所述目的基因元件为如本发明的第四方面所述的核酸。
在一些实施方案中，所述启动子元件可为本领域常规，优选选自CMV、CAG、PGK、EF1α、Ctsk和Lp1。
本发明的第六方面提供了一种胞嘧啶碱基编辑系统，其包括：sgRNA和如本发明的第二方面所述的胞嘧啶碱基编辑器。
在一些具体实施方案中，所述sgRNA的靶序列如SEQ ID NO:11～24或SEQ ID NO:83～93任一所示。
本发明的第七方面提供了一种药物组合物，所述药物组合物包括如本发明的第一方面所述的腺嘌呤脱氨酶、如本发明的第二方面所述的胞嘧啶碱基编辑器、如本发明的第三方面所述的融合蛋白或者如本发明的第六方面所述的胞嘧啶碱基编辑系统，以及药学上可接受的载体。
本发明的第八方面提供了一种碱基编辑方法，所述碱基编辑方法包括：
在靶细胞中表达如本发明的第一方面所述的腺嘌呤脱氨酶、如本发明的第二方面所述的胞嘧啶碱基编辑器、如本发明的第三方面所述的融合蛋白或者如本发明的第六方面所述的胞嘧啶碱基编辑系统，使所述靶细胞发生碱基编辑；所述碱基编辑包括将C编辑为T、将C编辑为G。
在一些实施方案中，所述靶细胞的来源为分离的细胞系；所述细胞系优选为哺乳动物细胞系。
在一些具体实施方案中，所述分离的细胞系为293T细胞、HELA细胞、U2OS细胞、NIH3T3细胞或N2A细胞。
本发明中，所述碱基编辑方法可为非治疗目的。
本发明的第九方面提供了一种构建动物模型、农作物育种或制造碱基编辑设备的方法，所述方法包括使用如本发明的第一方面所述的腺嘌呤脱氨酶、如本发明的第二方面所述的胞嘧啶碱基编辑器、如本发明的第三方面所述的融合蛋白、如本发明的第四方面所述的核酸、如本发明的第五方面所述的基因表达盒或者如本发明的第六方面所述的胞嘧啶碱基编辑系统。
本发明的第十方面提供了一种用于基因疗法的药物组合物，所述药物组合物包括如本发明的第一方面所述的腺嘌呤脱氨酶、如本发明的第二方面所述的胞嘧啶碱基编辑器、如本发明的第三方面所述的融合蛋白、如本发明的第四方面所述的核酸、如本发明的第五方面所述的基因表达盒或者如本发明的第六方面所述的胞嘧啶碱基编辑系统；所述基因疗法为治疗基因突变导致的疾病的基因疗法。
本发明的第十一方面提供了一种基因治疗的方法，所述方法为使用如本发明的第一方面所述的腺嘌呤脱氨酶、如本发明的第二方面所述的胞嘧啶碱基编辑器、如本发明的第三方面所述的融合蛋白、如本发明的第四方面所述的核酸、如本发明的第五方面所述的基因 表达盒或者如本发明的第六方面所述的胞嘧啶碱基编辑系统对基因突变导致的疾病进行治疗。
本发明的第十二方面提供了一种重组表达载体，所述重组表达载体包含如本发明的第四方面所述的核酸或如本发明的第五方面所述的基因表达盒。
本发明的第十三方面提供了一种转化体，所述转化体包含如本发明的第四方面所述的的核酸、如本发明的第五方面所述的基因表达盒或如本发明的第十二方面所述的重组表达载体。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明的腺嘌呤脱氨酶完全避免了腺嘌呤编辑，并极大提高了对胞嘧啶的编辑，实现单个碱基C-G到G-C的颠换编辑或C-G到T-A编辑。包含该腺嘌呤脱氨酶的碱基编辑器可高精度、高效、安全地介导胞嘧啶编辑，indels降低、体积更小，展示出较高的编辑活性和较低的脱靶事件，因此具备极高的安全性，将极大促进其在基因编辑领域的广泛应用。
附图说明
图1为根据Tad-8e晶体结构设计构建体(PDB:6VPC)。
图2为30个ABE8e突变体构建在293T上FANCF site 1位点实现的A3、A4和C6碱基编辑对比。
图3为ADS-BE1、CGBE1、APO1-nCas9-XRCC1在293T上12个内源位点展现的C>G碱基编辑对比。
图4为ADS-BE1、CGBE1、APO1-nCas9-XRCC1不同位置平均C>G碱基编辑对比(统计12个内源位点)。
图5为ADS-BE1、CGBE1、APO1-nCas9-XRCC1、ABE8e产生的indels对比(统计12个内源位点)。
图6A、图6B和图6C为ADS-BE1、CGBE1、APO1-nCas9-XRCC1、ABE8e在ABE site 23位点产生的非依赖性脱靶对比。
图7为ADS-BE1、CGBE1、APO1-nCas9-XRCC1、ABE8e在目标位点ABE site 23位点产生的C到G效率对比。
图8为nSaCas9-R-loop图谱。
图9为15个ADS-BE2系列和eADS-BE2在293T上FGF6-sg4位点实现的C到T碱基编辑对比示意图。
图10为2个ADS-BE2系列和2个eADS-BE2系列在293T上Site A和FGF6-sg4位点实现的C到T碱基编辑对比示意图。
图11为4个ADS-BE2系列、3个eADS-BE以及对照BE4max-PU、BE4max-YE1-PU，BE4max-YEE-PU、eA3A-PU、A3G5.13-PU在293T上8个内源位点实现C到T碱基编辑示意图。
图12为eADS-BE2.1、eADS-BE2.6、ADS-BE2.5和BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU、A3G5.13-PU不同位置平均C到T碱基编辑对比(统计8个内源位点)示意图。
图13为eADS-BE2.1、eADS-BE2.6、ADS-BE2.5和BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU、A3G5.13-PU产生的indels对比(统计8个内源位点)示意图。
图14为eADS-BE2.1、eADS-BE2.6、ADS-BE2.5和BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU、A3G5.13-PU在VEGFA site 2位点产生的非依赖性脱靶对比示意图。
图15为eADS-BE2.1、eADS-BE2.6、ADS-BE2.5和BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU、A3G5.13-PU在VEGFA site 2位点产生的C到T效率对比示意图。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例中使用的bNLS的氨基酸序列如下：
Figure PCTCN2022143408-appb-000001
TadA8e的氨基酸序列如下：
Figure PCTCN2022143408-appb-000002
连接子Linker1的氨基酸序列如下：
Figure PCTCN2022143408-appb-000003
连接子Linker18的氨基酸序列为：PAP。
连接子Linker15的氨基酸序列为：PAPAPAP(SEQ ID NO:9)。
连接子Linker13的氨基酸序列为下：PAPAP(SEQ ID NO:10)。
spCas9n(D10A)的氨基酸序列如下：
Figure PCTCN2022143408-appb-000004
Figure PCTCN2022143408-appb-000005
UGI的氨基酸序列如下：
Figure PCTCN2022143408-appb-000006
2×UGI的氨基酸序列如下：
Figure PCTCN2022143408-appb-000007
2×UGI之间的连接子Linker0的氨基酸序列为：SGGSGGSGGS(SEQ ID NO:8)。
P2A的氨基酸序列为：MTNFSLLKQAGDVEENPGP(SEQ ID NO:7)。
实施例1：ABE8e单点突变ABE8e-N46L和ABE8e-N46P可消除编辑A到G的编辑和产生高效C到G编辑
1.1质粒设计及构建
1.1.1根据ABE8e与底物DNA结合的晶体结构，设计了30个ABE8e的单点突变，同时设计了1个来自于人的内源性靶点FANCF site1(表2)；
1.1.2 30个ABE8e单点突变序列按表1中进行合成，以ABE8e为载体(Addgene#138489)，之后进行无缝克隆组装(试剂盒购于：Vazyme ClonExpress MultiS One Step Cloning Kit，C113-01)，靶点即按表2进行合成两条oligo，正链加CACC，反链加上AAAC，连接至已经用BbsI酶切好的U6-sgRNA-EF1α-GFP上；质粒U6-sgRNA-EF1α-GFP的核苷酸序列如SEQ ID NO:136所示，其中，靶向靶序列的sgRNA的编码序列用连续的N表示。
1.1.3将1.1.1与1.1.2中构建的质粒经sanger测序，确保完全正确。
表1 TadA-8e单点突变序列
单点突变体 密码子 单点突变体 密码子
V28A gca V82G ggc
V28C tgc F84A gct
V28G gga F84I atc
V28P cct F84L ctg
V28S agc F84M atg
V28T acc R107N aac
V30A gca N108A gca
V30F ttc N108G gga
V30M atg N108R aga
N46A gca R111A gcc
N46G gga L145A gcc
N46L ctg L145F ttc
N46P cct L145G gga
N46R aga F148A gca
E59A gca Y149A gca
ABE8e的各个功能嵌段排列次序为：bNLS+TadA8e+Linker1+spCas9n(D10A)+bNLS。
ADS-BE1的各个功能嵌段排列次序为：bNLS+TadA8e(N46L)+Linker1+spCas9n(D10A)+bNLS。
构建体中bNLS的氨基酸序列为SEQ ID NO:1的2-19位，省略第一位的M。
构建体中TadA-8e的氨基酸序列为SEQ ID NO:2的2-167位，省略第一位的M。
构建体中spCas9n(D10A)的氨基酸序列为SEQ ID NO:4的2-1368位，省略第一位的M。
表2实施例中使用的靶点及其序列
Figure PCTCN2022143408-appb-000008
Figure PCTCN2022143408-appb-000009
1.2细胞转染
第1天用HEK293T(HEK293T细胞为ATCC CRL-3216细胞系)细胞铺种24孔板：
(1)消化HEK293T细胞，按照2×10 5个/孔接种24孔板；
注：细胞复苏后，一般需传代2次后方可用于转染实验；
第2天转染：
(2)观察各孔细胞状态；
注：要求转染前细胞密度应为70％-90％，且状态正常；
(3)质粒转染；
使用上述步骤1中新构建的各个ABE8e突变体质粒：U6-sgRNA-EF1α-GFP＝750ng:250ng的质粒用量，转染试剂为PEI(每1μg质粒加3μL PEI)，共转染HEK293T宿主，以ABE8e作为对照；设置n＝3孔/组。
1.3基因组提取及扩增子文库的准备
转染后72h，用天根细胞基因组提取试剂盒(DP304)提取细胞基因组DNA。之后用Hi-Tom Gene Editing Detection Kit(诺禾致源)操作流程，设计相对应的鉴定引物(如表3所示)，即在正向鉴定引物5’端加上搭桥序列5’-ggagtgagtacggtgtgc-3’(SEQ ID NO:53)，反向鉴定引物5’端加上搭桥序列5’-gagttggatgctggatgg-3’(SEQ ID NO:54)，即得到一轮PCR产物，之后利用一轮PCR产物作为模板，进行二轮PCR产物，然后混在一起进行切胶回收纯化后送公司进行测序(测序公司：苏州金唯智生物科技有限公司)。
表3所用靶点的鉴定引物
Figure PCTCN2022143408-appb-000010
Figure PCTCN2022143408-appb-000011
Figure PCTCN2022143408-appb-000012
1.4深度测序结果分析与统计
利用BE-analyzer网站(http://www.rgenome.net/be-analyzer/#！)分析深度测序结果，即统计A到G、C到T、C到G、C到A的比率，并用graphpad prism 9.1.0进行统计作图，如表4和图2所示。
表4靶点FANCF site 1的编辑效率结果(单位：％)
Figure PCTCN2022143408-appb-000013
Figure PCTCN2022143408-appb-000014
Figure PCTCN2022143408-appb-000015
根据二代测序结果发现，相对于ABE8e，ABE8e-N46A、ABE8e-N46G、ABE8e-N46L、ABE8e-N46P和ABE8e-N46R均完全废除A到G的编辑(图2和表4)，其中ABE8e-N46L和ABE8e-N46P同时展现出高效的胞嘧啶C6编辑(C到G+C+A)，并且产物更多以C到G为主，产生C到G编辑分别为43.93％和43.87％；而3’端的C7和C8几乎不产生编辑(表4)，展现出较高的精准性，故选择ABE8e-N46L命名为ADS-BE1(ABE Derived Single Base Editor)，作为一种不依赖于胞嘧啶脱氨酶、尿嘧啶糖苷酶、DNA修复因子等多种融合物的新型迷你CGBE。
实施例2：ADS-BE1工作特性描述
2.1质粒设计及构建
2.1.1设计12个来自于人的内源性靶点SSH2sg10、TIM3-sg4、ABE site 8、EGFR-sg4、HBG-sg14、PPP1R12C site 5、FANCF-sg15、ABE site 23、PPP1R12C site 7、FANCF-sg17、Hp53-sg1和Ox40-sg1进行工作特性描述(表2)，构建方法同1.1.2；
2.1.2将2.1.1中构建的质粒经sanger测序，确保完全正确。
2.2细胞转染
第1天用HEK293T(HEK293T细胞为ATCC CRL-3216细胞系)细胞铺种24孔板：
(1)消化HEK293T细胞，按照2×10 5个/孔接种24孔板；
注：细胞复苏后，一般需传代2次后方可用于转染实验；
第2天转染：
(2)观察各孔细胞状态；
注：要求转染前细胞密度应为70％-90％，且状态正常；
(3)质粒转染量如下：
2.1中新构建的质粒：U6-sgRNA-EF1α-GFP＝750ng:250ng的质粒用量，转染试剂为 PEI(每1μg质粒加3μL PEI)，共转染HEK293T宿主，以ABE8e、CGBE1(Addgene#140252)和APO1–nCas9–XRCC1(Addgene#165444)作为对照；设置n＝3孔/组。
2.3基因组提取及扩增子文库的准备
转染后72h，用天根细胞基因组提取试剂盒(DP304)提取细胞基因组DNA。之后用Hi-Tom Gene Editing Detection Kit(诺禾致源)操作流程，设计相对应的鉴定引物(如表3所示)，即在正向鉴定引物5’端加上如SEQ ID NO:53所示的搭桥序列，反向鉴定引物5’端加上如SEQ ID NO:54所示的搭桥序列，即得到一轮PCR产物，之后利用一轮PCR产物作为模板，进行二轮PCR产物，然后混在一起进行切胶回收纯化后送公司进行测序(测序公司：苏州金唯智生物科技有限公司)。
2.4深度测序结果分析与统计
利用BE-analyzer网站进分析深度测序结果，即统计A到G、C到T、C到G、C到A的比率，并用graphpad prism 9.1.0进行统计作图。
表5 ADS-BE1、CGBE1、APO1-nCas9-XRCC1、ABE8e在293T上12个内源位点展现的C>G碱基编辑效率对比(单位：％)
表5-1
Figure PCTCN2022143408-appb-000016
表5-2
Figure PCTCN2022143408-appb-000017
Figure PCTCN2022143408-appb-000018
表5-3
Figure PCTCN2022143408-appb-000019
表5-4
Figure PCTCN2022143408-appb-000020
表5-5
Figure PCTCN2022143408-appb-000021
Figure PCTCN2022143408-appb-000022
表5-6
Figure PCTCN2022143408-appb-000023
表5-7
Figure PCTCN2022143408-appb-000024
表5-8
Figure PCTCN2022143408-appb-000025
表5-9
Figure PCTCN2022143408-appb-000026
表5-10
Figure PCTCN2022143408-appb-000027
Figure PCTCN2022143408-appb-000028
表5-11
Figure PCTCN2022143408-appb-000029
表5-12
Figure PCTCN2022143408-appb-000030
表6 ADS-BE1、CGBE1、APO1-nCas9-XRCC1、ABE8e不同位置平均C>G碱基编辑对比(统计12个内源位点)(单位：％)
Figure PCTCN2022143408-appb-000031
结果表明(图3和表5)，以CGBE1、APO-nCas9-XRCC1(两个传统CGBE)和ABE8e为对照，对于FANCF-sg15、PPP1R12C site7、Hp53-sg1和Ox40-sg1靶点，ADS-BE1展现精准编辑特性，更偏好编辑C5/C6，根据12个靶点不同位置的平均C到G效率统计(图4和表6)，对于C5位置，ADS-BE1、CGBE1和APO-nCas9-XRCC1三种CGBE平均效率分别为27.87％、17.96％和7.72％；对于C6位置，三者平均效率分别为32.23％、41.36％和26.26％，而对于旁观者C7、C8、C4，CGBE1产生C到G平均效率为11.04％、11.39％和9.55％；APO-nCas9-XRCC1产生C到G平均效率为6.29％、6.1％和4.45％，ADS-BE1产生C到G平均效率仅为2.15％、0.7％和0.5％；说明ADS-BE1对C5/C6之外的位置不会产生有效编辑，此外，ADS-BE1对于motif(目标C的背景序列)也有偏好，对于C5和C6位置的TC序列靶点，ADS-BE1与传统CGBE1和APO-nCas9-XRCC1效率持平，但是对于GC和CC序列靶点，ADS-BE1编辑效率要高于传统CGBE1和APO-nCas9-XRCC1；但是对AC序列靶点，ADS-BE1极其不敏感，编辑效率远远低于传统CGBE1和APO-nCas9-XRCC1。由上述结果得出：C5/C6极窄窗口和motif序列偏好赋予ADS-BE1高精度编辑性能。
实施例3：ADS-BE1安全性评价
3.1质粒设计及构建
3.1.1为了进一步描述ADS-BE1的安全性，再次以ABE8e为对照，目标位点为ABE site 23，设计4个Cas9非依赖性脱靶检测靶点(表2)，合成两条oligo，正链加CACC，反链加上AAAC，连接至已经用BbsI酶切好的nSaCas9-R-loop载体上(图8)；
3.1.2将1.1中构建的质粒经sanger测序，确保完全正确；
3.2.细胞转染
第1天用HEK293(HEK293T细胞为ATCC CRL-3216细胞系)细胞铺种24孔板：
(1)消化HEK293T细胞，按照2×10 5个/孔接种24孔板；
注：细胞复苏后，一般需传代2次后方可用于转染实验；
第2天转染：
(2)观察各孔细胞状态；
注：要求转染前细胞密度应为70％-90％，且状态正常；
(3)质粒转染量如下：
ADS-BE1/CGBE1/APO-nCas9-XRCC1/ABE8e：3.1中新构建的质粒：ABE site 23靶点质粒＝400ng:300ng:300ng的质粒用量，转染试剂为PEI(每1μg质粒加3μL PEI)，共转染HEK293T宿主，设置n＝3孔/组。
3.3基因组提取及扩增子文库的准备
转染后72h，用天根细胞基因组提取试剂盒(DP304)提取细胞基因组DNA。之后用Hi-Tom Gene Editing Detection Kit(诺禾致源)操作流程，设计相对应的鉴定引物(如表3所示)，即在正向鉴定引物5’端加上如SEQ ID NO:53所示的搭桥序列，反向鉴定引物5’端加上如SEQ ID NO:54所示的搭桥序列，即得到一轮PCR产物，之后利用一轮PCR产物作为模板，进行二轮PCR产物，后混在一起进行切胶回收纯化后进行送公司进行测序(测序公司：苏州金唯智生物科技有限公司)。
3.4深度测序结果分析与统计
利用BE-analyzer网站分析深度测序结果，即统计C到T、C到G、C到A的比率和indels，并用graphpad prism 9.1.0进行统计作图。
表7 ADS-BE1、CGBE1、APO1-nCas9-XRCC1、ABE8e产生的indels对比(统计12个内源位点)(单位：％)
Figure PCTCN2022143408-appb-000032
表8 ADS-BE1、CGBE1、APO1-nCas9-XRCC1、ABE8e产生的非依赖性脱靶对比(单位：％)
Figure PCTCN2022143408-appb-000033
Figure PCTCN2022143408-appb-000034
表9 ADS-BE1、CGBE1、APO1-nCas9-XRCC1、ABE8e目标位点ABE site23产生的C-G效率对比(单位：％)
Figure PCTCN2022143408-appb-000035
根据indels(插入和缺失)数据统计结果(图5和表7)表明：ABE8e产生indels平均效率为5.25％，ADS-BE1产生indels平均效率为8.1％，CGBE1产生indels平均效率为15.88％，APO-nCas9-XRCC1产生indels平均效率为26.93％，因此，ADS-BE1产生的indels远远低于传统CGBE系列，与原始的ABE8e持平；根据Cas9非依赖性脱靶评价(图6A、图6B、图6C和表8)，在4个R-loop位点检测发现ADS-BE1引起极低的C的编辑(C到G，C到T，C到A)，这些微量的脱靶编辑事件展现出ADS-BE1较高的安全性；同时，目标位点产生高效的C到G编辑(图7和表9)。
实施例4结构导向设计和linker改造获得高精度的eADS-BE2系列以及宽窗口的ADS-BE2.5
根据TadA-8e结合底物DNA的晶体结构，推测8个氨基酸可能影响TadA-8e与底物DNA的非特异性结合，进而改变ADS-BE2的编辑窗口，通过改变氨基酸的疏水性或者极性得到11种构建，并改变Linker长度获得ADS-BE2-Linker18、ADS-BE2-Linker15、ADS-BE2-Linker13和eADS-BE2构建。
4.1质粒设计及构建
4.1.1根据ABE8e与底物DNA结合的晶体结构，设计了11个ADS-BE2系列突变体(如表10所示)，同时设计了1个来自于人的内源性靶点FANCF site1(如表11所示)。
4.1.2以BE4max(Addgene#112093)为模板，利用PCR仪(Veriti 96孔型梯度PCR仪，Applied Biosystems)和无缝克隆组装(试剂盒为Vazyme ClonExpress MultiS One Step Cloning Kit，C113-01)将P2A-2×UGI引入到ADS-BE1载体中获得ADS-BE2，合成11个ADS-BE2系列突变序列，以ADS-BE2为载体进行无缝克隆组装，ADS-BE2-Linker18、ADS-BE2-Linker15、ADS-BE2-Linker13、eADS-BE2系列直接通过无缝克隆组装改变Linker长度获得。靶点即按表11进行合成两条oligo，正链加CACC，反链加上AAAC，连接至已经用BbsI酶切好的U6-sgRNA-EF1α-GFP上。
4.1.3将4.1.1与4.1.2中构建的质粒经sanger测序，确保完全正确，得到ADS-BE2系列。
表10 ADS-BE2系列组合突变序列
组合突变体 密码子
N46L&A48M ctg&atg
N46L&I49F ctg&ttc
N46L&L51A ctg&gcc
N46L&L51W ctg&tgg
N46L&E27R ctg&aga
N46L&P29A ctg&gca
N46L&P29S ctg&agc
N46L&G31S ctg&agc
N46L&H57A ctg&gca
N46L&H57S ctg&agc
N46L&P86S ctg&agc
表11所用靶点及序列
Figure PCTCN2022143408-appb-000036
Figure PCTCN2022143408-appb-000037
表中，Oligo-up为正向引物，Oligo-dn为反向引物。
表12设计的碱基编辑器
名称 功能嵌段排列次序
ABE8e bNLS+TadA8e+Linker1+spCas9n(D10A)+bNLS
ADS-BE2 bNLS+TadA8e(N46L)+Linker1+spCas9n(D10A)+bNLS+P2A+2×UGI+bNLS
ADS-BE2.1 bNLS+TadA8e(N46L&A48M)+Linker1+spCas9n(D10A)+bNLS+P2A+2×UGI+bNLS
ADS-BE2.2 bNLS+TadA8e(N46L&I49F)+Linker1+spCas9n(D10A)+bNLS+P2A+2×UGI+bNLS
ADS-BE2.3 bNLS+TadA8e(N46L&L51A)+Linker1+spCas9n(D10A)+bNLS+P2A+2×UGI+bNLS
ADS-BE2.4 bNLS+TadA8e(N46L&L51W)+Linker1+spCas9n(D10A)+bNLS+P2A+2×UGI+bNLS
ADS-BE2.5 bNLS+TadA8e(N46L&E27R)+Linker1+spCas9n(D10A)+bNLS+P2A+2×UGI+bNLS
ADS-BE2.6 bNLS+TadA8e(N46L&P29A)+Linker1+spCas9n(D10A)+bNLS+P2A+2×UGI+bNLS
ADS-BE2.7 bNLS+TadA8e(N46L&P29S)+Linker1+spCas9n(D10A)+bNLS+P2A+2×UGI+bNLS
ADS-BE2.8 bNLS+TadA8e(N46L&G31S)+Linker1+spCas9n(D10A)+bNLS+P2A+2×UGI+bNLS
ADS-BE2.9 bNLS+TadA8e(N46L&H57A)+Linker1+spCas9n(D10A)+bNLS+P2A+2×UGI+bNLS
ADS-BE2.10 bNLS+TadA8e(N46L&H57S)+Linker1+spCas9n(D10A)+bNLS+P2A+2×UGI+bNLS
ADS-BE2.11 bNLS+TadA8e(N46L&P86S)+Linker1+spCas9n(D10A)+bNLS+P2A+2×UGI+bNLS
ADS-BE2-L18 bNLS+TadA8e(N46L)+Linker18+spCas9n(D10A)+bNLS+P2A+2×UGI+bNLS
ADS-BE2-L15 bNLS+TadA8e(N46L)+Linker15+spCas9n(D10A)+bNLS+P2A+2×UGI+bNLS
ADS-BE2-L13 bNLS+TadA8e(N46L)+Linker13+spCas9n(D10A)+bNLS+P2A+2×UGI+bNLS
eADS-BE2 bNLS+TadA8e(N46L)+spCas9n(D10A)+bNLS+P2A+2×UGI+bNLS
eADS-BE2.1 bNLS+TadA8e(N46L&A48M)+spCas9n(D10A)+bNLS+P2A+2×UGI+bNLS
eADS-BE2.6 bNLS+TadA8e(N46L&P29A)+spCas9n(D10A)+bNLS+P2A+2×UGI+bNLS
4.2细胞转染
第1天用HEK293T(HEK293T细胞为ATCC CRL-3216细胞系)细胞铺种24孔板：
(1)消化HEK293T细胞，按照2×10 5cell/孔接种24孔板。
注：细胞复苏后，一般需传代2次后方可用于转染实验。
第2天转染：
(2)观察各孔细胞状态。
注：要求转染前细胞密度应为70％-90％，且状态正常。
(3)质粒转染如下：
使用上述步骤1中新构建的各个质粒：U6-sgRNA-EF1α-GFP＝750ng:250ng的质粒用量，转染试剂为PEI(每1μg质粒加3μL PEI)，共转染HEK293T宿主，以ADS-BE2或者ADS-BE2.1/2.6作为对照；设置n＝3孔/组。
4.3基因组提取及扩增子文库的准备
转染后72h，用天根细胞基因组提取试剂盒(DP304)提取细胞基因组DNA。之后用Hi-Tom Gene Editing Detection Kit(诺禾致源)操作流程，设计相对应的鉴定引物(如表13所示)，即在正向鉴定引物5’端加上搭桥序列5’-ggagtgagtacggtgtgc-3’(SEQ ID NO:53)，反向鉴定引物5’端加上搭桥序列5’-gagttggatgctggatgg-3’(SEQ ID NO:54)，即得到一轮PCR产物，之后利用一轮PCR产物作为模板，进行二轮PCR产物，后混在一起进行切胶回收纯化后进行送公司进行测序(测序的服务提供商为苏州金唯智生物科技有限公司)。
表13所用靶点的鉴定引物
Figure PCTCN2022143408-appb-000038
Figure PCTCN2022143408-appb-000039
表中，F为正向鉴定引物，R为反向鉴定引物。
4.4深度测序结果分析与统计
利用BE-analyzer网站(http://www.rgenome.net/be-analyzer/#！)分析深度测序结果，即统计C到T的比率，并用graphpad prism 9.1.0进行统计作图，如表14、表15和图9和图10所示。
表14 15个ADS-BE2系列和eADS-BE2在293T上FGF6-sg4位点实现的C到T碱基编辑对比(单位，％)
Figure PCTCN2022143408-appb-000040
Figure PCTCN2022143408-appb-000041
表中，Rep.1、Rep.2和Rep.3为一式三份。
表15-1 2个ADS-BE2系列和2个eADS-BE2系列在293T上Site A位点实现的C到T碱基编辑对比(单位，％)
Figure PCTCN2022143408-appb-000042
表15-2 2个ADS-BE2系列和2个eADS-BE2系列在293T上FGF6-sg4位点实现的C到T碱基编辑对比(单位，％)
Figure PCTCN2022143408-appb-000043
表15中，Rep.1、Rep.2和Rep.3为一式三份。
根据二代测序结果发现，ADS-BE2编辑范围为C5、C6和C7，C7编辑效率为20％。而ADS-BE2.1和ADS-BE2.6编辑范围仅限于C5和C6，C7编辑效率分别为2.2％和1.4％， 分别降低8.1倍和13.3倍，一定程度降低旁观者胞嘧啶。意外的是，ADS-BE2.5却极大的扩展了胞嘧啶的编辑范围，C7和C8位置的编辑效率分别是ADS-BE2的3.2倍和29.4倍。
实施例5 eADS-BE2.1、eADS-BE2.6和宽窗口ADS-BE2.5工作特性描述
5.1质粒设计及构建
5.1.1设计8个来自于人的内源性靶点EGFR-sg39、LAG3-sg4、PDCD1site 2、HBG-sg14、VEGFA site 2、FANCF-sg17、Site A、FGF6-sg4进行工作特性描述，构建方法同实施例4的4.1.2，对照组BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU、A3G5.13-PU分别以BE4max(Addgene#112093)、YE1-BE4max(#138155)、YE1-BE4max(#138155)、eA3A(Addgene#131315)、A3G5.13(#138155)为模板，将后者(SG 2) 3S-2×UGI更换为P2A-2×UGI作为公平对照，构建方法同4.1.2。
5.1.2将5.1.1中构建的质粒经sanger测序，确保完全正确。
5.2细胞转染
第1天用HEK293T(HEK293T细胞为ATCC CRL-3216细胞系)细胞铺种24孔板：
(1)消化HEK293T细胞，按照2×10 5cell/孔接种96孔板。
注：细胞复苏后，一般需传代2次后方可用于转染实验。
第2天转染：
(2)观察各孔细胞状态。
注：要求转染前细胞密度应为70％-90％，且状态正常。
(3)质粒转染量如下：
5.1中新构建的质粒：U6-sgRNA-EF1α-GFP＝750ng:250ng的质粒用量，转染试剂为PEI(每1μg质粒加3μL PEI)，共转染HEK293T宿主，以BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU和A3G5.13-PU作为对照；设置n＝3孔/组。
5.3基因组提取及扩增子文库的准备
转染后72h，用天根细胞基因组提取试剂盒(DP304)提取细胞基因组DNA。之后用Hi-Tom Gene Editing Detection Kit(诺禾致源)操作流程，设计相对应的鉴定引物(如表12所示)，即在正向鉴定引物5’端加上搭桥序列5’-ggagtgagtacggtgtgc-3’(SEQ ID NO:53)，反向鉴定引物5’端加上搭桥序列5’-gagttggatgctggatgg-3’(SEQ ID NO:54)，即得到一轮PCR产物，之后利用一轮PCR产物作为模板，进行二轮PCR产物，后混在一起进行切胶回收纯化后进行送公司进行测序(测序的服务提供商为苏州金唯智生物科技有限公司)。
5.4深度测序结果分析与统计
利用BE-analyzer网站进分析深度测序结果，即统计C到T、Indels的比率，并用graphpad  prism 9.1.0进行统计作图，如表16～表18和图11-图13所示。
表16-1 4个ADS-BE2系列、3个eADS-BE以及对照BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU和A3G5.13-PU在293T上EGFR-sg39内源位点实现C到T碱基编辑(单位，％)
Figure PCTCN2022143408-appb-000044
表16-2 4个ADS-BE2系列、3个eADS-BE以及对照BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU和A3G5.13-PU在293T上LAG3sg4内源位点实现C到T碱基编辑(单位，％)
Figure PCTCN2022143408-appb-000045
Figure PCTCN2022143408-appb-000046
表16-3 4个ADS-BE2系列、3个eADS-BE以及对照BE4max-PU、BE4max-YE1-PU、 BE4max-YEE-PU、eA3A-PU和A3G5.13-PU在293T上HBG-sg14内源位点实现C到T碱基编辑(单位，％)
Figure PCTCN2022143408-appb-000047
Figure PCTCN2022143408-appb-000048
表16-4 4个ADS-BE2系列、3个eADS-BE以及对照BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU和A3G5.13-PU在293T上FANCF-sg17内源位点实现C到T碱基编辑(单位，％)
Figure PCTCN2022143408-appb-000049
表16-5 4个ADS-BE2系列、3个eADS-BE以及对照BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU和A3G5.13-PU在293T上VEGFA site 2内源位点实现C到T碱基编辑(单位，％)
Figure PCTCN2022143408-appb-000050
Figure PCTCN2022143408-appb-000051
Figure PCTCN2022143408-appb-000052
表16-6 4个ADS-BE2系列、3个eADS-BE以及对照BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU和A3G5.13-PU在293T上Site A内源位点实现C到T碱基编辑(单位，％)
Figure PCTCN2022143408-appb-000053
Figure PCTCN2022143408-appb-000054
表16-7 4个ADS-BE2系列、3个eADS-BE以及对照BE4max-PU、BE4max-YE1-PU、 BE4max-YEE-PU、eA3A-PU和A3G5.13-PU在293T上FGF6-sg4内源位点实现C到T碱基编辑(单位，％)
Figure PCTCN2022143408-appb-000055
Figure PCTCN2022143408-appb-000056
表16-8 4个ADS-BE2系列、3个eADS-BE以及对照BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU和A3G5.13-PU在293T上PDCD1site 2内源位点实现C到T碱基编辑(单位，％)
Figure PCTCN2022143408-appb-000057
表16中，Rep.1、Rep.2和Rep.3为一式三份。
表17 eADS-BE2.1、eADS-BE2.6、ADS-BE2.5和BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU、A3G5.13-PU不同位置平均C到T碱基编辑对比(统计8个内源位点)(单位，％)
Figure PCTCN2022143408-appb-000058
表18 eADS-BE2.1、eADS-BE2.6、ADS-BE2.5和BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU、A3G5.13-PU产生的indels对比(统计8个内源位点)(单位，％)
Figure PCTCN2022143408-appb-000059
由测序结果可知，ADS-BE2-Linker18、ADS-BE2-Linker15和ADS-BE2-Linker13也是一定程度降低了C7的编辑效率，而删除Linker的eADS-BE2不仅展现最低的C7编辑，还进一步降低C6的编辑，使得编辑更偏好于单个碱基C5。eADS-BE2.1和eADS-BE2.6在内源性测试靶点Site A和FGF6-sg4上的编辑位点更加集中在C5/C6，对于Site A靶点C5位置，eADS-BE2.1和eADS-BE2.6编辑效率分别是原来的1.7倍和2倍，对于FGF6-sg4靶点C6位置，编辑效率分别是原来的1.2倍和1.1倍，而邻近C7分别降低6.6倍和3.6倍，极大降低了旁观者胞嘧啶编辑，具备高精度的特性。
在12个内源性靶点上的再次验证表明，相对于对照BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU、3G5.13-PU、eADS-BE2.1和eADS-BE2.6依然展现精准编辑特性，偏好C5/C6位置产生C到T的编辑；而ADS-BE2.5则展示宽窗口高活性的特点， 部分靶点(如HBG-sg14、FANCF-sg17、Site A)效率和效率甚至超过经典的BE4max-PU，根据12个靶点不同位置的平均C到T效率统计，以对照组中编辑表现相对较精准的BE4max-YEE-PU为对比，其主要编辑窗口C4、C5、C6、C7和C8的平均效率分别为10.7％、63.4％、57.2％、37.6％和11.2％，而eADS-BE2.1和eADS-BE2.6在C5位置轻微降低，平均效率为52.1％和52.7％，而C4位置平均效率分别为8.4％和9.3％，C6位置平均效率分别为24.4％和21.4％，C7位置平均效率分别为4％和5.4％，C8位置平均效率分别为0.3％和0.4％；ADS-BE2.5编辑范围为C2-C9，主要编辑活性窗口为C4-C8，这也与传统的BE4max保持一致。
实施例6 eADS-BE2.1，eADS-BE2.6和ADS-BE2.5安全性评价
6.1质粒设计及构建
6.1.1为了进一步描述eADS-BE2.1、eADS-BE2.6和ADS-BE2.5的安全性，再次以BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU、A3G5.13-PU为对照，目标位点为VEGFA site 2，设计6个Cas9非依赖性脱靶检测靶点(如表11所示)，合成两条oligo，正链加CACC，反链加上AAAC，连接至已经用BbsI酶切好的nSaCas9-R-loop载体(如图8所示)上。
6.1.2将6.1.1中构建的质粒经sanger测序，确保完全正确。
6.2细胞转染
第1天用HEK293T(HEK293T细胞为ATCC CRL-3216细胞系)细胞铺种24孔板：
(1)消化HEK293T细胞，按照2×10 5cell/孔接种96孔板。
注：细胞复苏后，一般需传代2次后方可用于转染实验。
第2天转染：
(2)观察各孔细胞状态
注：要求转染前细胞密度应为70％-90％，且状态正常。
(3)质粒转染量如下：
BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU、A3G5.13-PU、eADS-BE2.1、eADS-BE2.6和ADS-BE2.5:3.1中新构建的质粒:VEGFA site 2靶点质粒＝400ng:300ng:300ng的质粒用量，转染试剂为PEI(每1μg质粒加3μL PEI)，共转染HEK293T宿主，设置n＝3孔/组。
6.3基因组提取及扩增子文库的准备
转染后72h，用天根细胞基因组提取试剂盒(DP304)提取细胞基因组DNA。之后用Hi-Tom Gene Editing Detection Kit(诺禾致源)操作流程，设计相对应的鉴定引物(如表12所示)，即在正向鉴定引物5’端加上搭桥序列5’-ggagtgagtacggtgtgc-3’(SEQ ID NO:53)， 反向鉴定引物5’端加上搭桥序列5’-gagttggatgctggatgg-3’(SEQ ID NO:54)，即得到一轮PCR产物，之后利用一轮PCR产物作为模板，进行二轮PCR产物，后混在一起进行切胶回收纯化后进行送公司进行测序(测序的服务提供商为苏州金唯智生物科技有限公司)。
6.4深度测序结果分析与统计
利用BE-analyzer网站分析深度测序结果，即统计C到T比率，并用graphpad prism 9.1.0进行统计作图。如表19和表20、图14和图15所示。
表19 eADS-BE2.1、eADS-BE2.6、ADS-BE2.5和BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU、A3G5.13-PU在VEGFA site2位点产生的非依赖性脱靶对比(单位，％)
Figure PCTCN2022143408-appb-000060
Figure PCTCN2022143408-appb-000061
表20 eADS-BE2.1、eADS-BE2.6、ADS-BE2.5和BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU、A3G5.13-PU在VEGFA site2位点产生的C到T效率对比(单位，％)
Figure PCTCN2022143408-appb-000062
表19和表20中，Rep.1、Rep.2和Rep.3为一式三份。
根据以上数据统计结果表明，对照组BE4max-PU、BE4max-YE1-PU、BE4max-YEE-PU、eA3A-PU和A3G5.13-PU产生indels平均效率分别为6.2％、3.4％、1.5％、4.5％和3.9％，ADS-BE2.5、eADS-BE2.1和eADS-BE2.6产生indels平均效率分别为4.6％、1.8％和1.2％，宽窗口ADS-BE2.5产生indels明显低于经典的BE4max-PU；eADS-BE2.1和eADS-BE2.6在保证高精度的同时也保持较低水平的indels。根据Cas9非依赖性脱靶评价，在6个R-loop位点检测发现，ADS-BE2.5产生的非依赖性脱靶事件也是低于传统的BE4max-PU，特别是在R-loop1和R-loop3位点降低幅度最大，而eADS-BE2.1和eADS-BE2.6引起极低的C到T编辑，微量的脱靶编辑事件和低水平的indel表明eADS-BE2.1和eADS-BE2.6同时兼容高精度和高安全性。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。
参考文献：
[1]Rees HA,Liu DR.Base editing:Precision chemistry on the genome and transcriptome of living cells.Nat Rev Genet,2018,19:770-788.
[2]Komor AC,Kim YB,Packer MS,et al.,Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature,2016,533:420-424.
[3]Gaudelli NM,Komor AC,Rees HA,et al.,Programmable base editing of a*t to g*c in genomic DNA without DNA cleavage.Nature,2017,551:464-471.
[4]Komor AC,Zhao KT,Packer MS,et al.,Improved base excision repair inhibition and bacteriophage mu gam protein yields c:G-to-t:A base editors with higher efficiency and product purity.Sci Adv,2017,3:eaao4774.
[5]Kurt IC,Zhou R,Iyer S,et al.,Crispr c-to-g base editors for inducing targeted DNA transversions in human cells.Nat Biotechnol,2021,39:41-46.
[6]Zhao D,Li J,Li S,et al.,Glycosylase base editors enable c-to-a and c-to-g base changes.Nat Biotechnol,2021,39:35-40.
[7]Koblan LW,Arbab M,Shen MW,et al.,Efficient c*g-to-g*c base editors developed using crispri screens,target-library analysis,and machine learning.Nat Biotechnol,2021.
[8]Chen L,Park JE,Paa P,et al.,Programmable c:G to g:C genome editing with crispr-cas9-directed base excision repair proteins.Nat Commun,2021,12:1384.
[9]Li S,Yuan B,Cao J,et al.,Docking sites inside cas9 for adenine base editing diversification and rna off-target elimination.Nat Commun,2020,11:5827.
[10]Kim HS,Jeong YK,Hur JK,et al.,Adenine base editors catalyze cytosine conversions in human cells.Nat Biotechnol,2019,37:1145-1148.
[11]Grunewald J,Zhou R,Iyer S,et al.,Crispr DNA base editors with reduced rna off-target and self-editing activities.Nat Biotechnol,2019,37:1041-1048.

Claims (11)

  1. 一种腺嘌呤脱氨酶，其特征在于，所述腺嘌呤脱氨酶的氨基酸序列为SEQ ID NO:2经突变的序列，所述突变的位点为第28位、第30位、第46位和中的一位或两位。
  2. 如权利要求1所述的腺嘌呤脱氨酶，其特征在于，所述突变选自以下一种或多种：
    (1)第28位氨基酸残基V替换为A或G；
    (2)第30位氨基酸残基V替换为F；
    (3)第46位氨基酸残基N替换为A、G、L或P；
    较佳地，(3)中，第46位氨基酸残基N替换为L或P；和/或，所述突变的位点为第46位，以及第27位、第29位或第48位；
    更佳地，所述突变选自以下任一：
    (4)第46位氨基酸残基N替换为L，并且第27位氨基酸残基E替换为R；
    (5)第46位氨基酸残基N替换为L，并且第29位氨基酸残基P替换为A；和
    (6)第46位氨基酸残基N替换为L，并且第48位氨基酸残基A替换为M。
  3. 一种胞嘧啶碱基编辑器，其特征在于，所述胞嘧啶碱基编辑器包括核酸酶和如权利要求1或2所述的腺嘌呤脱氨酶；
    较佳地，所述核酸酶与所述腺嘌呤脱氨酶通过连接子连接，所述连接子优选如SEQ ID NO:3、SEQ ID NO:9或SEQ ID NO:10所示；和/或，所述核酸酶为Cas蛋白及其变体，所述Cas蛋白优选选自酿酒酵母来源的spCas9、金黄色葡萄球菌来源的SaCas9、毛螺菌科细菌来源的LbCas12a和酸胺球菌属细菌来源的enAsCas12a；所述Cas蛋白变体优选选自VQR-spCas9、VRER-spCas9、spRY、spNG、SaCas9-KKH和SaCas9-NG；和/或，所述胞嘧啶碱基编辑器还包括尿嘧啶DNA糖基化酶抑制剂(UGI)；
    更佳地，所述核酸酶为spCas9，所述spCas9的氨基酸序列优选如SEQ ID NO:4所示；和/或，所述UGI通过连接子与所述核酸酶连接，所述UGI的拷贝数为至少一个，例如一个、两个或三个。
  4. 一种融合蛋白，其特征在于，所述融合蛋白自N端至C端依次包含信号肽序列、如权利要求1或2所述的腺嘌呤脱氨酶、核酸酶和信号肽序列；所述核酸酶如权利要求3所述；
    较佳地，所述信号肽序列为核定位信号序列或polyA信号序列，所述核定位信号序列优选如SEQ ID NO:1所示；所述polyA信号序列优选为BGH polyA；和/或，所述融合蛋白还包括UGI，所述UGI如权利要求3所述；
    更佳地，所述融合蛋白自N端至C端依次包含信号肽序列、腺嘌呤脱氨酶、核酸酶、信号肽序列、UGI和信号肽序列；所述UGI优选通过氨基酸序列如SEQ ID NO:7所示的 连接子与其N端的信号肽序列连接；当UGI为至少两个拷贝时，UGI之间优选通过如SEQ ID NO:8所示的连接子连接。
  5. 一种分离的核酸，其特征在于，所述核酸编码如权利要求1或2所述的腺嘌呤脱氨酶、如权利要求3所述的胞嘧啶碱基编辑器或者如权利要求4所述的融合蛋白。
  6. 一种基因表达盒，其特征在于，所述基因表达盒包括启动子元件和目的基因元件；
    其中，所述目的基因元件为如权利要求5所述的核酸；
    较佳地，所述启动子元件选自CMV、CAG、PGK、EF1α、Ctsk和Lp1。
  7. 一种胞嘧啶碱基编辑系统，其特征在于，其包括：sgRNA和如权利要求3所述的胞嘧啶碱基编辑器；
    较佳地，所述sgRNA的靶序列如SEQ ID NO:11～24或SEQ ID NO:83～93任一所示。
  8. 一种药物组合物，其特征在于，所述药物组合物包括如权利要求1或2所述的腺嘌呤脱氨酶、如权利要求3所述的胞嘧啶碱基编辑器、如权利要求4所述的融合蛋白或者如权利要求7所述的胞嘧啶碱基编辑系统，以及药学上可接受的载体。
  9. 一种碱基编辑方法，其特征在于，所述碱基编辑方法包括：
    在靶细胞中表达如权利要求1或2所述的腺嘌呤脱氨酶、如权利要求3所述的胞嘧啶碱基编辑器、如权利要求4所述的融合蛋白或者如权利要求7所述的胞嘧啶碱基编辑系统，使所述靶细胞发生碱基编辑；
    所述碱基编辑包括将C编辑为T、将C编辑为G；
    较佳地，所述靶细胞的来源为分离的细胞系；所述细胞系优选为哺乳动物细胞系；
    更佳地，所述分离的细胞系为293T细胞、HELA细胞、U2OS细胞、NIH3T3细胞或N2A细胞。
  10. 一种构建动物模型、农作物育种或制造碱基编辑设备的方法，所述方法包括使用如权利要求1或2所述的腺嘌呤脱氨酶、如权利要求3所述的胞嘧啶碱基编辑器、如权利要求4所述的融合蛋白、如权利要求5所述的核酸、如权利要求6所基因表达盒或者如权利要求7所述的胞嘧啶碱基编辑系统。
  11. 一种用于基因疗法的药物组合物，其特征在于，所述药物组合物包括如权利要求1或2所述的腺嘌呤脱氨酶、如权利要求3所述的胞嘧啶碱基编辑器、如权利要求4所述的融合蛋白、如权利要求5所述的核酸、如权利要求6所基因表达盒或者如权利要求7所述的胞嘧啶碱基编辑系统；
    所述基因疗法为治疗基因突变导致的疾病的基因疗法。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112143753A (zh) * 2020-09-17 2020-12-29 中国农业科学院植物保护研究所 一套腺嘌呤碱基编辑器及其相关生物材料与应用
WO2021158921A2 (en) * 2020-02-05 2021-08-12 The Broad Institute, Inc. Adenine base editors and uses thereof
CN113699135A (zh) * 2021-08-10 2021-11-26 国家卫生健康委科学技术研究所 一种无pam限制的腺嘌呤碱基编辑器融合蛋白及应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021158921A2 (en) * 2020-02-05 2021-08-12 The Broad Institute, Inc. Adenine base editors and uses thereof
CN112143753A (zh) * 2020-09-17 2020-12-29 中国农业科学院植物保护研究所 一套腺嘌呤碱基编辑器及其相关生物材料与应用
CN113699135A (zh) * 2021-08-10 2021-11-26 国家卫生健康委科学技术研究所 一种无pam限制的腺嘌呤碱基编辑器融合蛋白及应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
RICHTER MICHELLE F.; ZHAO KEVIN T.; ETON ELLIOT; LAPINAITE AUDRONE; NEWBY GREGORY A.; THURONYI BENJAMIN W.; WILSON CHRISTOPHER; KO: "Author Correction: Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity", NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP US, NEW YORK, vol. 38, no. 7, 20 May 2020 (2020-05-20), New York, pages 901 - 901, XP037187567, ISSN: 1087-0156, DOI: 10.1038/s41587-020-0562-8 *
YAN DAQI, REN BIN, LIU LANG, YAN FANG, LI SHAOFANG, WANG GUIRONG, SUN WENXIAN, ZHOU XUEPING, ZHOU HUANBIN: "High-efficiency and multiplex adenine base editing in plants using new TadA variants", MOLECULAR PLANT, vol. 14, no. 5, 1 May 2021 (2021-05-01), pages 722 - 731, XP093006106, ISSN: 1674-2052, DOI: 10.1016/j.molp.2021.02.007 *

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