An adenine base editor capable of achieving precise deletion of small DNA fragments and construction and application thereof

Abstract

The application discloses an adenine base editor capable of realizing precise deletion of a small DNA fragment and construction and application thereof. The adenine single-base editor comprises a fusion protein ABE8e-EndoV, and the fusion protein comprises, from N terminus to C terminus, an adenine deaminase, a nickase variant of a Cas nuclease and an isosine I base deletion repair endonuclease in sequence. The adenine base editor for precise deletion of a small DNA fragment has the characteristics of not only high efficient A-G base substitution, but also 9-13 bp small fragment deletion from the deaminated A base to the cleavage site interval of the Cas nuclease protein. The adenine base editor is particularly suitable for mining and research of cis-acting elements in genomes of animals and plants, and has wide application prospects in single-base substitution of target genes and saturation mutation screening.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. More specifically, it relates to an adenine single-base editor, pYL-ABE8e-EndoV, that can both efficiently replace AG bases in target sites and precisely delete small 9-13bp DNA fragments in target sites, as well as its construction and application. Background Art

[0002] The spatiotemporal expression of eukaryotic genes is often regulated by a complex interplay of cis-acting elements and trans-acting factors. Cis-acting elements are typically found in the promoter region, 5' and 3' UTRs, miRNA coding sequences, and introns of target genes. The TATA box, typically located -25 to -30 bp upstream of the transcription start site, controls the accuracy and frequency of transcription. Other well-studied elements within promoters include the GC box and the CAAT box. Within introns, numerous studies have revealed a branch point between the 5' and 3' ends of the intron. This branch point is an adenosine base, typically located 20-30 bp upstream of the 3' splice junction. Deletion or mutation of this base often results in reduced splicing efficiency or inability to splice the precursor RNA (Simpson et al., 1996). These cis-acting elements are often composed of AT-rich sequences, making the development of precise and predictable tools for studying these AT-rich cis-acting elements crucial.

[0003] Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated endonuclease (CRISPR / Cas) are acquired immune systems formed by bacteria and archaea during evolution, which can quickly and accurately identify and degrade invading foreign DNA. Based on the number and functional diversity of Cas proteins in the CRISPR / Cas system, the CRISPR / Cas system can be divided into two major categories (Class 1 and Class 2), which contain five subcategories (Type I to Type V) (Makarova et al., 2017). Class 1 includes three types: Type I, Type III, and Type IV. These require the simultaneous participation of multiple Cas proteins to form a complex to function together, which poses great difficulties in designing and constructing vectors. Class 2 includes two types: Type II and Type V. They only require a single Cas protein and guide RNA (gRNA) to form a complex to achieve cleavage of the target site in the genome. Therefore, Type II and Type V CRISPR / Cas systems from different families of bacteria and archaea have been explored and used (Mohanraju et al., 2016). The representative of Type II is the Cas9 (SpCas9) system from Streptococcus pyogenes (Mali et al., 2013), and the main representative of Type V is the CRISPR / Cas12a (also known as Cpf1) system from Francisella novicida (Zetsche et al., 2015).

[0004] Although CRISPR / Cas9-mediated genome editing can efficiently knock out target genes by creating double-strand breaks (DSBs) at the target site through non-homologous end repair (NHEJ), CRISPR / Cas9-mediated genome editing mainly causes random deletions of 1-3bp or sometimes large fragments, making it difficult to accurately study the function of the target element. In addition, although CRISPR / Cas12a can cause deletions of larger fragments, the deleted fragments are also random. In addition, although Gao Caixia's research group combined wild-type SpCas9 with cytosine deaminase APOBEC, uracil glycosylase (UDG) and apurinic pyrimidine site lyase (AP lyase) based on the principles of cytosine deamination and base excision repair (BER), they established a predictable multinucleotide targeted deletion system (AFIDs). The cytosine deaminase APOBEC used in the AFIDs system has great advantages for CG-rich sequences, but has great limitations for AT-rich cis-acting elements. In addition, the system uses SpCas9 that recognizes NGG-PAM, which limits the scope of use of AFIDs.

[0005] In adenine base editors (ABEs), the A base is deaminated to inosine I, which is then recognized as a G base at the DNA level. DNA repair and replication then allow for the A-to-G base substitution. Because the enzyme activity for inosine base deletion repair is low in eukaryotic cells, the A-to-G substitution in the ABE base editing system produces virtually no AY or indel byproducts. However, existing studies have shown that the deamination of the A base to an I base occurs due to natural hydrolysis and nitrosative stress in cells exposed to endogenous and / or exogenous agents, triggering the AER repair pathway (Kuraoka et al., 2015). In the AER pathway, endonuclease V (EndoV) hydrolyzes the second 3' phosphodiester bond to deoxyinosine in the DNA chain. An EndoV-dependent AER pathway has been identified in Escherichia coli models for deoxyinosine removal from DNA. However, the AER pathway in mammalian cells remains largely uncharacterized. Biochemical studies of mammalian EndoV have shown that it has high deoxyinosine 3' endonuclease activity in single-stranded DNA containing deoxyinosine, but its activity in double-stranded DNA is very low. To date, the AER pathway based on I:T mismatches in ABE systems remains poorly understood.

[0006] In summary, the development of adenine base editors suitable for cis-acting elements, miRNA coding sequences, conserved elements in intronic regions, broad targeting, and precise deletion of new small DNA fragments that can adapt to high-AT polynucleotide sequence environments is an important direction for the optimization of gene editing technology. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the shortcomings of the CRISPR / Cas9 system-mediated gene editing in the prior art, which mainly causes random deletion of small fragments of 1-3bp, limiting its accurate study of the functions of cis-acting elements, miRNA coding sequences, and conserved elements in intron regions. Therefore, it is necessary to invent an adenine single-base editor that can accurately achieve DNA deletion of about 10bp.

[0008] The first objective of the present invention is to provide an adenine base editor fusion protein ABE8e-EndoV that can achieve precise deletion of small DNA fragments.

[0009] The second object of the present invention is to provide a nucleotide sequence encoding the fusion protein ABE8e-EndoV.

[0010] The third object of the present invention is to provide a plasmid vector containing the nucleotide sequence.

[0011] The fourth object of the present invention is to provide the use of the fusion protein ABE8e-EndoV or the nucleotide sequence or the plasmid vector in the preparation of an adenine base editor.

[0012] The fifth object of the present invention is to provide an adenine base editor that can achieve precise deletion of small DNA fragments.

[0013] The sixth object of the present invention is to provide a method for constructing the adenine base editor that can achieve precise deletion of small DNA fragments.

[0014] The seventh object of the present invention is to provide the application of the adenine base editor that can achieve precise deletion of small DNA fragments in gene editing of organisms.

[0015] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0016] An adenine base editor fusion protein, ABE8e-EndoV, that can precisely delete small DNA fragments. From the N-terminus to the C-terminus, it contains, in order: adenine deaminase, a nickase variant protein of Cas nuclease, and an inosine I base deletion repair endonuclease;

[0017] In the fusion protein ABE8e-EndoV of the present invention, adenine deaminase is used for A-to-G base editing, the nickase variant protein of the Cas nuclease is used to cut the target sequence, and the inosine I base deletion repair nuclease plays a key role in the inosine selective excision repair (AER) pathway. When the fusion protein ABE8e-EndoV is used for gene editing, based on the I:T base mismatch repair (AER) pathway in ABE8e-EndoV, A is first deaminated and hydrolyzed to inosine I by adenine deaminase, resulting in an I:T base mismatch in the DNA duplex, and the second phosphodiester bond at the 3' end of inosine is hydrolyzed by the inosine I base deletion repair nuclease. After DNA repair and replication, A-to-G base replacement is achieved; in addition, A-to-Deletions mutation is achieved through non-homologous end joining repair, resulting in precise deletion of a small fragment of 9-13bp from the 5'-deaminated A base to the nuclease cleavage site. Theoretically, all adenine deaminases or their mutants, nickase variant proteins of Cas nuclease variants and nucleases with inosine I base deletion repair can achieve the purpose of the present invention and should all be within the scope of protection of the present invention.

[0018] Preferably, the adenine deaminase is an adenine deaminase mutant.

[0019] Further preferably, the adenine deaminase mutant is TadA8e, whose amino acid sequence is shown in SEQ ID NO. 1. The adenine deaminase TadA8e has high activity and high A-to-G base editing efficiency.

[0020] Preferably, the nickase variant of the Cas nuclease is a nickase variant of the Cas9 protein.

[0021] Further preferably, the nickase variant of the Cas9 protein is the SpCas9 nickase variant SpGn, whose amino acid sequence is shown in SEQ ID NO.2, and can recognize concise NG-PAM.

[0022] Preferably, the inosine I base deletion repair endonuclease is EndoV, and its amino acid sequence is shown in SEQ ID NO.3, or an amino acid sequence with 80% homology to the sequence shown in SEQ ID NO.3.

[0023] Preferably, it further comprises two nuclear localization signals fused to the N-terminus and C-terminus of ABE8e-EndoV, respectively.

[0024] Further preferably, the amino acid sequence of the nuclear localization signal is shown as SEQ ID NO.4.

[0025] Preferably, it further comprises a protein linker sequence that respectively connects adenine deaminase TadA8e and SpCas9 variant SpGn, and SpCas9 variant SpGn and inosine I base deletion repair key enzyme EndoV; preferably, the protein linker sequence is a flexible linker sequence.

[0026] Preferably, the adenine base editor fusion protein ABE8e-EndoV that can achieve precise deletion of small DNA fragments comprises, from N-terminus to C-terminus, a nuclear localization signal bpNLS, adenine deaminase TadA8e, a flexible linker sequence, SpCas9 variant SpGn, a flexible linker sequence, the inosine I base deletion repair key enzyme EndoV and the nuclear localization signal bpNLS, and its full amino acid sequence is shown in SEQ ID NO.5.

[0027] The present invention also provides a nucleotide encoding any of the above-mentioned adenine base editor fusion proteins ABE8e-EndoV that can achieve precise deletion of small DNA fragments.

[0028] Preferably, the nucleotide sequence is shown as SEQ ID NO.6.

[0029] The present invention also provides a plasmid vector containing any one of the above-mentioned nucleotide sequences.

[0030] The present invention also provides the use of any of the above-mentioned adenine base editor fusion proteins ABE8e-EndoV that can achieve precise deletion of small DNA fragments, or any of the above-mentioned nucleotides or plasmid vectors in the preparation of an adenine base editor that can precisely delete small DNA fragments.

[0031] The present invention also provides an adenine base editor that can achieve precise deletion of small DNA fragments. The adenine base editor for precise deletion of small DNA fragments contains a nucleotide sequence encoding any of the above-mentioned adenine base editor fusion proteins ABE8e-EndoV for precise deletion of small DNA fragments. Specifically, the nucleotide sequence encoding any of the above-mentioned adenine base editor fusion proteins ABE8e-EndoV for precise deletion of small DNA fragments is constructed onto a transformation vector to obtain the result.

[0032] Preferably, the transformation vector is a plant transformation vector, including but not limited to pCAMBIA1300 and vectors modified therefrom, such as pYLCRISPR / Cas9Pubi-H.

[0033] More preferably, the plant transformation vector is the binary vector pYLCRISPR / Cas9Pubi-H.

[0034] The present invention also provides a method for constructing the DNA small fragment precise deletion adenine base editor, first preparing a complete fusion DNA sequence encoding the DNA small fragment precise deletion adenine base editor fusion protein ABE8e-EndoV, then inserting it into an expression vector, transforming the host bacteria, extracting the positive plasmid, sequencing, and obtaining a stable DNA small fragment precise deletion adenine base editor;

[0035] Preferably, the adenine base editor pYL-ABE8e-EndoV is obtained by inserting it between Pst I and BamH I of the binary vector pYLCRISPR / Cas9Pubi-H.

[0036] As a preferred embodiment, the method for constructing an adenine base editor capable of achieving precise deletion of small DNA fragments comprises the following steps:

[0037] S1. Gene fragment 1 encoding bpNLS-TadA8e-linker 1 and gene fragment 2 encoding linker2-EndoV-bpNLS were synthesized separately. A restriction enzyme site was added to the 5' end of gene fragment 1 and another restriction enzyme site was added to the 3' end of gene fragment 2 by PCR reaction; gene fragment 3 encoding SpGn was synthesized;

[0038] S2. Using overlapping PCR, the C-terminus of gene fragment 1 with the restriction enzyme cleavage site was ligated to the N-terminus of gene fragment 3 to generate gene fragment 4, which contained the fusion protein bpNLS-TadA8e-linker 1-SpGn. The C-terminus of gene fragment 4 was then ligated to the N-terminus of gene fragment 2 with the restriction enzyme cleavage site to generate gene fragment 5, which contained the fusion protein bpNLS-TadA8e-linker 1-SpGn-linker 2-EndoV-bpNLS.

[0039] S3. Insert gene fragment 5 between the two corresponding restriction sites of the plant vector pYLCRISPR / Cas9Pubi-H, transform the host bacteria, extract the positive plasmid, and sequence to obtain a stable and efficient ABE base editor for precise deletion of small DNA fragments.

[0040] Preferably, the insertion of the gene fragment 5 into the vector in step S3 is achieved by Gibson assembly technology.

[0041] Preferably, the host bacteria in step S3 is Escherichia coli Top10F'.

[0042] The present invention also provides the application of the adenine base editor for precise deletion of small DNA fragments in genome editing in organisms. The adenine base editor for precise deletion of small DNA fragments is particularly suitable for functional studies of cis-acting elements, miRNAs, and key elements of intron splicing, and can simultaneously compare the functional differences caused by target base substitutions and fragment deletions. Therefore, the present invention also provides the mining and research of cis-acting elements in the adenine base editor animal and plant genomes, and the application of precise deletion of small fragments of genomic DNA in organisms.

[0043] Preferably, the mining and research of cis-acting elements in the genome are specifically performed as follows:

[0044] (1) Identify the cis-acting element to be studied, and design and synthesize the sgRNA expression cassette element according to the target site;

[0045] (2) integrating the sgRNA expression cassette element into the adenine base editor for precise deletion of the small DNA fragment, thereby obtaining a vector that can achieve precise deletion of the target DNA fragment and can achieve adenine base replacement in the target DNA fragment;

[0046] (3) Transforming host cells with the adenine base editing vector that precisely deletes the target gene DNA fragment, and screening to obtain cells in which the corresponding target DNA fragment is precisely deleted or the adenine base in the target DNA fragment is replaced.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] (1) When the fusion protein ABE8e-EndoV of the present invention is used for gene editing, based on the I:T base mismatch repair (AER) pathway in ABE8e-EndoV, A is first deaminated and hydrolyzed into inosine I by adenine deaminase, resulting in an I:T base mismatch in the DNA double strand. The second phosphodiester bond at the 3' end of inosine is hydrolyzed by the inosine I base deletion repair nuclease, and the A-to-G base replacement is achieved through DNA repair and replication; in addition, the A-to-Deletions mutation is achieved through non-homologous end joining repair, resulting in a precise deletion of a 9-13bp small fragment from the 5'-deaminated A base to the nuclease cleavage site. The DNA small fragment precise deletion adenine base editor is particularly suitable for functional studies of key elements such as cis-acting elements, miRNAs, and intron splicing. In addition, it is also suitable for the replacement of key amino acids in target genes, large-scale saturation mutations, etc.

[0049] (2) The present invention is based on the DNA small fragment precise deletion adenine base editor fusion protein ABE8e-EndoV constructed by the present invention. The DNA small fragment precise deletion adenine base editor pYL-ABE8e-EndoV tool can recognize concise NG-PAM and has a high efficiency of A-to-G replacement efficiency. The base replacement activity window is mainly concentrated between A3-A8. In addition, it also has high efficiency of DNA small fragment precise deletion activity at the target site. The range of DNA small fragments deleted is from the deaminated A base to 9-13bp of the Cas nuclease cleavage site. Compared with other currently available editing systems, ABE8e-EndoV editing has more advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This diagram shows the structure of the pYL-ABE8e-EndoV vector, a DNA base editor for precise adenine deletion. The diagram shows the pCAMBIA1300 vector backbone; the polynucleotide sequence of the ABE8e-EndoV fusion protein is expressed using the Pubi constitutive promoter; TadA8e, an adenine deaminase with high A-to-G base editing efficiency; SpGn, a SpCas9 variant capable of recognizing NG-PAM; and EndoV, endonuclease V, which plays a key role in the inosine selective excision repair (AER) pathway.

[0051] Figure 2 Figures 1 and 2 show the editing efficiency and editing window of pYL-ABE8e-EndoV. (a) ABE8e-EndoV editing efficiency at five target sites. The total editing efficiency is the ratio of plants with mutations at the target site to the total number of transformants in the previous T0 generation. The A-to-G editing efficiency is the ratio of plants with A-to-G base substitutions at the target site to the total number of transformants in the previous T0 generation. The indels editing efficiency is the ratio of plants with small DNA deletions at the target site (including plants with both deletions and base substitutions). The PAM in the target site is underlined and highlighted in bold. (b) The average A-to-G editing window and average editing efficiency of ABE8e-EndoV.

[0052] Figure 3ABE8e-EndoV demonstrates efficient and predictable DNA deletion. (a) ABE8e-EndoV generates precise deletions from the 5'-deaminated A base of the target site to the SpGn nick site, as well as the efficiency. The PAM and precise editing efficiency are highlighted in light black, with black bars representing the deleted nucleotides and the SpGn nick site highlighted. (b) Sanger sequencing chromatograms of several typical ABE8e-EndoV mutation types. Het, heterozygous mutation; del, deletion mutation.

[0053] Figure 4 This figure shows off-target analysis of ABE8e-EndoV. Candidate off-target sites were analyzed using the CRISPR-GE off-target subroutine, and candidate off-target sites with one to three base variations in the target sequence were selected. The PAM of the target is underlined and highlighted in bold, and bases that differ between the candidate off-target sites and the target are highlighted in bold. The analysis results show that ABE8e-EndoV has a low off-target efficiency.

[0054] Figure 5 A schematic diagram of the repair pathway for ABE8e-EndoV-mediated precise deletion of adenine base editing in small DNA fragments. The I:T base mismatch repair pathway in the ABE8e-EndoV system begins with the deamination and hydrolysis of A to inosine I by the adenine deaminase TadA8e, resulting in an I:T base mismatch in the DNA duplex. The second phosphodiester bond at the 3' end of inosine is then hydrolyzed by EndoV, leading to A-to-deletion mutations and A-to-G base substitutions through DNA repair and replication. DETAILED DESCRIPTION

[0055] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0056] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0057] Example 1 Construction of the DNA base editor pYL-ABE8e-EndoV for precise adenine deletion of small fragments

[0058] The protein and polynucleotide sequences of the N-terminal nuclear localization signal bpNLS1, deaminase TadA8e, 32 amino acid linker1, and SpCas9 variant SpGn used in the present invention have been reported and published in the early stage of this project (Tan et al., 2020, Plant Biotechnology Journal, 20:934–943). The amino acid sequence of bpNLS1-TadA8e-linker1-SpGn is as follows ( aa1 to aa1584 of SEQ ID NO.5 ), and the polynucleotide sequence of bpNLS1-TadA8e-linker-SpGn1 is shown in ( Bases 4 to 4752 of SEQ ID NO.6 ). In addition, the main function of EndoV, the key enzyme for inosine deletion repair, was mainly referenced (Kuraoka et al., 2015, Biomolecules, 5: 2194-2206). Then, the amino acid sequence of EndoV from Escherichia coli was searched in the public data of NCBI. Subsequently, the 32 amino acid linker2, EndoV amino acid sequence and C-terminal nuclear localization signal bpNLS2 (linker2-EndoV-bpNLS2) were handed over to Wuhan Genecreate Company for direct synthesis according to the optimized nucleic acid sequence of rice codon preference (the amino acid sequence of linker2-EndoV-bpNLS2 is shown in the figure). SEQ ID NO.5 The amino acids 1585 to 1860 are shown in , the polynucleotide of linker2-EndoV-bpNLS2 is as follows SEQ ID NO.6 Bases 4753 to 5583 The optimized synthesized linker2-EndoV-bpNLS2 was ligated to the C-terminus of bpNLS1-TadA8e-linker1-SpGn by overlapping PCR to form a complete fusion of bpNLS1-TadA8e-linker1-SpGn-linker2-EndoV-bpNLS2. This fusion was then cloned into the binary vector pYLCRISPR / Cas9Pubi-H (Ma et al., 2015, Molecular Plant, 8:1274-1284) between Pst I and BamH I using the Gibson assembly method. This resulted in a DNA base editor for precise adenine deletion of a small fragment, named pYL-ABE8e-EndoV. The ABE8e-SpG adenine base editor previously developed by the applicant (Tan et al., 2020, Plant Biotechnology Journal, 20:934–943) was used as a control for editing efficiency comparison.

[0059] Amino acid sequence of adenine deaminase TadA8e: (SEQ ID NO.1)

[0060] SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN

[0061] Amino acid sequence of SpCas9 variant SpGn: (SEQ ID NO.2)

[0062]

[0063] Amino acid sequence of EndoV, a key enzyme in inosine base deletion repair (SEQ ID NO. 3)

[0064] DLASLRAQQIELASSVIREDRLDKDPPDLIAGADVGFEQGGEVTRAAMVLLKYPSLELVEYKVARIATTMPYIPGFLSFREYPALLAAWEMLSQKPDLVFVDGHGISHPRR LGVASHFGLLVDVPTIGVAKKRLCGKFEPLSSEPGALAPLMDKGEQLAWVWRSKARCNPLFIATGHRVSVDSALAWVQRCMKGYRLPEPTRWADAVASERPAFVRYTANQP

[0065] Amino acid sequence of nuclear localization signal (SEQ ID NO.4)

[0066] KRTADGSEFESPKKKRKV

[0067] The complete amino acid sequence of the fusion protein (SEQ ID NO.5)

[0068]

[0069] The complete nucleotide sequence encoding the fusion protein (SEQ ID NO.6)

[0070]

[0071]

[0072]

[0073] The specific procedures are as follows:

[0074] The primers used in the construction process are shown in Table 1:

[0075] Table 1 Primers used for basic vector transformation of pYL-ABE8e-EndoV

[0076]

[0077] (1) Using primers F-ABE8e-EndoV-1 / R-ABE8e-EndoV-1 (SEQ ID NO. 7 and SEQ ID NO. 8) and ABE8e-SpG, which had been developed in our laboratory earlier, as a template, a fragment with a PstI restriction site was amplified and named fragment D1.

[0078] PCR system (15 μl): 2× Phanta Max Buffer 7.5 μl, 10 mmol / L dNTPs Mix 0.35 μl, Phanta Max Polymerase 0.35 μl, ABE8e-SpG 10 ng, 10 μmol / L F-ABE8e-EndoV-1 0.35 μl, 10 μmol / L R-ABE8e-EndoV-1 0.35 μl, and ddH2O to make up to 15 μl.

[0079] PCR program: pre-denaturation at 95°C for 2 min, 28 PCR cycles (95°C for 10 s, 56°C for 15 s, 72°C for 4 min), and extension at 72°C for 5 min.

[0080] (2) Using primers F-ABE8e-EndoV-2 / R-ABE8e-EndoV-2 (SEQ ID NO. 9 and SEQ ID NO. 10) and the chemically synthesized linker2-EndoV-bpNLS2 target plasmid as a template, the linker2-EndoV-bpNLS2 fragment was amplified and named fragment D2.

[0081] PCR system (15 μl): 2× Phanta Max Buffer 7.5 μl, 10 mmol / L dNTPs Mix 0.35 μl, Phanta Max Polymerase 0.35 μl, linker2-EndoV-bpNLS2 10 ng, 10 μmol / L F-ABE8e-EndoV-2 0.35 μl, 10 μmol / L R-ABE8e-EndoV-2 0.35 μl, and ddH2O to make up to 15 μl.

[0082] PCR program: pre-denaturation at 95°C for 2 min, 28 PCR cycles (95°C for 10 s, 56°C for 15 s, 72°C for 30 s), and extension at 72°C for 5 min.

[0083] (3) Using primers F-ABE8e-EndoV-1 / R-ABE8e-EndoV-2 (SEQ ID NO. 7 and SEQ ID NO. 10), fragments D1 and D2 amplified in the first round were used as templates to amplify the fusion fragment of bpNLS1-TadA8e-linker1-SpGn-linker2-EndoV-bpNLS2 (referred to as TadA8e-SpGn-EndoV fusion fragment).

[0084] PCR system (50 μl): 2×Phanta Max Buffer 25 μl, 10 mmol / L dNTPs Mix 1.0 μl, Phanta Max Polymerase 1.0 μl, 0.5 μl each of the bpNLS1-TadA8e-linker1-SpGn fragment D1 amplified in the first round and the linker2-EndoV-bpNLS2 fragment D2 amplified in the first round, 10 μmol / L F-ABE8e-EndoV-1 1.0 μl, 10 μmol / L R-ABE8e-EndoV-2 1.0 μl, and ddH2O to make up to 50 μl.

[0085] PCR program: pre-denaturation at 95°C for 2 min, 28 PCR cycles (95°C for 10 s, 56°C for 15 s, 72°C for 4.5 min), and extension at 72°C for 10 min.

[0086] (4) Purify the PCR product of the amplified TadA8e-SpGn-EndoV fusion DNA fragment using the Genstar purification kit. Digest pYLCRISPR / Cas9Pubi-H (Ma et al., 2015, Molecular Plant, 8:1274-1284) with Pst I and BamH I: 10× Faster digest buffer, Pst I 0.5μl, BamH I 0.5μl, pYLCRISPR / Cas9Pubi-H 300ng, ddH2O to 10μl, react at 37℃ for 1h, gel-recover the vector backbone and use it in Gibson assembly reaction (NEB#E5510S): 2× Mix 5μl, eCBE-Cas9n-NG-2×UGI fusion fragment 60ng, gel-recovered vector backbone 90ng, ddH2O to 10μl, react at 50℃ for 50min. Take 1.5 μl of Gibson's ligation product and electroporate to transform E. coli Top10F'. Screen the transformed single clones on kanamycin-resistant (Kana) LB plates. Send the positive clones for sequencing to obtain the pYL-ABE8e-EndoV basic vector plasmid ( Figure 1 ).

[0087] Example 2 pYL-ABE8e-EndoV has the functions of precise deletion of small DNA fragments and adenine base substitution

[0088] With reference to the literature previously published by the inventors' team (Ma et al., 2015, Molecular Plant, 8: 1274-1284; Ma and Liu, 2016, Current Protocols in Molecular Biology, 115: 31.6.1-31.6.21; Zeng Dongchang et al., 2018, Science China: Life Sciences, 48: 783-794), small nuclear RNA gene promoters (OsU6a, OsU6b, OsU6c, and OsU3) were constructed to drive sgRNA expression cassettes for different targets. These sgRNAs were inserted into the pYL-ABE8e-EndoV binary vector (Ma et al., 2015, Molecular Plant, 8: 1274-1284) using the Golden Gate assembly method. Rice was transformed, and the target sites of the transformants were sequenced to analyze the editing performance of pYL-ABE8e-EndoV. The specific operations are as follows:

[0089] 1. Target design of TS1 to TS5

[0090] The OsMSP1 gene (Os01g0917500), OsChalk5 gene (Os05g0156900), and OsCKS2 gene (Os01g0197700) were selected from the rice genome as target genes. Two target sites (SEQ ID NOs. 11 and 12) were designed in the CDS region of OsMSP1, one target site each in the promoter region and 3' UTR of OsChalk5 (SEQ ID NOs. 13 and 14), and one target site (SEQ ID NO. 15) was designed in the intron region of OsCKS2. These five target sites, all containing A- or T-rich sequences, were used to test the editing performance of pYL-ABE8e-EndoV.

[0091] Table 2 Five targets used to test the editing performance of pYL-ABE8e-EndoV

[0092]

[0093] 2. Primer design for sgRNA expression cassettes of TS1 to TS5

[0094] Using the online program CRISPR-GE developed by our research group (http: / / skl.scau.edu.cn / ) (Xie et al., 2018, Molecular Plant, 11:720-735), we entered the PrimerDesign subroutine, the PrimerDesign-V branch program, selected the corresponding promoter, checked the checkbox, selected method 2, and clicked design. Five target primers gR-T# and U#-T# were automatically generated (Table 3).

[0095] 3. Overlapping PCR splicing of sgRNA expression cassettes from T1 to T5

[0096] According to our previously published literature (Ma et al., 2015, Molecular Plant, 8: 1274-1284; Ma and Liu, 2016, Current Protocols in Molecular Biology, 115: 31.6.1-31.6.21; Zeng Dongchang et al., 2018, Science China: Life Sciences, 48: 783-794), a small RNA promoter-driven sgRNA expression cassette with Bsa I restriction sites on both sides was obtained by two rounds of PCR.

[0097] Table 3 Sequences of first-round PCR target primers gR-T# and U#-T#

[0098]

[0099]

[0100] In the first round of PCR, the target sequence was introduced downstream of the OsU6 / OsU3 promoter and upstream of the sgRNA sequence using the designed U#-T# / gR-T# primers (SEQ ID NO. 16 to SEQ ID NO. 25, Table 3). In a single PCR system, the UF primer (SEQ ID NO. 26, Table 3) was paired with the gR-T# primer to amplify the promoter sequence containing the target; the gR-R primer (SEQ ID NO. 27, Table 3) was paired with the U#-T# primer to amplify the sgRNA sequence containing the target. PCR system (20 μl): 2× Phanta Max Buffer 10.0 μl, 10 mmol / L dNTPs Mix 0.4 μl, Phanta Max Polymerase 0.3 μl, pYLgRNA-OsU6 / 3 (plasmid containing promoter and sgRNA) (Ma et al., 2015, Molecular Plant, 8:1274-1284) 3 ng, 10 μmol / L UF 0.4 μl, 10 μmol / L gR-T# 0.2 μl, 10 μmol / L gRNA-R 0.4 μl, 10 μmol / L U#-T# 0.2 μl, ddHO to 20 μl. PCR program: initial denaturation at 95°C for 1 min, 28 PCR cycles (95°C for 10 s, 58°C for 15 s, 72°C for 20 s), and extension at 72°C for 1 min.

[0101] Table 4 Universal primers for constructing multiple sgRNA expression cassettes

[0102]

[0103]

[0104] Note 1: The Bsa I digestion ends are designed to be non-palindromic sequences, which can produce efficient ligation (Golden Gateligation).

[0105] Note 2: If more than 8 sgRNA expression cassettes are connected, more sets of Pgs and Pps primers need to be designed by yourself, each set containing complementary non-palindromic Bsa I restriction endonucleases.

[0106] Table 5 Second-round PCR primer combinations for assembling different numbers of sgRNA expression cassettes

[0107]

[0108] In the second round of PCR, primers Pps and Pgs (SEQ ID NO. 28 to SEQ ID NO. 42, Tables 4 and 5) were used to construct the small RNA promoter-driven sgRNA expression cassette. Bsa I restriction sites were added to both sides of the PCR product. SgRNA expression cassette vectors T1 to T5 were constructed. PCR system (50 μl): 2× Phanta Max Buffer 25.0 μl, 10 mmol / L dNTPs Mix 1.0 μl, Phanta Max Polymerase 1.0 μl, 10× diluted PCR product from the previous round 1.0 μl, primers for each of the five targets, 1.0 μl each of 10 μmol / L Pps-L / Pgs-2 (T1), Pps-2 / Pgs-3 (T2), Pps-3 / Pgs-4 (T3), Pps-4 / Pgs-5 (T4), and Pps-5 / Pgs-R (T5), and ddHO to 50 μl. PCR procedure was the same as for the first round. Purify the second-round PCR product using the Genstar purification kit.

[0109] 4. Construction of DNA small fragment deletion adenine base editor vectors containing sgRNA expression cassettes with different targets

[0110] Using the "Golden Gate" cloning method based on Bsa I digestion and ligation, a "cut-and-ligate" approach (Ma and Liu, 2016, Current Protocols in Molecular Biology, 115:31.6.1-31.6.21; Zeng et al., 2018, Science China: Life Sciences, 48:783-794) was used to assemble TS1 to TS5. Two sets of small nuclear RNA promoter-driven sgRNA expression cassettes were cloned into the binary vector pYL-ABE8e-EndoV. A 15 μl reaction system consisted of 10× CutSmart Buffer (1.5 μl), 10 mmol / L ATP (1.5 μl), 80–100 ng of pYL-ABE8e-EndoV plasmid, 10–15 ng of purified sgRNA expression cassette, 10 units of Bsa I-HF, 35 units of T4 DNA ligase, and ddH2O to make up to 15 μl. The enzyme digestion and ligation reaction was performed using a PCR instrument with a variable temperature cycle: 37°C for 10 min, followed by 10-12 cycles (37°C for 5 min, 10°C for 3 min, and 20°C for 5 min); and finally 37°C for 3 min. After dialyzing the ligation product, it was electroporated into DH10B cells and screened on kanala-resistant (Kan) LB plates. Colony PCR was performed using the primer pair SP-L1 / SP-R (SEQ ID NO. 53 and SEQ ID NO. 54, Table 3) according to the literature (Ma and Liu, 2016, Current Protocols in Molecular Biology, 115:31.6.1-31.6.21; Zeng Dongchang et al., 2018, Science China: Life Sciences, 48:783-794). Positive clones were screened and finally confirmed by sequencing using primer SP-L1.

[0111] 5. pYL-ABE8e-EndoV has high editing efficiency

[0112] Using Agrobacterium-mediated transformation of rice (japonica Zhonghua 11) calli, the five target vectors containing sgRNAs driven by different small RNA promoters TS1-TS5 were transformed into rice calli. Following the plant genetic transformation process, T0 rice plants were grown. Leaf DNA from T0 transformed plants was used as a template to amplify the DNA fragments at the target editing sites using amplification (amp) and sequencing (seq) primers (SEQ ID NO. 45 to SEQ ID NO. 59, Table 6). Direct Sanger sequencing was performed, and the editing efficiency of pYL-ABE8e-EndoV was calculated by aligning the sequencing results with the reference sequence. T0 generation results showed stable editing in ABE8e-EndoV-TS1-TS5, with an average overall editing efficiency of 62.7%. Moreover, the average A-to-G substitution efficiency of ABE8e-EndoV was almost the same as that of ABE8e-SpG (59.7%), indicating that the addition of the inosine base deletion repair enzyme EndoV did not affect the deamination efficiency of ABE8e-SpG. Figure 2 a). In addition to detecting A-to-G base editing, ABE8e-EndoV also produced an average of 17.3% monoallelic insertion or deletion mutations. This result indicates that EndoV indeed plays a key role in inosine mismatch repair. In terms of editing window, the A-to-G base editing window of ABE8e-EndoV is mainly concentrated in A3-A8. Compared with ABE8e-SpGn (A4-A8, which can be expanded to A4-A11), ABE8e-EndoV has a narrower editing window ( Figure 2 b).

[0113] 6. pYL-ABE8e-EndoV can produce predictable and precise deletion of small DNA fragments

[0114] CRISPR / Cas9 has two nuclease domains, HNH and RuvC, which cut the target chain and the non-target chain respectively, causing double-strand breaks in DNA and generating random insertion and deletion mutations. In the genomes of plants and animals, there are a large number of cis-acting elements composed of small fragments that also play a key role. The need for CRISPR / Cas9 to recognize NGG-PAM and the characteristics of random insertions and deletions is challenging to apply to the study of the functions of specific small fragments. In the present invention, ABE8e-EndoV detected an average of 17.3% insertion or deletion mutations in the target site. We further analyzed these indels and found that ABE8e-EndoV precisely deleted the small fragment from the 5'-deaminated A base to the SpGn cleavage site in TS1 (33.3%), TS3 (33.3%), TS4 (55.5%) and TS5 (60%) ( Figure 3a). The deletion window of ABE8e-EndoV mainly focuses on the A5-A8 to SpGn cleavage site, resulting in a 9-13 bp deletion ( Figure 3 b) These results demonstrate that ABE8e-EndoV, in addition to its highly efficient A-to-G base substitutions, can also predictably and precisely delete small target DNA fragments. ABE8e-EndoV can be applied to functional studies of key A / T-rich elements. Furthermore, ABE8e-EndoV utilizes the CRISPR / Cas9 variant SpGn (which recognizes NG-PAM) with a broader targeting range, further facilitating precise base substitutions and deletions at the target site.

[0115] Table 6 Target amplification and sequencing primers for T0 generation transformed plants

[0116]

[0117] 7. pYL-ABE8e-EndoV has low off-target efficiency

[0118] Using the online program CRISPR-GE webpage developed by our research group (http: / / skl.scau.edu.cn / ) (Xie et al., 2018, Molecular plant, 11:720-735), we entered the off-target detection subroutine and entered the target sequences of TS1, TS2, TS3, TS4 and TS5. From these five targets, off-target sites with ≤3 base mismatches were selected for off-target efficiency analysis. TS5 did not have an off-target site with ≤3 base mismatches, so TS5 selected three candidate off-target sites containing 4 base mismatches. Using resistant callus genomic DNA as a template, PCR amplified potential off-target sites for high-throughput sequencing analysis. The results showed that except for one transformant detected mutation in ABE8e-EndoV in TS2-off1, no off-target sites were detected at other sites ( Figure 4 The results of ABE8e-EndoV off-target analysis also showed that the addition of the EndoV domain did not result in a high frequency of off-targets.

[0119] 8. pYL-ABE8e-EndoV-mediated precise deletion of small DNA fragments and adenine base editing pathway

[0120] In the present invention, ABE8e-EndoV plays an important role in the I:T base mismatch repair pathway. In ABE8e-EndoV editing, A is first deaminated and hydrolyzed to inosine I by adenine deaminase TadA8e, and the second phosphodiester bond at the 3' end of inosine is hydrolyzed by EndoV. After DNA repair and replication, A-to-G base substitution (56.7%) is achieved. In addition, A-to-deletions (17.3%) mutations are achieved through non-homologous end joining repair ( Figure 5 ).

[0121] In summary, the new DNA small fragment precise deletion adenine base editor pYL-ABE8e-EndoV developed by the present invention has more advantages than the ABE editing system. First, pYL-ABE8e-EndoV has broad targeting (recognition of NG-PAM). In addition, it can also achieve efficient A-to-G base substitution editing. The most outstanding advantage is that pYL-ABE8e-EndoV can achieve efficient 9-13bp DNA small fragment precise deletion at the target site. Therefore, pYL-ABE8e-EndoV is more widely applicable to cis-acting elements, miRNA coding sequences, conserved elements in intron regions, and high AT polynucleotide sequence environments. In addition, it is also suitable for gene function screening, large-scale saturation mutagenesis, and alternative splicing operations.

[0122] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An adenine base editor fusion protein ABE8e-EndoV that can achieve precise deletion of small DNA fragments, characterized in that: From N-terminus to C-terminus, it contains the nuclear localization signal bpNLS, adenine deaminase TadA8e, flexible linker sequence, SpCas9 variant SpGn, flexible linker sequence, inosine I base deletion repair key enzyme EndoV and nuclear localization signal bpNLS, and its full amino acid sequence is shown in SEQ ID NO.

5.

2. A nucleotide encoding the adenine base editor fusion protein ABE8e-EndoV that can achieve precise deletion of small DNA fragments as described in claim 1.

3. The nucleotide according to claim 2, characterized in that The nucleotide sequence is shown in SEQ ID NO.

6.

4. A plasmid vector containing the nucleotide sequence according to claim 2 or 3.

5. Use of the adenine base editor fusion protein ABE8e-EndoV capable of achieving precise deletion of small DNA fragments as described in claim 1, or the nucleotide as described in claim 2 or 3, or the plasmid vector as described in claim 4 in the preparation of an adenine base editor capable of achieving precise deletion of small DNA fragments.

6. A base editor capable of precisely deleting adenine from small DNA fragments, characterized in that: The method is prepared by constructing a nucleotide sequence encoding the adenine base editor fusion protein ABE8e-EndoV according to claim 1 onto a transformation vector.

7. The adenine base editor according to claim 6, characterized in that The transformation vector is a plant transformation vector.

8. The adenine base editor according to claim 7, characterized in that The plant transformation vector is a binary vector pYLCRISPR / Cas9Pubi-H.

9. The method for constructing a DNA base editor for precise adenine deletion of a small fragment according to any one of claims 6 to 8, characterized in that: First, prepare the complete fusion DNA sequence encoding the adenine base editor fusion protein ABE8e-EndoV that precisely deletes small DNA fragments, then insert it into the expression vector, transform the host bacteria, extract the positive plasmid, and sequence it to obtain a stable adenine base editor that precisely deletes small DNA fragments.

10. The method according to claim 9, characterized in that: For insertion into the binary vector pYLCRISPR / Cas9Pubi-H Pst I and BamH I, and obtained the adenine base editor pYL-ABE8e-EndoV.

11. Use of the DNA base editor for precise adenine deletion of small fragments according to any one of claims 6 to 8 in genome editing in an organism.

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