A highly accurate base editor that expands editing range and improves editing efficiency

Abstract

The present invention relates to the field of genetic engineering in biotechnology, and specifically to a high-precision base editor that expands the editing range and improves editing efficiency. The editor of the present invention constructs a CDA1Δ-SpRY-BE3 high-precision editor with a wider editing range and higher editing efficiency by fusing the Cas9 variant SpRY that is not restricted by PAM with the cytosine deaminase CDA1 of different truncation lengths. The editor can effectively edit any cytosine on the genome while maintaining high editing accuracy. Tests in yeast and rice cells showed that compared with the existing standard editor CDA1-SpRY-BE3, the editor of the present invention achieved a significant improvement in editing efficiency at multiple targets, proving its application value in higher eukaryotes. The present invention provides a more efficient and accurate tool for the treatment of genetic diseases and crop breeding.

Description

Technical Field

[0001] The present invention belongs to the category of nucleic acid editing technology, more specifically, it relates to the field of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, and specifically to a high-precision base editor that expands the editing range and improves editing efficiency. Background Art

[0002] In the current biotechnology field, CRISPR-mediated base editing technology has attracted much attention for its ability to achieve efficient nucleotide replacement without causing DNA double-strand breaks (DSBs) and without the need for additional repair templates. This technology has shown great application prospects in the treatment of genetic diseases caused by point mutations and in crop precision breeding. However, existing base editors have the problem of a wide editing window (for example, C4-C 17 ), within this window, not only the target bases but also non-target bases may be edited, which will inevitably introduce unexpected mutations. This non-specific editing not only poses a threat to the safety of disease treatment, but also increases the difficulty of screening in plant engineering breeding.

[0003] In response to this technical bottleneck, the preliminary research work of the present invention has made important progress based on the classic base editor CDA1-BE3. Based on the classic base editor CDA1-BE3 mediated by cytosine deaminase PmCDA1 (hereinafter referred to as CDA1) from sea lamprey (Petromyzon marinus), by truncating the carbon end of CDA1 to varying degrees (CDA1Δ) and removing its linker sequence with SpCas9 nickase, a high-precision base editor that reduces the editing window to 1-2 nucleotides was successfully developed (Nature Communications 2019, Vol. 10, Article 439; US2022 / 0154163). Although this high-precision base editor significantly improves the specificity of editing, the reduction of its editing window (from the original 7 nucleotides to 2 nucleotides) leads to a reduction in the number of editable bases, that is, the editing range is significantly reduced, which has become a limiting factor for the further development of this technology. Summary of the invention

[0004] In response to the problems such as the narrow editing range of existing precision base editors, the present invention aims to propose a base editor that expands the editing range and improves efficiency. While maintaining the high editing accuracy of the original technology, the editor of the present invention achieves a significant expansion of the editing range, so that any cytosine position in the genome can be edited efficiently and accurately. The improved base editor of the present invention significantly improves its practicality and application value in the field of genetic disease treatment and crop genetic improvement, bringing new progress and innovation to the relevant technical fields.

[0005] In order to achieve the above-mentioned invention objectives, the present invention proposes an innovative base editor optimization solution. The specific technical solution includes but is not limited to the following points:

[0006] First, in view of the limitation of the editing range of the existing high-precision base editor CDA1Δ-nCas9(D10A)-UGI (CDA1Δ-BE3), the present invention adopts an innovative strategy, that is, replacing the Cas9 protein with the reported Cas9 variant SpRY that is less dependent on the PAM sequence (Science 2020, Vol. 368, No. 6488, pp. 290-296). Through this replacement, the optimized base editor CDA1Δ-nSpRY(D10A)-UGI (CDA1Δ-SpRY-BE3) was successfully constructed, in which CDA1Δ used fragments truncated to 195 / 194 / 193 / 192 / 190 / 188 amino acids.

[0007] Wherein, the CDA1Δ195-SpRY-BE3 consists of SEQ ID NO:4 and SEQ ID NO:1 connected in sequence;

[0008] The CDA1Δ194-SpRY-BE3 consists of SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0009] The CDA1Δ193-SpRY-BE3 consists of SEQ ID NO: 6 and SEQ ID NO: 1 connected in sequence;

[0010] The CDA1Δ192-SpRY-BE3 consists of SEQ ID NO:7 and SEQ ID NO:1 connected in sequence;

[0011] The CDA1Δ190-SpRY-BE3 consists of SEQ ID NO: 8 and SEQ ID NO: 1 connected in sequence;

[0012] The CDA1Δ188-SpRY-BE3 consists of SEQ ID NO: 9 and SEQ ID NO: 1 connected in sequence.

[0013] Furthermore, under the premise of maintaining the original editing accuracy, in order to improve the editing efficiency of CDA1Δ-SpRY-BE3, the present invention screened a large number of DNA interaction factors (DNA Binding Domains, DBDs) and fused them to the nitrogen end of the optimized base editing complex CDA1Δ194-SpRY-BE3 to form a new DBDs-CDA1Δ194-SpRY-BE3 editor. The editing efficiency of various DBDs-CDA1Δ194-SpRY-BE3 editors was evaluated, and DNA interaction factors that can significantly improve the editing efficiency were screened out.

[0014] Specifically, DBDs-CDA1Δ194-SpRY-BE3 are 2xTAL-CDA1Δ194-SpRY-BE3, VP64-CDA1Δ194-SpRY-BE3, and HMGB1-CDA1Δ194-Sp respectively. RY-BE3, CHD1-CDA1Δ194-SpRY-BE3, H1-CDA1Δ194-SpRY-BE3, HMGN1-CDA1Δ194-SpRY-BE3, p65-CDA1Δ194-SpRY- BE3, HSF1-CDA1Δ194-SpRY-BE3, Oct4-CDA1Δ194-SpRY-BE3, Sox2-CDA1Δ194-SpRY-BE3, Klf4-CDA1Δ194-SpRY-B E3, cMyc-CDA1Δ194-SpRY-BE3, p53DD-CDA1Δ194-SpRY-BE3, HU-CDA1Δ194-SpRY-BE3, HLP-CDA1Δ194-SpRY-BE3.

[0015] Specifically, the 2xTAL-CDA1Δ194-SpRY-BE3 consists of SEQ ID NO: 12, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0016] The VP64-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 13, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0017] The HMGB1-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 14, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0018] The CHD1-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO:15, SEQ ID NO:5 and SEQ ID NO:1 connected in sequence;

[0019] The H1-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 16, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0020] The HMGN1-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 17, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0021] The p65-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 18, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0022] The HSF1-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 19, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0023] The Oct4-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO:20, SEQ ID NO:5 and SEQ ID NO:1 connected in sequence;

[0024] The Sox2-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO:21, SEQ ID NO:5 and SEQ ID NO:1 connected in sequence;

[0025] The Klf4-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO:22, SEQ ID NO:5 and SEQ ID NO:1 connected in sequence;

[0026] The cMyc-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO:23, SEQ ID NO:5 and SEQ ID NO:1 connected in sequence;

[0027] The p53DD-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO:24, SEQ ID NO:5 and SEQ ID NO:1 connected in sequence;

[0028] The HU-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO:25, SEQ ID NO:5 and SEQ ID NO:1 connected in sequence;

[0029] The HLP-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO:26, SEQ ID NO:5 and SEQ ID NO:1 connected in sequence.

[0030] Furthermore, the present invention screens out two or more highly efficient DBDs (such as DBD1, DBD2, etc.), then combines these DBDs in different orders (such as DBD1-DBD2 and DBD2-DBD1), and fuses them to the nitrogen terminus of the base editing complex CDA1Δ194-SpRY-BE3, and tests their editing efficiency to identify the best combination that can maximize the editing efficiency.

[0031] Among them, the DBDs-CDA1Δ194-SpRY-BE3 with two or more DBDs is 4xTAL-CDA1Δ194-SpRY-BE3, 6xTAL-CDA1Δ194-SpRY-BE3, HMGN1-VP64-CDA1Δ194-SpRY-BE3 and / or VP64-HMGN1-CDA1Δ194-SpRY-BE3.

[0032] Specifically, the 4xTAL-CDA1Δ194-SpRY-BE3 consists of SEQ ID NO: 12, SEQ ID NO: 12, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0033] The 6xTAL-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 12, SEQ ID NO: 12, SEQ ID NO: 12, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0034] The HMGN1-VP64-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0035] The VP64-HMGN1-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence.

[0036] Finally, the present invention tested the editing efficiency of the final base editor in the higher eukaryotic organism rice, and compared it with the existing base editor version Rice.CDA1-SpRY-BE3 and the improved versions Rice.CDA1Δ194-SpRY-BE3 and Rice.HMGN1-VP64-CDA1Δ194-SpRY-BE3 to verify the actual effect of the optimization scheme of the present invention in higher eukaryotic cells.

[0037] Wherein, the Rice.CDA1-SpRY-BE3 is composed of SEQ ID NO:27, SEQ ID NO:10 and SEQ ID NO:2 connected in sequence;

[0038] The Rice.CDA1Δ194-SpRY-BE3 consists of SEQ ID NO:27, SEQ ID NO:11 and SEQ ID NO:2 connected in sequence;

[0039] The Rice.HMGN1-VP64-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:11 and SEQ ID NO:2 connected in sequence.

[0040] In a specific embodiment, the base editor of the present invention also includes sgRNA or an sgRNA expression vector.

[0041] In a second aspect, the present invention also protects a recombinant vector containing the base editor described above.

[0042] In a third aspect, the present invention also protects recombinant microorganisms containing the base editor described above.

[0043] Fourthly, the present invention also protects the use of the base editor described above in the preparation of gene-edited products.

[0044] In a fifth aspect, the present invention also protects the use of the base editor contained in improving the scope and / or efficiency of gene editing for non-disease treatment purposes.

[0045] In a sixth aspect, the present invention also protects the use of the recombinant vector described above in the preparation of gene-edited products.

[0046] In a seventh aspect, the present invention also protects the use of the recombinant vector described above in improving the scope and / or efficiency of gene editing for non-disease treatment purposes.

[0047] In an eighth aspect, the present invention also protects the use of the recombinant microorganisms described above in the preparation of gene-edited products or in improving the scope and / or efficiency of gene editing for non-disease treatment purposes.

[0048] In a ninth aspect, the present invention also protects a method for preparing the base editor described above, the method comprising the following steps:

[0049] (1) Replace nCas9(D10A) with SpRY in CDA1-nCas9(D10A)-UGI;

[0050] (2) CDA1 was truncated to 195 / 194 / 193 / 192 / 190 / 188 amino acids, namely CDA1Δ195, CDA1Δ194, CDA1Δ193, CDA1Δ192, CDA1Δ190, and CDA1Δ188.

[0051] Wherein, the CDA1Δ195 sequence is shown in SEQ ID NO:4; the CDA1Δ194 sequence is shown in SEQ ID NO:5;

[0052] The CDA1Δ193 sequence is shown in SEQ ID NO:6; the CDA1Δ192 sequence is shown in SEQ ID NO:7;

[0053] The CDA1Δ190 sequence is shown in SEQ ID NO:8; the CDA1Δ188 sequence is shown in SEQ ID NO:9.

[0054] Furthermore, DNA interacting factors were screened and fused to the nitrogen terminus of the optimized base editing complex CDA1Δ194-SpRY-BE3 to form a new editor DBDs-CDA1Δ194-SpRY-BE3.

[0055] Furthermore, two or more efficient DBDs were combined in different orders and fused to the nitrogen terminus of the base editing complex CDA1Δ194-SpRY-BE3.

[0056] Ninthly, the present invention also protects a method for improving the scope and / or efficiency of gene editing for non-disease treatment purposes, wherein the method uses the base editor described above for in vitro editing.

[0057] Technical Effects

[0058] The present invention successfully constructed a series of precise base editors CDA1Δ-SpRY-BE3 with low PAM dependence by fusing the Cas9 variant SpRY, which is almost not restricted by PAM sequence, with CDA1 proteins of different lengths (i.e., CDA1Δ, where the truncated fragments are 195 / 194 / 193 / 192 / 190 / 188 amino acids, respectively). In the editing test of 9 endogenous non-NGG PAM sites in the model eukaryotic organism Saccharomyces cerevisiae, the following experimental results were obtained (see Figure 1 ).

[0059] Compared with the traditional base editor CDA1-BE3 that does not use Cas9 variants, CDA1-BE3 showed almost no editing activity in all the test sites. In contrast, the base editor CDA1Δ-SpRY-BE3 of the present invention not only showed significant editing efficiency, with an editing efficiency of more than 50% in specific test sites, but also maintained the original editing accuracy (the editing window is strictly limited to 1-2 nucleotides; it mainly targets the 18th cytosine upstream of the PAM sequence, i.e., C -18 ), while achieving a significant expansion of the editing range. The editing range has been greatly expanded from specific sites containing NGG (N represents any base) PAM sequences to a wide range of sites covering almost any NNN PAM sequence, thereby greatly enhancing the application potential and practical scope of the editor of the present invention.

[0060] The base editor CDA1Δ-SpRY-BE3 containing truncated CDA1 showed varying degrees of improvement in editing efficiency compared to the full-length control group, with the highest increase reaching 6.66 times, fully demonstrating the significant advantages of the technology of the present invention in improving editing efficiency.

[0061] In order to further verify the expansion capability of the CDA1Δ-SpRY-BE3 base editor in terms of editing range, this study tested its ability to precisely edit any cytosine base C in the genome. The operation of the editing site followed a simple four-step design process:

[0062] (1) Determine the target cytosine base C and its flanking sequence;

[0063] (2) The target cytosine base C is located at the 18th position upstream of the PAM sequence, based on which the spacer sequence and PAM sequence are determined, and the corresponding sgRNA is designed;

[0064] (3) Select a matching base editor based on the PAM sequence;

[0065] (4) By testing different combinations of CDA1 truncated versions, the combination that achieves the best precision editing effect is screened out. The simplicity and effectiveness of the method of the present invention are verified by editing a randomly selected endogenous target site containing three consecutive cytosines (C) in Saccharomyces cerevisiae (see Figure 2). In addition, using a screening platform based on the yeast endogenous marker gene Can1, the precision editing ability of the editor of the present invention at non-NGG PAM target sites has also been confirmed (see Figure 2 ). These experimental results consistently show that the CDA1Δ-SpRY-BE3 base editor has broad and precise editing capabilities.

[0066] Using whole genome sequencing technology, the present invention comprehensively evaluated the DNA off-target effects of the CDA1Δ-SpRY-BE3 base editor across the genome. Experimental data showed that compared with CDA1-BE3 and CDA1-SpRY-BE3, the use of a truncated version of the deaminase significantly reduced DNA off-target events across the genome. In particular, when the CDA1 protein was truncated to 188 amino acids, the observed off-target rate was almost reduced to the level of the control group (see Figure 3 ). This result fully demonstrates the remarkable effectiveness of the technology of the present invention in improving editing specificity and reducing off-target risks, and further enhances its safety and reliability in precise gene editing applications.

[0067] In order to enhance the interaction between base editors and DNA molecules, and thus enhance the editing efficiency of CDA1Δ-SpRY-BE3 base editors, the present invention screened out two proteins that can independently improve the editing efficiency from a variety of DNA binding proteins, namely HMGN1 and VP64 (see Figure 4 ). Fusing these two proteins to the amino terminus of CDA1Δ194-SpRY-BE3 not only significantly improved the editing efficiency, but also maintained the original accuracy of the editing process.

[0068] The present invention further optimized the structure of the base editor CDA1Δ-SpRY-BE3 by simultaneously fusing two DNA binding proteins, HMGN1 and VP64, to its amino terminus, with HMGN1 located at the amino terminus of the fusion protein (forming an HMGN1-VP64 structure), thus constructing a HMGN1-VP64-CDA1Δ-SpRY-BE3 composite structure. This structural optimization strategy greatly improved the editing efficiency. Experimental results showed that the editing efficiency could be increased by up to 23 times compared with the original editor (see Figure 5 ). Importantly, this structural optimization does not cause any damage to the original high editing accuracy of the editor, ensuring that the editing process maintains a high degree of accuracy while improving efficiency.

[0069] In order to further verify the enhanced effect of the HMGN1-VP64-CDA1Δ-SpRY-BE3 composite base editor on editing efficiency, the present invention implemented a detailed test scheme. The scheme involves editing tests on 36 endogenous targets of Saccharomyces cerevisiae, and CDA1Δ-SpRY-BE3, CDA1Δ-SpRY-BE3 fused with HMGN1 alone (HMGN1-CDA1Δ-SpRY-BE3), and CDA1Δ-SpRY-BE3 fused with VP64 alone (VP64-CDA1Δ-SpRY-BE3) are used as control groups. The experimental results show that among the 36 targets tested, HMGN1-VP64-CDA1Δ-SpRY-BE3 achieved an average 4-fold increase in editing efficiency compared with CDA1Δ-SpRY-BE3, which significantly demonstrates the ability of the present invention technology to improve editing efficiency.

[0070] In order to evaluate the performance of the improved base editor HMGN1-VP64-CDA1Δ-SpRY-BE3 in the present invention in more complex eukaryotic cells, its editing ability was tested in rice cells. As a control, the editing effects of the existing standard base editor CDA1-SpRY-BE3 and its truncated version CDA1Δ194-SpRY-BE3 were also investigated. Experimental data show that among the five rice target sites tested, compared with the existing standard base editor CDA1-SpRY-BE3, the two improved versions of the present invention, CDA1Δ194-SpRY-BE3 and HMGN1-VP64-CDA1Δ-SpRY-BE3, both showed an improvement in editing efficiency, especially the HMGN1-VP64-CDA1Δ-SpRY-BE3 version, which was more significantly improved, with an editing efficiency increased by 1.7 to 8.3 times (see Figure 7 ). These results fully demonstrate the application potential of the improved base editor of the present invention in higher eukaryotes and the significant improvement of its performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Schematic diagram of the base editor vector that fuses nSpRY and truncated cytosine deaminase CDA1Δ, where CDA1-BE3 is a traditional cytosine base editor that can only recognize NGG PAM; nSpCas9 is replaced with nSpRY that recognizes more types of PAM in CDA1-SpRY-BE3; CDA1Δ-SpRY-BE3 is a series of base editors obtained by truncating the carbon terminus of CDA1 in the structure and removing the XTEN linker between cytosine deaminase and nSpRY.

[0072] Figure 2Achieve precise base editing of CDA1Δ-SpRY-BE3 at non-NGG PAM target sites.

[0073] Figure 3 Achieve precise editing of any C in multiple C sites on the genome for CDA1Δ-SpRY-BE3; Figure 3 Figure a shows the design strategy for editing any cytosine in the genome. Figure 3 Figure b shows the design strategy of Figure a, which selects three sgRNAs with three consecutive cytosine sites in yeast genomic DNA for precise editing. Figure 3 Figure c in the figure is the test result of Figure b; Figure 3 Figure d shows the results of drug screening tests.

[0074] Figure 4 The DNA off-target status of the candidate base editors in the whole genome; Figure 4 Figure a shows the insertion and deletion situation in the whole genome. Figure 4 Figure b shows the nucleotide variation in the whole genome. Figure 4 Figure c shows the specific categories of single nucleotide variations.

[0075] Figure 5 Improve the editing efficiency of CDA1Δ194-SpRY-BE3 for the DNA-binding factors HMGN1 and VP64.

[0076] Figure 6 Co-fusion of HMGN1 and VP64 to the N-terminus of CDA1Δ-SpRY-BE3 significantly improved the editing efficiency.

[0077] Figure 7 This is a general test of the efficiency improvement achieved by fusing HMGN1 and VP64 to the N-terminus of CDA1Δ194-SpRY-BE3.

[0078] Figure 8 For the application of related strategies in rice. DETAILED DESCRIPTION

[0079] The present invention is further described in detail below with reference to the examples. The reagents or instruments used without indicating the manufacturer are all regarded as conventional products that can be purchased on the market.

[0080] The present invention provides a series of base editors, wherein the base editors contain one or more of the following: CDA1-SpRY-BE3, CDA1Δ195-SpRY-BE3, CDA1Δ194-SpRY-BE3, CDA1Δ193-SpRY-BE3, CDA1Δ192-SpRY-BE3, CDA1Δ190-SpRY-BE3, CDA1Δ188-SpRY-BE3, 2xTAL-CDA1Δ19 4-SpRY-BE3, 4xTAL-CDA1Δ194-SpRY-BE3, 6xTAL-CDA1Δ194-SpRY-BE3, VP64-CDA1Δ194-SpRY-BE3, HMG B1-CDA1Δ194-SpRY-BE3, CHD1-CDA1Δ194-SpRY-BE3, H1-CDA1Δ194-SpRY-BE3, HMGN1-CDA1Δ194-SpRY-B E3, p65-CDA1Δ194-SpRY-BE3, HSF1-CDA1Δ194-SpRY-BE3, Oct4-CDA1Δ194-SpRY-BE3, Sox2-CDA1Δ194- SpRY-BE3, Klf4-CDA1Δ194-SpRY-BE3, cMyc-CDA1Δ194-SpRY-BE3, p53DD-CDA1Δ194-SpRY-BE3, HU-CDA 1Δ194-SpRY-BE3, HLP-CDA1Δ194-SpRY-BE3, HMGN1-VP64-CDA1Δ194-SpRY-BE3, VP64-HMGN1-CDA1Δ194 -SpRY-BE3, Rice.CDA1-SpRY-BE3, Rice.CDA1Δ194-SpRY-BE3, Rice.HMGN1-VP64-CDA1Δ194-SpRY-BE3.

[0081] Wherein, the CDA1-SpRY-BE3 is composed of SEQ ID NO:3 and SEQ ID NO:1 connected in sequence;

[0082] The CDA1Δ195-SpRY-BE3 consists of SEQ ID NO:4 and SEQ ID NO:1 connected in sequence;

[0083] The CDA1Δ194-SpRY-BE3 consists of SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0084] The CDA1Δ193-SpRY-BE3 consists of SEQ ID NO: 6 and SEQ ID NO: 1 connected in sequence;

[0085] The CDA1Δ192-SpRY-BE3 consists of SEQ ID NO:7 and SEQ ID NO:1 connected in sequence;

[0086] The CDA1Δ190-SpRY-BE3 consists of SEQ ID NO: 8 and SEQ ID NO: 1 connected in sequence;

[0087] The CDA1Δ188-SpRY-BE3 consists of SEQ ID NO:9 and SEQ ID NO:1 connected in sequence;

[0088] The 2xTAL-CDA1Δ194-SpRY-BE3 consists of SEQ ID NO: 12, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0089] The 4xTAL-CDA1Δ194-SpRY-BE3 consists of SEQ ID NO: 12, SEQ ID NO: 12, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0090] The 6xTAL-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 12, SEQ ID NO: 12, SEQ ID NO: 12, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0091] The VP64-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 13, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0092] The HMGB1-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 14, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0093] The CHD1-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO:15, SEQ ID NO:5 and SEQ ID NO:1 connected in sequence;

[0094] The H1-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 16, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0095] The HMGN1-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 17, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0096] The p65-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 18, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0097] The HSF1-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 19, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0098] The Oct4-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO:20, SEQ ID NO:5 and SEQ ID NO:1 connected in sequence;

[0099] The Sox2-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO:21, SEQ ID NO:5 and SEQ ID NO:1 connected in sequence;

[0100] The Klf4-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO:22, SEQ ID NO:5 and SEQ ID NO:1 connected in sequence;

[0101] The cMyc-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO:23, SEQ ID NO:5 and SEQ ID NO:1 connected in sequence;

[0102] The p53DD-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO:24, SEQ ID NO:5 and SEQ ID NO:1 connected in sequence;

[0103] The HU-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO:25, SEQ ID NO:5 and SEQ ID NO:1 connected in sequence;

[0104] The HLP-CDA1Δ194-SpRY-BE3 consists of SEQ ID NO:26, SEQ ID NO:5 and SEQ ID NO:1 connected in sequence;

[0105] The HMGN1-VP64-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0106] The VP64-HMGN1-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence;

[0107] The Rice.CDA1-SpRY-BE3 is composed of SEQ ID NO:27, SEQ ID NO:10 and SEQ ID NO:2 connected in sequence;

[0108] The Rice.CDA1Δ194-SpRY-BE3 consists of SEQ ID NO:27, SEQ ID NO:11 and SEQ ID NO:2 connected in sequence;

[0109] The Rice.HMGN1-VP64-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:11 and SEQ ID NO:2 connected in sequence.

[0110] The YPDA medium in the following examples is prepared from 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, 0.12 g / L adenine hemisulfate and water, and the solid medium needs to be supplemented with 15 g / L agarose.

[0111] The defective culture medium in the following examples is prepared by 6.7 g / L YNB, 20 g / L glucose, an appropriate amount of SC defective amino acid mixture lacking uracil and leucine (SC-LU) and water, and the solid culture medium needs to be supplemented with 15 g / L agarose.

[0112] Example 1 Construction of vector

[0113] 1. Construction method of fusion protein vector:

[0114] (1) Design the primers required for constructing the vector, as shown in Table 1;

[0115] (2) PCR amplification using Phanta Max Super-Fidelity DNA Polymerase (Vazyme), corresponding primer pairs and DNA template;

[0116] (3) Use restriction endonucleases to digest the plasmid vector as the backbone. The reaction conditions are: 37°C, 4h;

[0117] (4) The PCR amplification products and enzyme digestion products were identified by agarose gel electrophoresis for fragment size, and the gel was cut and recovered;

[0118] (5) Use the OK Clon DNA Ligation Kit II (Accurate) to seamlessly clone and connect the purified linear vector and fragment;

[0119] (6) Transform into E. coli DH5α competent cells, spread onto resistant LB medium, and select single clones for sequencing verification;

[0120] (7) Transfer the correctly sequenced colonies to 5 mL of liquid resistant LB medium and culture at 37°C with shaking at 225 rpm for 12-18 hours;

[0121] (8) Use the OMEGA plasmid miniprep kit to extract the plasmid.

[0122] 2. GoldenGate method to construct all gRNA expression vectors:

[0123] (1) Design the primers required for constructing the vector, as shown in Table 2;

[0124] (2) Mix the corresponding primer pairs in a 1:1 ratio, heat to 95°C, and then gradually anneal to 25°C to obtain primer annealing products;

[0125] (3) Use class IIs restriction endonuclease and T4 DNA ligase (NEB) to prepare a working system and mix them. Add the primer annealing product and vector backbone into the system for GoldenGate ligation. The reaction conditions are: 37°C, 2 min; 16°C, 30 s, 37°C, 5 min after 50 cycles, and 85°C 5 min to terminate the reaction.

[0126] (4) The GoldenGate reaction product was transformed into E. coli DH5α competent cells, spread on resistant LB medium, and single clones were selected for sequencing verification;

[0127] (5) Transfer the correctly sequenced colonies to 5 mL of liquid resistant LB medium and culture at 37°C with shaking at 225 rpm for 12-18 hours;

[0128] (6) Use the OMEGA plasmid miniprep kit to extract the plasmid.

[0129] 3. Construction by T4 DNA ligase method Figure 5 and Figure 6 Medium carrier:

[0130] (1) Design the primers required for constructing the vector, as shown in Table 1;

[0131] (2) PCR amplification using Phanta Max Super-Fidelity DNA Polymerase (Vazyme), corresponding primer pairs and DNA template;

[0132] (3) Use restriction endonucleases to digest the plasmid vector used as the backbone and the PCR amplification product obtained in the previous step. The reaction conditions are: 37°C, 4h;

[0133] (4) The size of the digested products was determined by agarose gel electrophoresis, and the fragments were cut and recovered;

[0134] (5) Use T4 DNA ligase (NEB) to connect the purified linear vector and fragment. The reaction conditions are: 22°C, 2h;

[0135] (6) Transform into E. coli DH5α competent cells, spread onto resistant LB medium, and select single clones for sequencing verification;

[0136] (7) Transfer the correctly sequenced colonies to 5 mL of liquid resistant LB medium and culture at 37°C with shaking at 225 rpm for 12-18 hours;

[0137] (8) Use the OMEGA plasmid miniprep kit to extract the plasmid.

[0138]

[0139] The specific construction process of each expression vector is as follows:

[0140] (A1) Construction of CDA1-SpRY-BE3 vector

[0141] The primer sets for constructing CDA1-SpRY-BE3 in Table 1 were used, wherein the four primer sets CDA1-SpRY-BE3-F1 / R1 to F4 / R4 were used to perform PCR amplification with pJT45-GalL-CDA1-BE3 (Addgene, Plasmid #145038) as templates, and the obtained fragments were ligated with pJT45-GalL-CDA1-BE3 digested with restriction endonucleases NruI / NcoI to obtain the intermediate vector CDA1-SpRY-BE3a with D1135L / S1136W / G1218K / E1219Q / R1335Q / T1337R mutations; and the two primer sets CDA1-SpRY-BE3-F5 / R5 and F6 / R6 were used to perform PCR amplification with the intermediate vector CDA1-SpRY-BE3a as templates, and the fragments were ligated with restriction endonucleases SpeI / NdeI. The digested CDA1-SpRY-BE3a was connected to obtain the intermediate vector CDA1-SpRY-BE3b with A61R mutation; finally, PCR amplification was performed using two sets of primers CDA1-SpRY-BE3-F7 / R7 to F8 / R8 with CDA1-SpRY-BE3b as a template, and the obtained fragment was connected with CDA1-SpRY-BE3b digested with restriction endonucleases NruI / NcoI to obtain CDA1-SpRY-BE3 vector, such as Figure 1 shown.

[0142] (A2) Construction of CDA1Δs-SpRY-BE3 series vectors

[0143] The primer set for constructing CDA1Δs-SpRY-BE3 in Table 1 was used, wherein CDA1Δs-SpRY-BE3-F1 / R1 was amplified by PCR using CDA1-SpRY-BE3 prepared in Example 1 as a template to obtain fragment 1; using the same primer set, CDA1Δs-SpRY-BE3-F was used as a forward primer, CDA1Δ195-SpRY-BE3-R, CDA1Δ194-SpRY-BE3-R, CDA1Δ192-SpRY-BE3-R, CDA1Δ190-SpRY-BE3-R, CDA1Δ188-SpRY-BE3-R were used as reverse primers, and CDA1Δ prepared in Example 1 was used as a template to amplify CDA1Δ of corresponding length fragments, and different CDA1Δ and fragment 1 were co-incubated with restriction endonucleases SpeI / SbfI The digested CDA1-SpRY-BE3 can be ligated to obtain CDA1Δs-SpRY-BE3 series vectors, such as Figure 1 shown.

[0144] (A3) Construction of VP64-HMGN1-CDA1Δ194-SpRY-BE3

[0145] (1) The primer set for constructing Factors-CDA1Δ194-SpRY-BE3 in Table 1 was used, wherein CDA1Δ194-SpRY-BE3-F1 / R(AvrII) was amplified by PCR using CDA1Δ194-SpRY-BE3 prepared in Example 2 as a template to obtain a fragment, and the fragment was amplified by PCR using the primer pair CDA1Δ194-SpRY-BE3-F2 / R(AvrII) as a template to obtain a fragment having an AvrII restriction site, which was then ligated with CDA1Δ194-SpRY-BE3 digested with restriction endonucleases SpeI / SbfI to obtain CDA1Δ194-SpRY-BE3(AvrII).

[0146] (2) 2xTAL-F, VP64-F, HMGB1-F, CHD1-F, H1-F, HMGN1-F, p65-F, HSF1-F, Oct4-F, Sox2-F, Klf4-F, cMyc-F, p53DD-F, HU-F, and HLP-F were grouped with the universal reverse primer Factor-R, and PCR amplification was performed using the synthesized corresponding DNA fragments as templates to obtain the corresponding fragments; the amplified fragments digested with restriction endonucleases AvrII / NheI were ligated with CDA1Δ194-SpRY-BE3 (AvrII) digested with restriction endonucleases AvrII to obtain 2xTAL-CDA1Δ194-SpRY-BE3, VP64-CDA1Δ194-SpRY-BE3, HMGB1-CDA1Δ194- SpRY-BE3, CHD1-CDA1Δ194-SpRY-BE3, H1-CDA1Δ194-SpRY-BE3, HMGN1-CDA1Δ194-SpRY-BE3, p65-CDA1Δ194-SpRY-BE3, HSF1-CDA1Δ194-SpRY-BE3, Oct4-CDA1Δ194-SpRY-BE3, Sox2-CDA1Δ194-SpRY-BE3, Klf4-CDA1Δ194-SpRY-BE3, cMyc-CDA1Δ194-SpRY-BE3, p53DD-CDA1Δ194-SpRY-BE3, HU-CDA1Δ194-SpRY-BE3, HLP-CDA1Δ194-SpRY-BE3, a total of 15 single factor vectors.

[0147] (3) The 2xTAL fragment digested with restriction endonucleases AvrII / NheI was ligated with 2xTAL-CDA1Δ194-SpRY-BE3 digested with restriction endonucleases AvrII to obtain 4xTAL-CDA1Δ194-SpRY-BE3; the 2xTAL fragment digested with restriction endonucleases AvrII / NheI was ligated with 4xTAL-CDA1Δ194-SpRY-BE3 digested with restriction endonucleases AvrII / NheI to obtain 6xTAL-CDA1Δ194-SpRY-BE3; The HMGN1 fragment digested with restriction endonuclease AvrII / NheI is connected with VP64-CDA1Δ194-SpRY-BE3 digested with restriction endonuclease AvrII to obtain HMGN1-VP64-CDA1Δ194-SpRY-BE3; the VP64 fragment digested with restriction endonuclease AvrII / NheI is connected with HMGN1-CDA1Δ194-SpRY-BE3 digested with restriction endonuclease AvrII to obtain VP64-HMGN1-CDA1Δ194-SpRY-BE3.

[0148] (A4) Construction of Rice.Fusion-CDA1Δ194-SpRY-BE3

[0149] (1) Using the primer set for constructing the rice editor in Table 1, PCR amplification was performed using Rice.CDA1-SpRY-BE3-F1 / R1 with the synthetic rice codon-optimized SpRY fragment (SEQ ID NO: 29) as a template to obtain fragment 1; PCR amplification was performed using Rice.CDA1-SpRY-BE3-F2 / R2 with the synthetic rice codon-optimized 2xUGI fragment (SEQ ID NO: 30) as a template, and the obtained fragment and fragment 1 were PCR amplified with pH-A3A-PBE (Addgene, Plasmid) digested with restriction endonucleases MluI / SacI. #119774) to obtain the intermediate vector Rice.CDA1-SpRY-BE3a containing 2xUGI and ten point mutations including L111R / D1135L / S1136W / G1218K / E1219Q / N1317R / A1322R / R1333P / R1335Q / T1337R.

[0150] (2) Using Rice.CDA1-SpRY-BE3-F3 / R3 and the synthetic rice codon-optimized PmCDA1 (SEQ ID NO: 10) as a template for PCR amplification to obtain fragment 2; using Rice.CDA1-SpRY-BE3-F4 / R4 and the synthetic rice codon-optimized SpRY fragment as a template, the obtained fragment and fragment 2 are connected with Rice.CDA1-SpCas9-BE3a digested with restriction endonucleases AvrII / SbfI to obtain vector Rice.CDA1-SpRY-BE3; using Rice.CDA1Δ194-SpRY-BE3-F1 / R1 to F3 / R3 primer pairs, PCR amplification is performed respectively with Rice.CDA1-SpRY-BE3 as a template, and the three obtained fragments are connected together with Rice.CDA1-SpRY-BE3 digested with restriction endonucleases BssHII / SbfI to obtain Rice.CDA1Δ194- SpRY-BE3; use two pairs of primers Rice.Fusion-CDA1Δ194-SpRY-BE3-F1 / R1 and Rice.Fusion-CDA1Δ194-SpRY-BE3-F3 / R3 to perform PCR amplification with Rice.CDA1Δ194-SpRY-BE3 as template to obtain fragment 3 and fragment 4, use Rice.Fusion-CDA1Δ194-SpRY-BE3-F2 / R2 to perform PCR amplification with the synthetic rice codon-optimized HMGN1-VP64 fragment as template, and connect the three fragments together with fragments 3 and 4 to Rice.CDA1Δ194-SpRY-BE3 digested with restriction endonucleases NcoI / SbfI to obtain Rice.Fusion-CDA1Δ194-SpRY-BE3.

[0151] (A5) Construction of gRNA expression vector

[0152] (1) Use the primer set for constructing the gRNA expression vector backbone in Table 2, use sgRNA-scaffold-F1 / R1 to perform PCR amplification with the ccdB expression frame as a template, and use the obtained fragment as a template to perform PCR amplification with the sgRNA-scaffold-F2 / R1 primer pair. The obtained fragment is ligated with pJT303-SNR52-sgRNA-Can1-3 digested with restriction endonucleases AatII / KpnI to obtain the gRNA expression vector backbone.

[0153] (2) Use the primer set for constructing gRNA expression vector in Table 2. Take PolyC-1-NGN as an example for all yeast gRNA expression vectors. Use the primer PolyC-1-NGN-F / R in Table 2 for annealing. The annealing product is mixed with the gRNA expression vector backbone. Use restriction endonuclease AarI and T4 DNA ligase for Golden Gate ligation to obtain the corresponding gRNA expression vector.

[0154] (3) Use the primer set for constructing a series of rice target vectors in Table 2. All rice gRNA expression frames are on the rice editor. Take the construction of the CDA1-SpRY-BE3 vector targeting rice target 1 as an example. Use the primers Rice.site1-F / R in Table 2 for annealing. The obtained annealing product is mixed with CDA1-SpRY-BE3 and ligated with restriction endonuclease BsaI and T4 DNA ligase Golden Gate to obtain the CDA1-SpRY-BE3 vector targeting rice target 1.

[0155]

[0156] Example 2 Transformation and induction of Saccharomyces cerevisiae and high-throughput sequencing analysis

[0157] In order to detect the editing status of the editor in yeast, this example uses the above-mentioned (A1) and (A2) vectors and the sgRNA vector in (A5) to co-transform and induce in yeast. After DNA is extracted, the editing efficiency is obtained by high-throughput sequencing of the target fragments, and analysis and visualization are performed. The specific method is as follows:

[0158] 1. Yeast transformation:

[0159] (1) Saccharomyces cerevisiae BY4743 was cultured on YPDA medium at 28°C for 2-3 days;

[0160] (2) Wash and collect the yeast cells with sterile ddH2O, add 100 mM LiAc, and incubate at 28°C for 10 min.

[0161] (3) After centrifugation for 5 seconds, the supernatant was removed, and the cells were mixed with 0.5-1 μg of the plasmid DNA obtained in Example 1, 240 μL of 50% PEG3350, 36 μL of 1M LiAc, 50 μL of 2 mg / mL salmon sperm DNA, and 20 μL of sterile water in a centrifuge tube and incubated at 42°C for 1.5 h;

[0162] (4) After centrifugation for 5 seconds, remove the supernatant and culture on defective solid medium SC-LU (yeast synthetic medium lacking uracil and leucine) at 28°C for 2-3 days;

[0163] (5) Pick a single clone and culture it in defective liquid culture medium SC-LU at 28°C and 225 rpm for 18-20 hours, and confirm the positivity by PCR amplification.

[0164] 2. Yeast induction:

[0165] (1) Pick 3-5 positive colonies and culture them in 3 mL of defective liquid medium SC-LU containing 2% glucose at 28°C for 18-20 hours;

[0166] (2) Pipette 0.8 mL of bacterial solution, centrifuge and discard the supernatant, wash three times with sterile water to remove residual glucose, then resuspend in 5 mL SC-LU liquid induction medium containing 2% galactose and 1% raffinose, and culture at 28°C, 225 rpm for 20 h;

[0167] (3) Pipette 0.5 mL of bacterial solution, centrifuge briefly and discard the supernatant to obtain the induced bacteria.

[0168] 3. Yeast DNA extraction:

[0169] (1) Add yeast cell lysis solution to the induced bacteria, resuspend the yeast cells and treat at 70°C for 10 min;

[0170] (2) Add three times the volume of anhydrous ethanol (Shanghai test) to the treated resuspension, shake and mix, centrifuge briefly, and discard the supernatant to obtain a precipitate containing yeast DNA;

[0171] (3) Wash the precipitate with 70% ethanol and dry it at room temperature;

[0172] (4) Add sterile water to the dried precipitate to obtain yeast DNA.

[0173] 4. Construction and analysis of next-generation sequencing libraries:

[0174] (1) PCR amplification was performed using Phanta Max Super-Fidelity DNA Polymerase, corresponding primer pairs and genomic DNA as templates, wherein the primers were a combination of the second-generation sequencing adapters in Table 3 and the amplification sequences in Table 4;

[0175] (2) PCR products were purified using the Cycle Pure kit (OMEGA);

[0176] (3) The purified products were subjected to PCR-free library construction, high-throughput sequencing, and data analysis (Boxon Biotechnology, Beijing, China). Sequencing was performed using the Illumina NovaSeq 6000 platform.

[0177] (4) On average, more than 100,000 reads were obtained for each sample. After data filtering, the FASTQ files were analyzed using the https: / / github.com / zfcarpe / Cas9Sequencing script;

[0178] (5) Use vector graphics tools GraphPad Prism 8 and Adobe Illustrator to draw vector graphics.

[0179]

[0180]

[0181] The results are as follows Figure 2 As shown, among all NGN, NAN, NTN, and NCN target sites with non-NGG PAMs, the editing efficiency of CDA1-BE3 that traditionally recognizes NGG PAMs is less than 1% for C > T in these sites;

[0182] CDA1-SpRY-BE3 was constructed by replacing nSpCas9 with nSpRY. It showed 5%-40% C > T editing efficiency in 4 targets with PAMs of NGN and NAN. Among 5 targets with PAMs of NCN and NTN, except for PolyC-7-NCN which showed 22%-27% editing efficiency, the remaining 4 targets showed 1.2%-3.3% C > T editing efficiency. In targets of all PAM types, the editing window was between C-19 and C-15.

[0183] The carbon terminus of CDA1 was further truncated and the XTEN linker was removed to construct a series of CDA1Δ-SpRY-BE3. Among all PAM type targets, this editor design strategy increased the C > T editing efficiency by at least 1.3 times based on CDA1-SpRY-BE3, as shown by PolyC-8-NCN, and at most 6.1 times, as shown by PolyC-6-NTN. In addition, except for PolyC-3-NAN, whose editing window was within the 2-nt range of C-19 and C-18, the editing window of this design strategy in all targets was precisely positioned at C-18.

[0184] Example 3 Canavanine resistance experiment and off-target test

[0185] In order to verify whether CDA1Δ-SpRY-BE3 can achieve precise editing of any C in the poly-C sites on the genome; the present invention designs a strategy for editing any cytosine in the genome, which is divided into four steps: first, the cytosine site to be edited in the genome must be determined; according to the determined cytosine site, it is designed to be located at the -18 position of the gRNA to determine the PAM sequence required by the gRNA; according to the different PAM sequences, for example, when the PAM is a non-standard NHN sequence, CDA1Δ-SpRY-BE3 is selected for editing to efficiently and directionally obtain products containing only the target cytosine editing ( Figure 3 (a in Figure 1);

[0186] Figure 3 Figure b in the figure selects a site with three consecutive cytosines in the yeast genomic DNA based on the strategy described in Figure a. In order to accurately edit the three cytosines respectively, three different sgRNAs are designed for subsequent testing; this part of the testing method is the same as that in Example 2.

[0187] Test results such as Figure 3 As shown in Figure c, when different cytosines are located at C-18, the cytosine at this site shows the highest C > T editing efficiency among the three cytosines; and taking cytosine C1 as an example, in the base editing process guided by sgRNA-C1, the proportion of the editing product T1C2C3 in all editing products has been significantly improved compared with CDA1-BE3 and CDA1-SpRY-BE3, proving that this target strategy and the aforementioned base editor design strategy can achieve effective and precise C>T editing of any cytosine in the genome;

[0188] In order to further eliminate the influence of the second-generation sequencing library construction process on the editing results, two consecutive cytosines were selected on the canavanine-sensitive gene Can1 for drug screening test, such as Figure 3 As shown in Figure d; when the sgRNA designed for C1 and C2 guides the test editor to perform C > T editing on the site simultaneously or separately, the termination codons TGA, TAG or TAA will be generated on Can1, resulting in canavanine resistance in Saccharomyces cerevisiae; further genotype analysis of the canavanine-resistant strains produced by different editors found that with the gradual truncation of cytosine deaminase CDA1, the proportion of target mutant strains in all mutant strains gradually increased, which was consistent with the results of second-generation sequencing.

[0189] Among them, the specific steps of the canavanine resistance experiment and off-target test are as follows:

[0190] (1) Transform and culture yeast using the selected Can1-1 / Can1-2 gRNA expression vector and candidate base editors.

[0191] (2) Simultaneously culturing a group of blank controls without any expression vector;

[0192] (3) Subsequently, the cells were transferred to a liquid induction medium containing 2% galactose and 1% raffinose and cultured at 28°C with shaking at 225 rpm for 20 h.

[0193] (4) Dilute the bacterial solution 10,000 times, spread the blank control on YPDA medium, and spread the remaining bacterial solution on SC-Arg solid medium containing 60 μg / mL L-canavanine, and culture at 28°C for 2-3 days;

[0194] (5) Colonies were picked from each culture dish and cultured in YPDA liquid medium at 28°C and 225 rpm for 20 h. Some colonies were selected for single clone sequencing.

[0195] (6) Take 0.5-1 mL of bacterial solution and use the yeast genomic DNA extraction kit (Solarbio) to extract yeast genomic DNA;

[0196] (7) Perform quality assessment on the extracted DNA samples and perform library construction, whole genome sequencing and bioinformatics analysis.

[0197] Figure 4 The DNA off-target status of the candidate base editing tool in the whole genome; Figure 4 Figure a shows the insertion and deletion situation across the entire genome. Taking untreated Saccharomyces cerevisiae as the blank control, all base editing tools showed insertion and deletion situations consistent with the blank control, indicating that CDA1-mediated base editing tools rarely or basically do not produce insertion and deletion in yeast editing.

[0198] Figure 4 Figure b shows the single nucleotide variation in the whole genome. Compared with the blank control, CDA1-BE3 as a negative control caused more single nucleotide variation in the whole genome; the single nucleotide variation type off-target of CDA1-SpRY-BE3 that replaced the nuclease increased further; with the truncation of CDA1, this type of off-target situation gradually decreased, proving that the CDA1Δ-SpRY-BE3 strategy can significantly reduce the off-target situation in the whole genome.

[0199] Figure 4 Figure c shows the specific categories of single nucleotide variations. As shown in the figure, most of the single nucleotide variations are of the type C>T / G>A, confirming that off-target effects throughout the genome are mainly caused by deaminases.

[0200] Example 4 Effects of DNA Interaction Factors on CDA1Δ-SpRY-BE3 Base Editor

[0201] 1. Under the premise of maintaining the original editing accuracy, in order to improve the editing efficiency of CDA1Δ-SpRY-BE3, the present invention screened 15 DNA interaction factors of four major categories, namely TAF, CAF, PTF and DBP, in two test sites respectively, and fused them to the nitrogen end of the optimized base editing complex CDA1Δ194-SpRY-BE3 to form a new editor DBDs-CDA1Δ194-SpRY-BE3, and tested the changes in editing efficiency and editing window.

[0202] The results are as follows Figure 5 As shown, both VP64 in TAF and HMGN1 in CAF fused to the N-terminus of CDA1Δ194-SpRY-BE3 exhibited optimal C>T editing efficiency, and the editing window was not significantly broadened.

[0203] 2. Further Figure 5 The two DNA binding factors HMGN1 and VP64 were combined and fused to the nitrogen end of CDA1Δ194-SpRY-BE3 in an arranged manner; the test results showed that the fusion proteins could significantly improve the C > T editing efficiency in the test site, and the editing window was only 2-nt, and the highest editing efficiency was still at C-18 ( Figure 6 ).

[0204] The efficiency improvement by fusing HMGN1 and VP64 to the N-terminus of CDA1Δ194-SpRY-BE3 was universally tested; the results are shown in Figure 7 As shown, the improvement of editing efficiency by single VP64 fusion is universal, and the additional fusion of HMGN1 can increase the editing efficiency to a higher level, about 2 times on average and up to 15 times.

[0205] Example 5 Application of base editor construction strategy in rice

[0206] In higher eukaryotic organism rice, the editing efficiency of the base editor of the present invention is tested, and compared with the existing base editor version CDA1-SpRY-BE3 and the improved version CDA1Δ194-SpRY-BE3 in step (1), wherein the base editor in rice requires Agrobacterium transformation and rice callus transformation, and then after DNA extraction, the editing efficiency is obtained by high-throughput sequencing of the target fragment, analysis and visualization. The specific methods of Agrobacterium transformation, rice callus transformation, and DNA extraction are as follows:

[0207] 1. Agrobacterium transformation:

[0208] (1) Place the Agrobacterium competent cells in a 37°C water bath and culture for 5 minutes;

[0209] (2) Figure 8 The corresponding vector plasmid DNA was added to the Agrobacterium competent cells, and then placed in an ice bath for 30 min, liquid nitrogen for 5 min, 37 °C water bath for 5 min, and ice bath for 5 min.

[0210] (3) Add 700 μL LB liquid medium (without antibiotics) and culture at 28°C with shaking at 225 rpm for 2–3 h.

[0211] (4) Take out the bacterial solution, centrifuge at 6000 rpm for 1 min, keep 100 μL of the supernatant, and resuspend the bacterial mass;

[0212] (5) Spread the bacterial solution on a resistant culture medium plate containing 50 mg / L kanamycin and 20 mg / L rifampicin, and culture it upside down at 28°C for 2-3 days. Perform colony PCR on the colonies and sequence the amplified products. The Agrobacterium with the correct sequencing results is the recombinant Agrobacterium containing the corresponding vector.

[0213] 2. Rice callus transformation:

[0214] (1) Soak the mature seeds of Ningjing No. 7 in NaCl for 2 h and rinse with sterile water 3 to 4 times;

[0215] (2) Place the treated seeds on callus induction medium and culture in the dark for 15 to 20 days;

[0216] (3) The recombinant Agrobacterium containing the corresponding vector was cultured in a liquid resistance medium containing 50 mg / L kanamycin and 20 mg / L rifampicin at 28°C and 225 rpm until the OD value was in the range of 0.6 to 1.0;

[0217] (4) Transformation into induced embryonic callus;

[0218] (5) Three days after transformation, callus tissues were transferred to N6 medium containing 2,4-dichlorophenoxyacetic acid, tyrosine, gelatin, sucrose, carbenicillin, and hygromycin for 30 days.

[0219] 3. Rice callus DNA extraction:

[0220] (1) Take new rice callus tissue, grind it with liquid nitrogen, 25 Hz, 90 s, and obtain ground samples;

[0221] (2) The CTAB method was used to extract callus genomic DNA.

[0222] The results are as follows Figure 8As shown, compared with CDA1-SpRY-BE3, which exhibited lower editing efficiency in targets with different PAMs, CDA1Δ194-SpRY-BE3 had a 1.4- to 4.5-fold increase in editing efficiency at C-18 under the truncation strategy; further, the addition of DNA binding-related factors could increase the editing efficiency in rice to 1.3 to 12.6 times that of the control.

[0223] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A base editor, characterized in that The base editor contains one or more of the following: HMGN1-VP64-CDA1Δ194-SpRY-BE3, VP64-HMGN1-CDA1Δ194-SpRY-BE3; The HMGN1-VP64-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence; The VP64-HMGN1-CDA1Δ194-SpRY-BE3 is composed of SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence.

2. The base editor according to claim 1, characterized in that The base editor also includes sgRNA or an sgRNA expression vector.

3. A recombinant vector containing the base editor according to claim 1 or 2.

4. A recombinant microorganism containing the base editor according to claim 1 or 2.

5. Use of the base editor according to claim 1 or 2 in the preparation of gene editing products.

6. Use of the base editor described in claim 1 or 2 to improve the scope and / or efficiency of gene editing for non-disease treatment purposes.

7. Use of the recombinant vector according to claim 3 or the recombinant microorganism according to claim 4 in the preparation of gene-edited products.

8. Use of the recombinant vector according to claim 3 or the recombinant microorganism according to claim 4 in improving the scope and / or efficiency of gene editing for non-disease treatment purposes.

9. The method for preparing a base editor according to claim 1, characterized in that: The method comprises the following steps: (1) Replace nCas9(D10A) with SpRY in CDA1-nCas9(D10A)-UGI; (2) CDA1 is truncated to 194 amino acids, namely CDA1Δ194; the sequence of CDA1Δ194 is shown in SEQ ID NO:5; (3) fusing two DNA interaction factors to the nitrogen terminus of the base editor in step (2) to construct DBDs-CDA1 Δ 194-SpRY-BE3, wherein the DBDs-CDA1 Δ 194-SpRY-BE3 is HMGN1-VP64-CDA1 Δ 194-SpRY-BE3 and / or VP64-HMGN1-CDA1 Δ 194-SpRY-BE3; The HMGN1-VP64-CDA1 Δ 194-SpRY-BE3 is composed of SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence; The VP64-HMGN1-CDA1 Δ 194-SpRY-BE3 is composed of SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 5 and SEQ ID NO: 1 connected in sequence.

10. A method for improving the scope or efficiency of gene editing for non-disease treatment purposes, characterized in that: The method utilizes the base editor described in claim 2 for in vitro editing.

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