CN119162152B - A highly accurate base editor that expands editing range and improves editing efficiency - Google Patents
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Abstract
本发明涉及生物技术中的基因工程领域，具体涉及一种扩大编辑范围且提高编辑效率的高精准碱基编辑器。本发明的编辑器通过融合不受PAM限制的Cas9变体SpRY与不同截短长度的胞嘧啶脱氨酶CDA1，构建了编辑范围更广、编辑效率更高的CDA1Δ‑SpRY‑BE3高精准编辑器，该编辑器在保持高编辑精度的同时，能够有效编辑基因组上的任意胞嘧啶。在酵母和水稻细胞中的测试显示，相较于现有标准编辑器CDA1‑SpRY‑BE3，本发明的编辑器在多个靶点上实现了编辑效率的显著提升，证明了其在高等真核生物中的应用价值，本发明为遗传疾病治疗和作物育种提供了更为高效精准的工具。
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
The invention belongs to the category of nucleic acid editing technology, and particularly relates to the technical field of regular clustered interval short palindromic repeat (Clustered Regularly Interspaced Short Palindromic Repeats, abbreviated as CRISPR), in particular to a high-precision base editor which enlarges the editing range and improves the editing efficiency.
Background
In the current biotechnology field, CRISPR-mediated base editing technology has been attracting attention as being able to achieve efficient nucleotide substitution without causing DNA double-strand breaks (DSBs) and without additionally providing repair templates. The technology has great application prospect in the aspect of treating genetic diseases caused by point mutation and accurate breeding of crops. However, the existing base editor has a problem of a wide editing window (e.g., C 4-C17) within which not only the target base but also the non-target base may be subjected to editing, thereby inevitably introducing unexpected mutation. The nonspecific editing not only threatens the safety of disease treatment, but also increases the difficulty for screening plant engineering breeding.
Aiming at the technical bottleneck, the prior research work of the invention makes important progress on the basis of classical base editors CDA1-BE 3. On the basis of classical base editing CDA1-BE3 mediated by cytosine deaminase PmCDA (hereinafter CDA 1) derived from sea lamprey (Petromyzon marinus), a highly accurate base editor (Nature Communications, volume 2019, volume 10 439; U.S. Pat. No. 4,202/0154163) was successfully developed that reduces the editing window to 1-2 nucleotides by making various degrees of truncations (CDA1Δ) to the CDA1 carbon end and removing its linker sequence (linker) to the SpCas9 nickase. Although this highly accurate base editor significantly improves the specificity of editing, the shrinking of its editing window (from 7 nucleotides to 2 nucleotides originally) results in a reduction in the number of editable bases, i.e., the editing range is significantly reduced, which is a limiting factor in the further development of the technology.
Disclosure of Invention
Aiming at the problems of narrow editing range and the like of the existing accurate base editor, the invention aims to provide the base editor which enlarges the editing range and improves the efficiency. The editor provided by the invention can realize the remarkable widening of the editing range while maintaining the high editing precision of the prior art, thereby being capable of editing any cytosine position in a genome efficiently and accurately. The improved base editor provided by the invention remarkably improves the practicality and application value in the fields of genetic disease treatment and crop genetic improvement, and brings new progress and innovation to the related technical fields.
In order to achieve the above object, the present invention proposes an innovative base editor optimization scheme. The specific technical scheme comprises, but is not limited to, the following points:
In a first aspect, the present invention employs an innovative strategy to replace Cas9 protein with the reported Cas9 variant SpRY that is less dependent on PAM sequence (Science 2020, volume 6488, pages 290-296) with respect to the limitations of the edit scope of the existing high precision base editor cda1Δ -nCas (D10A) -UGI (cda1Δ -BE 3). By this substitution, the optimized base editor CDA1Δ -nSpRY (D10A) -UGI (CDA1Δ -SpRY-BE 3) was successfully constructed, wherein CDA1Δ employs a fragment truncated to 195/194/193/192/190/188 amino acids.
Wherein CDA1Δ195-SpRY-BE3 consists of SEQ ID NO. 4 and SEQ ID NO.1 which are connected in sequence;
the CDA1Δ194-SpRY-BE3 consists of SEQ ID NO 5 and SEQ ID NO 1 which are connected in sequence;
The CDA1Delta193-SpRY-BE 3 consists of SEQ ID NO. 6 and SEQ ID NO. 1 which are connected in sequence;
The CDA1Δ192-SpRY-BE3 consists of SEQ ID NO 7 and SEQ ID NO 1 which are connected in sequence;
the CDA1Δ190-SpRY-BE3 consists of SEQ ID NO 8 and SEQ ID NO 1 which are connected in sequence;
the CDA1Delta188-SpRY-BE 3 consists of SEQ ID NO 9 and SEQ ID NO 1 which are connected in sequence.
Further, in order to improve the editing efficiency of CDA1 delta-SpRY-BE 3 on the premise of keeping the original editing accuracy, a large number of DNA interaction factors (DNA Binding Domains, DBDs) are screened and fused to the nitrogen end of an optimized base editing complex CDA1 delta 194-SpRY-BE3, so that a novel DBDs-CDA1 delta 194-SpRY-BE3 editor is formed. The editing efficiency of various DBDs-CDA1Delta194-SpRY-BE 3 editors is evaluated, and DNA interaction factors capable of remarkably improving the editing efficiency are screened.
Specifically, DBDs-CDA1Δ194-SpRY-BE3 are each 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.
Specifically, the 2xTAL-CDA1Δ194-SpRY-BE3 consists of SEQ ID NO. 12, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
The VP 64-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 13, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
the HMGB 1-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 14, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
the CHD 1-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 15, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
The H1-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 16, SEQ ID NO. 5 and SEQ ID NO.1 which are connected in sequence;
The HMGN-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO 17, SEQ ID NO 5 and SEQ ID NO 1 which are connected in sequence;
the p65-CDA1Δ194-SpRY-BE3 consists of SEQ ID NO 18, SEQ ID NO 5 and SEQ ID NO 1 which are connected in sequence;
The HSF 1-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO 19, SEQ ID NO 5 and SEQ ID NO 1 which are connected in sequence;
the Oct 4-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 20, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
the Sox 2-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 21, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
The Klf 4-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 22, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
the cMyc-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 23, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
The p53 DD-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 24, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
The HU-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 25, SEQ ID NO. 5 and SEQ ID NO.1 which are connected in sequence;
The HLP-CDA1Δ194-SpRY-BE3 consists of SEQ ID NO. 26, SEQ ID NO. 5 and SEQ ID NO.1 which are connected in sequence.
Further, the present invention screens out two or more high-efficiency DBDs (e.g., DBD1, DBD2, etc.), then combines these DBDs in different orders (e.g., DBD1-DBD2 and DBD2-DBD 1), fuses them to the nitrogen end of the base editing complex cda1Δ194-SpRY-BE3, respectively, and tests the editing efficiency thereof to identify the optimal combination that maximizes the improvement of editing efficiency.
Wherein, two or more DBDs-CDA1Δ194-SpRY-BE3 of DBDs are 4xTAL-CDA1Δ194-SpRY-BE3, 6xTAL-CDA1Δ194-SpRY-BE3, HMGN-VP 64-CDA1Δ194-SpRY-BE3 and/or VP64-HMGN1-CDA1Δ194-SpRY-BE3.
Specifically, the 4xTAL-CDA1Δ194-SpRY-BE3 consists of SEQ ID NO. 12, SEQ ID NO. 5 and SEQ ID NO.1 which are connected in sequence;
The 6xTAL-CDA1Δ194-SpRY-BE3 consists of SEQ ID NO. 12, SEQ ID NO. 5 and SEQ ID NO.1 which are connected in sequence;
The HMGN-VP 64-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO 17, SEQ ID NO 13, SEQ ID NO 5 and SEQ ID NO 1 which are connected in sequence;
VP64-HMGN1-CDA1Δ194-SpRY-BE3 consists of SEQ ID NO.13, SEQ ID NO. 17, SEQ ID NO. 5 and SEQ ID NO.1 which are connected in sequence.
Finally, the invention tests the editing efficiency of the finally determined base editor in the higher eukaryotic rice, and compares and analyzes the finally determined base editor with the current base editor version rice.CDA1-SpRY-BE3 and the modified version rice.CDAA1 delta 194-SpRY-BE3 and rice.HMGN1-VP 64-CDA1delta 194-SpRY-BE3 to verify the actual effect of the optimization scheme in the higher eukaryotic cells.
Wherein, the Rice. CDA1-SpRY-BE3 is composed of SEQ ID NO 27, SEQ ID NO 10 and SEQ ID NO 2 which are connected in sequence;
The Rice. CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO 27, SEQ ID NO 11 and SEQ ID NO 2 which are connected in sequence;
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 which are sequentially connected.
In a specific embodiment, the base editor of the invention further comprises an sgRNA or an sgRNA expression vector.
In a second aspect, the invention also provides a recombinant vector comprising a base editor as described hereinbefore.
In a third aspect, the invention also provides a recombinant microorganism comprising a base editor as described hereinbefore.
In a fourth aspect, the invention also provides the use of a base editor as described hereinbefore for the preparation of a gene-edited product.
In a fifth aspect, the invention also provides the use of a base editor as described for increasing the range and/or efficiency of gene editing for non-disease therapeutic purposes.
In a sixth aspect, the invention also provides the use of a recombinant vector as described hereinbefore for the preparation of a gene-edited product.
In a seventh aspect, the invention also provides the use of a recombinant vector as described hereinbefore for improving the range and/or efficiency of gene editing for non-disease therapeutic purposes.
In an eighth aspect, the invention also provides the use of a recombinant microorganism as described hereinbefore for the preparation of a gene-edited product or for the improvement of gene editing range and/or efficiency for non-disease therapeutic purposes.
In a ninth aspect, the present invention also provides a method for preparing a base editor as described above, comprising the steps of:
(1) nCas9 (D10A) in CDA1-nCas (D10A) -UGI was replaced with SpRY;
(2) CDA1 was truncated to 195/194/193/192/190/188 amino acids, cda1Δ195, cda1Δ194, cda1Δ193, cda1Δ192, cda1Δ190, cda1Δ188, respectively.
Wherein the CDA1Δ195 sequence is shown as SEQ ID NO. 4, and the CDA1Δ194 sequence is shown as SEQ ID NO. 5;
The CDA1Δ193 sequence is shown as SEQ ID NO. 6, and the CDA1Δ192 sequence is shown as SEQ ID NO. 7;
the CDA1Δ190 sequence is shown as SEQ ID NO. 8, and the CDA1Δ188 sequence is shown as SEQ ID NO. 9.
Further, the DNA interaction factor is screened and fused to the nitrogen end of the optimized base editing complex CDA1Δ194-SpRY-BE3, so that a DBDs-CDA1Δ194-SpRY-BE3 novel editor is formed.
Furthermore, two or more high-efficiency DBDs were combined in different orders and fused to the nitrogen end of the base editing complex CDA1Δ194-SpRY-BE3, respectively.
In a ninth aspect, the invention also provides a method of improving the range and/or efficiency of gene editing for non-disease therapeutic purposes, said method using the base editor as described hereinbefore for in vitro editing.
Technical effects
The invention successfully constructs a series of accurate base editors CDA1 delta-SpRY-BE 3 with low PAM dependency by fusing Cas9 variants SpRY which are hardly limited by PAM sequences with CDA1 proteins truncated in different lengths (namely CDA1 delta, wherein truncated fragments are 195/194/193/192/190/188 amino acids respectively). In the editorial test performed on 9 non-NGG PAM sites endogenous to the model eukaryotic saccharomyces cerevisiae, the following experimental results were obtained (see fig. 1).
In all test sites, CDA1-BE3 showed almost no editing activity compared to the traditional base editors CDA1-BE3 without Cas9 variant. In contrast, the base editor CDA1 delta-SpRY-BE 3 not only shows remarkable editing efficiency, but also achieves remarkable widening of the editing range while maintaining the original editing accuracy (the editing window is strictly limited in the range of 1-2 nucleotides; mainly aiming at cytosine at the 18 th position upstream of PAM sequence, namely C -18) in a specific test site. The editing range is limited from the original specific site containing NGG (N represents any base) PAM sequence to the wide site covering almost any NNN PAM sequence, thereby greatly improving the application potential and practical range of the editor.
The base editor CDA1Δ -SpRY-BE3 containing truncated CDA1 exhibits different degrees of improvement in editing efficiency compared with the full-length control group, wherein the highest improvement amplitude reaches 6.66 times, which fully proves the remarkable advantage of the technology in improving editing efficiency.
In order to further verify the expansion capability of the CDA1 delta-SpRY-BE 3 base editor over the editing range, the present study conducted a test for its precise editing capability for any cytosine base C on the genome. The editing site operation follows a four-step compact design flow:
(1) Determining a target cytosine base C and a flanking sequence thereof;
(2) Positioning a target cytosine base C at an 18 th position on the upstream of a PAM sequence, determining a spacer sequence and the PAM sequence according to the target cytosine base C, and designing corresponding sgRNA;
(3) Selecting a matched base editor according to the PAM sequence;
(4) And by testing different CDA1 truncated version combinations, the combination realizing the best and accurate editing effect is screened out. The simplicity and effectiveness of the method of the invention was verified by performing an editing test on randomly selected endogenous target sites containing three consecutive cytosines (C) in saccharomyces cerevisiae (see figure 2). In addition, the ability of the editor of the present invention to edit precisely at non-NGG PAM target sites was also demonstrated using a screening platform based on the yeast endogenous marker gene Can1 (see fig. 2). These experimental results agree that CDA 1. Delta. -SpRY-BE3 base editor has a broad and precise editing capability.
By adopting the whole genome sequencing technology, the invention comprehensively evaluates the DNA off-target effect of the CDA1 delta-SpRY-BE 3 base editor in the genome range. Experimental data shows that the use of a truncated version of deaminase significantly reduces genome-wide DNA off-target events compared to CDA1-BE3 and CDA1-SpRY-BE 3. Especially 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 fig. 3). This result fully demonstrates the remarkable success of the present technology in improving editing specificity and reducing risk of off-target, further enhancing its safety and reliability in precision gene editing applications.
In order to enhance the interaction between the base editor and the DNA molecule and further enhance the editing efficiency of the CDA1 delta-SpRY-BE 3 base editor, the present invention screens out two proteins from a plurality of DNA binding proteins that can independently enhance the editing efficiency, namely HMGN and VP64 (see FIG. 4). The two proteins are respectively fused to the amino terminal of CDA1Δ194-SpRY-BE3, so that the editing efficiency is remarkably improved, and the original precision of the editing process is maintained.
The invention further optimizes the structure of a base editor CDA1Δ -SpRY-BE3, and constructs a HMGN1-VP64-CDA1Δ -SpRY-BE3 composite structure by simultaneously fusing two DNA binding proteins HMGN and VP64 to the amino terminal of the proteins, wherein HMGN1 is positioned at the amino terminal of the fusion proteins (HMGN-VP 64 structure is formed). The structure optimization strategy greatly improves the editing efficiency, and experimental results show that compared with the original editor, the editing efficiency can be improved by 23 times (see fig. 5). Importantly, the structural optimization does not cause any damage to the original high editing accuracy of the editor, so that the efficiency of the editing process is improved, and meanwhile, the high accuracy is still maintained.
In order to deeply verify the enhancement effect of HMGN-VP 64-CDA1 delta-SpRY-BE 3 compound base editor in the aspect of editing efficiency, the invention implements a set of detailed test scheme. The protocol involved the editing test of 36 targets endogenous to Saccharomyces cerevisiae and CDA1Delta SpRY-BE3 and CDA1Delta SpRY-BE3 alone fused to HMGN1 (HMGN 1-CDA1Delta SpRY-BE 3) and CDA1Delta SpRY-BE3 alone fused to VP64 (VP 64-CDA1Delta SpRY-BE 3) were used as control groups. Experimental results show that, compared with CDA1 delta-SpRY-BE 3, HMGN1-VP64-CDA1 delta-SpRY-BE 3 achieves average 4-fold increase in editing efficiency in 36 targets tested, so that the capability of the technology in improving editing efficiency is remarkably demonstrated.
To evaluate the performance of the modified base editor HMGN-VP 64-CDA 1. Delta. -SpRY-BE3 of the present invention in more complex eukaryotic cells, an edit ability test in rice cells was performed. As a control, the editing effect of the existing standard base editor CDA1-SpRY-BE3 and its truncated version CDA1Δ194-SpRY-BE3 were also examined. Experimental data shows that, of the 5 rice target sites tested, the two improved versions CDA1Δ194-SpRY-BE3 and HMGN1-VP64-CDA1Δ SpRY-BE3 of the present invention both show a more significant improvement in editing efficiency, especially the HMGN1-VP64-CDA1Δ SpRY-BE3 version, compared to the existing standard base editor CDA1-SpRY-BE3 (see FIG. 7). These results fully demonstrate the potential of the improved base editor of the invention for use in higher eukaryotes and the significant improvement in its performance.
Drawings
FIG. 1 is a schematic diagram of a base editor vector fusing nSpRY with truncated cytosine deaminase CDA 1. Delta. Wherein CDA1-BE3 is a traditional cytosine base editor that recognizes only NGG PAM, nSpCas9 is replaced with nSpRY with more types of PAM recognition in CDA1-SpRY-BE3, CDA 1. Delta. -SpRY-BE3 is truncated at the carbon end of CDA1 in the structure and the XTEN linker between cytosine deaminase and nSpRY is removed, resulting in a series of base editors.
FIG. 2 shows that CDA1 delta-SpRY-BE 3 realizes precise base editing on a non-NGG PAM target.
FIG. 3 shows CDA1Δ -SpRY-BE3 for achieving precise editing of any C in multiple C sites on a genome, wherein a graph a in FIG. 3 shows a design strategy for editing any cytosine in the genome, b graph b in FIG. 3 shows a test result of selecting 3 sgRNAs with consecutive 3 cytosine sites in yeast genomic DNA for precise editing design based on the a graph design strategy, C graph in FIG. 3 shows a test result of b graph, and d graph in FIG. 3 shows a drug screening test result.
FIG. 4 shows the case of DNA off-target of the candidate base editor in the whole genome, wherein, the a diagram in FIG. 4 shows the case of whole genome-wide indels, the b diagram in FIG. 4 shows the case of whole genome but nucleotide variation, and the c diagram in FIG. 4 shows a specific category of single nucleotide variation.
FIG. 5 shows that DNA binding factor HMGN1 and VP64 enhance the editing efficiency of CDA1Δ194-SpRY-BE 3.
FIG. 6 shows that HMGN.sup.1 and VP.sup.64 are co-fused to the nitrogen terminus of CDA1Δ -SpRY-BE3 to significantly improve editing efficiency.
FIG. 7 is a universal test of efficiency improvement for HMGN and VP64 fusion at the nitrogen terminus of CDA1Δ194-SpRY-BE 3.
FIG. 8 shows the application of the relevant strategy to rice.
Detailed Description
The present invention will be described in further detail with reference to examples. The reagents or instrumentation used are not manufacturer specific and are considered to be commercially available conventional products.
The present invention provides a series of base editors comprising 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-CDA1Δ194-SpRY-BE3、4xTAL-CDA1Δ194-SpRY-BE3、6xTAL-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、HMGN1-VP64-CDA1Δ194-SpRY-BE3、VP64-HMGN1-CDA1Δ194-SpRY-BE3、Rice.CDA1-SpRY-BE3、Rice.CDA1Δ194-SpRY-BE3、Rice.HMGN1-VP64-CDA1Δ194-SpRY-BE3.
Wherein the CDA1-SpRY-BE3 consists of SEQ ID NO.3 and SEQ ID NO. 1 which are connected in sequence;
the CDA1Δ195-SpRY-BE3 consists of SEQ ID NO. 4 and SEQ ID NO. 1 which are connected in sequence;
the CDA1Δ194-SpRY-BE3 consists of SEQ ID NO 5 and SEQ ID NO 1 which are connected in sequence;
The CDA1Delta193-SpRY-BE 3 consists of SEQ ID NO. 6 and SEQ ID NO. 1 which are connected in sequence;
The CDA1Δ192-SpRY-BE3 consists of SEQ ID NO 7 and SEQ ID NO 1 which are connected in sequence;
the CDA1Δ190-SpRY-BE3 consists of SEQ ID NO 8 and SEQ ID NO 1 which are connected in sequence;
the CDA1Delta188-SpRY-BE 3 consists of SEQ ID NO 9 and SEQ ID NO 1 which are connected in sequence;
The 2 xTAL-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 12, SEQ ID NO. 5 and SEQ ID NO.1 which are connected in sequence;
The 4 xTAL-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 12, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
The 6xTAL-CDA1Δ194-SpRY-BE3 consists of SEQ ID NO. 12, SEQ ID NO. 5 and SEQ ID NO.1 which are connected in sequence;
The VP 64-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 13, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
the HMGB 1-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 14, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
the CHD 1-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 15, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
The H1-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 16, SEQ ID NO. 5 and SEQ ID NO.1 which are connected in sequence;
The HMGN-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO 17, SEQ ID NO 5 and SEQ ID NO 1 which are connected in sequence;
the p65-CDA1Δ194-SpRY-BE3 consists of SEQ ID NO 18, SEQ ID NO 5 and SEQ ID NO 1 which are connected in sequence;
The HSF 1-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO 19, SEQ ID NO 5 and SEQ ID NO 1 which are connected in sequence;
the Oct 4-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 20, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
the Sox 2-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 21, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
The Klf 4-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 22, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
the cMyc-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 23, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
The p53 DD-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 24, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
The HU-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO. 25, SEQ ID NO. 5 and SEQ ID NO.1 which are connected in sequence;
The HLP-CDA1Δ194-SpRY-BE3 consists of SEQ ID NO. 26, SEQ ID NO. 5 and SEQ ID NO. 1 which are connected in sequence;
The HMGN-VP 64-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO 17, SEQ ID NO 13, SEQ ID NO 5 and SEQ ID NO 1 which are connected in sequence;
VP64-HMGN1-CDA1Δ194-SpRY-BE3 consists of SEQ ID NO.13, SEQ ID NO. 17, SEQ ID NO. 5 and SEQ ID NO.1 which are connected in sequence;
The Rice. CDA1-SpRY-BE3 consists of SEQ ID NO 27, SEQ ID NO 10 and SEQ ID NO 2 which are connected in sequence;
The Rice. CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO 27, SEQ ID NO 11 and SEQ ID NO 2 which are connected in sequence;
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 which are sequentially connected.
YPDA medium in the following examples was prepared from 20g/L peptone, 10g/L yeast extract, 20g/L glucose, 0.12g/L adenine hemisulfate and water, and 15g/L agarose was added to the solid medium.
The defective media in the examples below were prepared from 6.7g/L YNB, 20g/L glucose, a suitable amount of SC-defective amino acid mixture lacking uracil and leucine (SC-L-U) and water, and the solid medium was supplemented with an additional 15g/L agarose.
EXAMPLE 1 construction of vectors
1. The fusion protein carrier construction method comprises the following steps:
(1) The primers required in the construction vector were designed as shown in Table 1;
(2) Performing PCR amplification by using Phanta Max Super-FIDELITY DNA Polymerase (Vazyme), corresponding primer pairs and a DNA template;
(3) Restriction enzyme is used for carrying out enzyme digestion on a plasmid vector serving as a framework, and the reaction condition is 37 ℃ for 4 hours;
(4) The PCR amplification product and the enzyme digestion product are respectively subjected to fragment size identification through agarose gel electrophoresis, and the digestion is recovered;
(5) Performing seamless cloning connection on the purified linear vector and the fragment by using OK Clon DNA connection kit II (Accurate);
(6) Transferring into E.coli DH5 alpha competent cells, coating onto a resistant LB medium, and selecting a monoclonal for sequencing verification;
(7) Transferring the colony with correct sequence to 5mL liquid resistant LB culture medium, and shake culturing at 37 ℃ and 225r/min for 12-18 hours;
(8) Plasmids were extracted using OMEGA plasmid miniprep kit.
The goldengate method constructs all gRNA expression vectors:
(1) The primers required in the construction vector were designed as shown in Table 2;
(2) Mixing the corresponding primer pairs in a ratio of 1:1, heating to 95 ℃, and then gradually annealing to 25 ℃ to obtain a primer annealing product;
(3) Preparing a working system by using IIs type restriction enzyme and T4 DNA ligase (NEB), mixing, adding a primer annealing product and a carrier skeleton into the system for GoldenGate connection, wherein the reaction conditions are 37 ℃ and 2min, 16 ℃ and 30s, and after 50 times of circulation, stopping the reaction at 37 ℃ and 5 min and 85 ℃ and 5 min;
(4) The GoldenGate reaction product is transformed into E.coli DH5 alpha competent cells, the competent cells are coated on a resistant LB culture medium, and monoclonal sequencing verification is carried out;
(5) Transferring the colony with correct sequence to 5mL liquid resistant LB culture medium, and shake culturing at 37 ℃ and 225r/min for 12-18 hours;
(6) Plasmids were extracted using OMEGA plasmid miniprep kit.
3. T4 DNA ligase construction of vectors in FIGS. 5 and 6:
(1) The primers required in the construction vector were designed as shown in Table 1;
(2) Performing PCR amplification by using Phanta Max Super-FIDELITY DNA Polymerase (Vazyme), corresponding primer pairs and a DNA template;
(3) Using restriction enzyme to enzyme cut the plasmid vector as skeleton and the PCR amplified product obtained in the last step, wherein the reaction condition is 37 ℃ for 4 hours;
(4) The enzyme cutting products are respectively identified in the size of fragments through agarose gel electrophoresis, and the fragments are cut into gel and recovered;
(5) Ligating the purified linear vector and fragment using T4 DNA ligase (NEB) at 22 ℃ for 2 hours;
(6) Transferring into E.coli DH5 alpha competent cells, coating onto a resistant LB medium, and selecting a monoclonal for sequencing verification;
(7) Transferring the colony with correct sequence to 5mL liquid resistant LB culture medium, and shake culturing at 37 ℃ and 225r/min for 12-18 hours;
(8) Plasmids were extracted using OMEGA plasmid miniprep kit.
The specific construction process of each expression vector is as follows:
(A1) Construction of CDA1-SpRY-BE3 vector
The primers of Table 1 were used to construct CDA1-SpRY-BE3, wherein four sets of primers CDA1-SpRY-BE3-F1/R1 to F4/R4 were used as templates, PCR was performed using pJT45-GalL-CDA1-BE3 (Addgene, plasmid # 145038)) as templates, and after obtaining fragments, ligation was performed with pJT45-GalL-CDA1-BE3 digested with restriction enzyme NruI/NcoI, to obtain intermediate vector CDA1-SpRY-BE3a having D1135L/S1136W/G1218K/E1219Q/T1337R mutation, PCR was performed using two sets of primers of CDA1-SpRY-BE3-F5/R5 and F6/R6 as templates, ligation was performed with CDA1-SpRY-BE3a digested with restriction enzyme NruI/NcoI, and obtaining fragments of CDA 1-37-BE 3 with CDA 1-37-B3 as templates, and finally, PCR was performed using two sets of primers of CDA1-SpRY-BE3 as templates, and PCR was performed using two sets of primers of CDA1-SpRY-BE 3-F3 as templates, and PCR was performed using two sets of primers of CDA 1-37-BE 3 to obtain fragments, and PCR was performed using two sets of primers of CDA 1-35R 3 and CDA 3-R8-R3 as template, and PCR was used to obtain the intermediate vector CDA 1-35-3B 3.
(A2) Construction of CDA 1. Delta. S-SpRY-BE3 series vector
The CDA1 delta s-SpRY-BE3 was amplified by PCR using the set of primers shown in Table 1, in which CDA1 delta s-SpRY-BE3-F1/R1 was used as a template to obtain fragment 1, CDA1 delta of the corresponding length fragment was amplified using CDA1-SpRY-BE3 prepared in example 1 as a template, CDA1 delta and fragment 1 were ligated with CDA1 delta 194-SpRY-BE 3-F as forward primers, CDA1 delta 194-SpRY-BE3-R, CDA1 delta 192-SpRY-BE3-R, CDA1 delta 190-SpRY-BE3-R, CDA delta 188-SpRY-BE3-R, respectively, using the same set of primers, CDA1 delta and fragment 1 together with restriction endonuclease SpeI/SbfI digested CDA1-SpRY-BE3, and the series of vectors shown in FIG. 1 delta 74-74 were obtained.
(A3) Construction of VP64-HMGN1-CDA1Δ194-SpRY-BE3
(1) The CDA1Δ194-SpRY-BE3 (AvrII) was obtained by PCR amplification using the primer set for constructing factor-CDA1Δ194-SpRY-BE3 in Table 1, wherein CDA1Δ194-SpRY-BE3-F1/R (AvrII) was obtained by using CDA1Δ194-SpRY-BE3 prepared in example 2 as a template, and PCR amplification was performed using the fragment as a template using the CDA1Δ194-SpRY-BE3-F2/R (AvrII) primer set, and after obtaining a fragment having the AvrII cleavage site, ligation was performed with CDA1Δ194-SpRY-BE3 digested with restriction enzyme SpeI/SbfI.
(2) And carrying out PCR amplification by using 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、HLP-F and a universal reverse primer Factor-R respectively, taking the synthesized corresponding DNA fragment as a template to obtain the corresponding fragment, and connecting the amplified fragment digested by using restriction enzyme AvrII/NheI with CDA1Delta194-SpRY-BE 3 (AvrII) digested by using restriction enzyme 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 single Factor vectors.
(3) A2 xTAL fragment digested with the restriction enzyme AvrII/NheI was ligated with 2xTAL-CDA1Δ194-SpRY-BE3 digested with the restriction enzyme AvrII to obtain 4xTAL-CDA1Δ194-SpRY-BE3, a 2xTAL fragment digested with the restriction enzyme AvrII/NheI was ligated with 4xTAL-CDA1Δ194-SpRY-BE3 digested with the restriction enzyme AvrII to obtain 6xTAL-CDA1Δ194-SpRY-BE3, a HMGN fragment digested with the restriction enzyme AvrII/NheI was ligated with VP64-CDA1Δ194-SpRY-BE3 digested with the restriction enzyme AvrII to obtain HMGN-VP 64-CDA1Δ194-SpRY-BE3, and a VP64 fragment digested with the restriction enzyme AvrII/NheI was ligated with the restriction enzyme CDA HMGN-CDA1Δ194-SpRY-BE3 to obtain HMGN-VP 64-CDA1Δ194-SpRY-BE3.
(A4) Construction of Rice. Fusion-CDA1Δ194-SpRY-BE3
(1) Using the primer set for constructing a rice editor shown in Table 1, a fragment 1 was obtained by PCR amplification using the rice. CDA1-SpRY-BE3-F1/R1 as a template with the synthesized rice codon-optimized SpRY fragment (SEQ ID NO: 29), and a fragment 1 was obtained by PCR amplification using the rice. CDA1-SpRY-BE3-F2/R2 as a template with the synthesized rice codon-optimized 2xUGI fragment (SEQ ID NO: 30), and an intermediate vector containing 2xUGI and having ten point mutations of L111R/D1135L/S1136W/G1218K/E1219Q/N1317R/A1322R/R1333P/R1335Q/T1337R was obtained by ligation with the restriction enzyme MluI/SacI-digested pH-A3A-PBE (Addgene, plasmid # 119774).
(2) PCR amplification was performed using Rice. CDA1-SpRY-BE3-F3/R3 with synthetic rice codon-optimized PmCDA1 (SEQ ID NO: 10) as a template to obtain fragment 2; A vector rice. CDA1-SpRY-BE3 can BE obtained by ligating the rice. CDA1-SpRY-BE3-F4/R4 with a synthetic rice codon optimized SpRY fragment as a template, obtaining a fragment together with fragment 2, a rice. CDA1-SpCas9-BE3a digested with restriction enzyme avrII/SbfI, a rice. CDA1-SpRY-BE 1 primer pair with rice. CDA 1. Delta 194-SpRY-BE3-F1/R1 to F3/R3, PCR amplification with rice. CDA1-SpRY-BE3 as a template, respectively, obtaining three fragments together with a restriction enzyme BssHII/SbfI digested with rice. CDA1-SpRY-BE3, obtaining rice. CDA 194-SpRY-BE3, a rice. CDA 194-194A 2/BRL 3, respectively, and a rice 36-194-BRL 3 fragment with a restriction enzyme Δ194-3496-3495, PCR with a rice. CDA 1-194-BRL 3 as a template, and a rice 36-194-BRL 3 fragment with a restriction enzyme Δ194-3496-34FI digested with a restriction enzyme Δ194-SpRY-BE3, respectively.
(A5) Construction of gRNA expression vectors
(1) PCR amplification was performed using the primer set for constructing the gRNA expression vector backbone in Table 2, using the sgRNA-scaffold-F1/R1 as a template and the ccdB expression cassette, and using the obtained fragment as a template, using the sgRNA-scaffold-F2/R1 primer pair, PCR amplification was performed, and the obtained fragment was ligated with pJT303-SNR52-sgRNA-Can1-3 digested with restriction enzymes AatII/KpnI, to obtain the gRNA expression vector backbone.
(2) Using the primer set for constructing the gRNA expression vector in Table 2, all yeast gRNA expression vectors were annealed using the primer PolyC-1-NGN-F/R in Table 2 as an example, and the annealed product was mixed with the skeleton of the gRNA expression vector, and Golden Gate ligation was performed using restriction enzymes AarI and T4 DNA ligase, to obtain the corresponding gRNA expression vector.
(3) Using the primer group for constructing the rice target series vector in table 2, taking the CDA1-SpRY-BE3 vector for constructing the targeting rice target 1 as an example, annealing by using the primer rice. Site1-F/R in table 2, mixing the obtained annealing product with the CDA1-SpRY-BE3, and connecting by using the restriction enzyme BsaI and the T4 DNA ligase Golden Gate, thereby obtaining the CDA1-SpRY-BE3 vector corresponding to the targeting rice target 1.
Example 2 transformation and Induction and high throughput sequencing analysis of Saccharomyces cerevisiae
In order to detect the editing situation of the editor in yeast, the embodiment uses the (A1) and (A2) vectors to respectively transform and induce together with the sgRNA vector in (A5) in yeast, extracts DNA, and then carries out high-throughput sequencing on target fragments, and analyzes and visualizes to obtain editing efficiency, and the specific method is as follows:
1. yeast transformation:
(1) Culturing for 2-3 days at 28deg.C on YPDA medium using Saccharomyces cerevisiae BY 4743;
(2) After washing with sterile ddH 2O and harvesting yeast cells, 100mM LiAc was added and incubated at 28℃for 10 min;
(3) After centrifugation for 5 seconds, the supernatant was removed and the cells were mixed with 0.5-1. Mu.g of the plasmid DNA obtained in example 1, 240. Mu.L of 50% PEG3350, 36. Mu.L of 1M LiAc, 50. Mu.L of 2mg/mL salmon sperm DNA and 20. Mu.L of sterile water in a centrifuge tube and incubated at 42℃for 1.5 h;
(4) Centrifuging for 5 seconds, removing supernatant, and culturing at 28deg.C on defective solid culture medium SC-L-U (yeast synthetic culture medium lacking uracil and leucine) for 2-3 days;
(5) The monoclonal was picked up, shake-cultured in a defective liquid medium SC-L-U at 28℃for 18-20 hours at 225r/min, and positive was confirmed by PCR amplification.
2. Yeast induction:
(1) 3-5 positive colonies are picked and cultured in 3mL of defect type liquid culture medium SC-L-U containing 2% glucose at 28 ℃ for 18-20 hours;
(2) Sucking 0.8 mL of bacterial liquid, centrifuging, discarding the supernatant, washing 3 times with sterile water to remove residual glucose, and then re-suspending in 5mL of SC-L-U liquid induction medium containing 2% galactose and 1% raffinose, and performing shake culture at 28 ℃ for 20 hours at 225 r/min;
(3) Sucking 0.5 mL of bacterial liquid, and briefly centrifuging to discard the supernatant to obtain the induced bacterial cells.
3. Yeast DNA extraction:
(1) Adding a yeast cell lysate into the induced thalli, and carrying out 70 ℃ treatment on the saccharomycete after being resuspended for 10 min;
(2) Adding three times of absolute ethyl alcohol (Shanghai test) into the treated heavy suspension, shaking, mixing, centrifuging briefly, and discarding supernatant to obtain precipitate containing yeast DNA;
(3) Washing the precipitate with 70% ethanol, and air drying at room temperature;
(4) Adding sterile water into the dried precipitate to obtain yeast DNA.
4. Library construction and analysis of second generation sequencing:
(1) PCR amplification was performed using Phanta Max Super-FIDELITY DNA Polymerase, corresponding primer pairs, and genomic DNA as templates, the primers being a combination of the second-generation sequencing adaptors of Table 3 and the amplified sequences of Table 4;
(2) PCR products were purified using the Cycle Pure kit (OMEGA);
(3) Performing PCR-free library construction, high-throughput sequencing and data analysis (Bokesen organism, beijing, china) on the purified product, wherein Illumina NovaSeq platforms are used for sequencing;
(4) Over 100,000 reads can be obtained per sample on average. After data filtering, the FASTQ file is analyzed by using https:// github.com/zfcarpe/Cas9Sequencing script;
(5) Vector graphics are drawn using vector graphics drawing tools GRAPHPAD PRISM and Adobe Illustrator.
As shown in fig. 2, in NGN, NAN, NTN, NCN targets of all PAM non-NGG, CDA1-BE3 that traditionally recognizes NGG PAM has editing efficiency of C > T below 1% in these sites;
The CDA1-SpRY-BE3 is constructed by replacing nSpCas9 with nSpRY, wherein the editing efficiency of C > T is 5-40% in 4 targets of NGN and NAN of PAM, the editing efficiency of C > T is 1.2-3.3% in the remaining 4 targets except PolyC-7-NCN in 5 targets of NCN and NTN of PAM, and the editing window is between C-19 and C-15 in all targets of PAM type;
The CDA1 carbon end is further truncated and the XTEN linker is removed, a series of CDA1 delta-SpRY-BE 3 is constructed, in all PAM type targets, the design strategy of the editor ensures that the C > T editing efficiency is improved by 1.3 times at least on the basis of CDA1-SpRY-BE3, as shown by PolyC-8-NCN, and is improved by 6.1 times at most, as shown by PolyC-6-NTN, in addition, the editing window of PolyC-3-NAN is in the range of 2-nt of C-19 and C-18, and the editing window of the design strategy in all targets is accurately positioned at the C-18 position.
Example 3 canavanine resistance test and off-target test
In order to verify whether CDA1 delta-SpRY-BE 3 can realize accurate editing of any C in multiple C sites on a genome, the invention designs a strategy for editing any cytosine in the genome, and comprises four steps of firstly determining the cytosine site to BE edited in the genome, designing the cytosine site to BE positioned at-18 positions of gRNA according to the determined cytosine site to determine a PAM sequence required by the gRNA, selecting CDA1 delta-SpRY-BE 3 for editing according to the difference of the PAM sequences, for example, when PAM is a nonstandard NHN sequence, so as to efficiently and directionally obtain a product only comprising target cytosine editing (a graph in figure 3);
Panel b of FIG. 3 shows that a site with 3 consecutive cytosines was selected in the yeast genomic DNA based on the strategy described in panel a, and 3 different sgRNAs were designed for subsequent testing in order to accurately edit the 3 cytosines, respectively, and this part of the test was performed in the same manner as in example 2.
The test result is shown in a graph C in FIG. 3, when different cytosines are respectively positioned at C-18 positions, the cytosine positioned at the position shows highest C > T editing efficiency in 3 cytosines, and in the base editing process guided by sgRNA-C 1, the ratio of an editing product T 1C2C3 to all editing products is obviously improved compared with CDA1-BE3 and CDA1-SpRY-BE3 by taking cytosine C 1 as an example, so that the target strategy and the design strategy of the base editor can realize effective and accurate C > T editing on any cytosine in a genome;
In order to further eliminate the influence of the second generation sequencing library building process on the editing result, two continuous cytosines are selected on the canavanine sensitive gene Can1 for drug screening test, as shown in a d diagram in fig. 3, when the sgRNA guide test editors designed for C1 and C2 edit the sites simultaneously or respectively for C > T, stop codons TGA, TAG or TAA Can be generated on Can1 to cause the saccharomyces cerevisiae to have canavanine resistance, genotype analysis is further carried out on canavanine resistant strains generated under the action of different editors, and the fact that the proportion of the target mutant strains to all mutant strains is gradually increased along with gradual truncation of the cytosine deaminase CDA1 is found to be consistent with the second generation sequencing result.
The canavanine resistance test and the off-target test specifically comprise the following steps:
(1) Selecting a Can1-1/Can1-2 gRNA expression vector and a candidate base editor to perform yeast transformation and culture,
(2) Simultaneously culturing a group of blank control without any expression vector;
(3) Subsequently, the cells were transferred to a liquid induction medium containing 2% galactose and 1% raffinose, and shake-cultured at 28℃for 20 hours at 225 r/min;
(4) Diluting the bacterial liquid by 10,000 times, coating blank control on YPDA culture medium, coating the rest bacterial liquid on SC-Arg solid culture medium containing 60 mug/mL L-canavanine, and culturing at 28 ℃ for 2-3 days;
(5) Colonies were picked from each dish, cultured in YPDA liquid medium, shake cultured at 28 ℃ for 20 hours at 225r/min, while part of colonies were selected for monoclonal sequencing;
(6) Sucking 0.5-1 mL bacterial liquid, and extracting yeast genome DNA by using a yeast genome DNA extraction kit (Solarbio);
(7) And (3) carrying out quality evaluation on the extracted DNA sample, and carrying out database construction, whole genome sequencing and bioinformatics analysis.
FIG. 4 is a graph of DNA off-target for candidate base editing tools across the whole genome, wherein graph a in FIG. 4 is a graph of whole genome indels, with untreated Saccharomyces cerevisiae as a control blank, all base editing tools exhibit indels consistent with the control blank, indicating that little or no indels are generated by CDA 1-mediated base editing tools in yeast editing.
Panel b in FIG. 4 shows that the single nucleotide variation in the whole genome range causes more single nucleotide variation in the whole genome range of CDA1-BE3 as a negative control compared with a blank control, the single nucleotide variation type off-target of CDA1-SpRY-BE3 replaced by nuclease is further increased, the off-target of the type is gradually reduced along with shortening of CDA1, and the fact that the CDA1 delta-SpRY-BE 3 strategy can obviously reduce the off-target of the whole genome is proved.
Panel C in FIG. 4 is a specific class of single nucleotide variation, as shown, most single nucleotide variation types are C > T/G > A, confirming that genome-wide decoys are mainly caused by deaminase.
Example 4 effect of DNA interaction factor on CDA 1. Delta. -SpRY-BE3 base editor
1. On the premise of keeping the original editing accuracy, in order to improve the editing efficiency of CDA1 delta-SpRY-BE 3, the invention respectively screens 15 DNA interaction factors of four classes of TAF, CAF, PTF and DBP in two test sites, fuses the 15 DNA interaction factors to the nitrogen end of an optimized base editing complex CDA1 delta 194-SpRY-BE3, forms a DBDs-CDA1 delta 194-SpRY-BE3 novel editor, and tests the changes of the editing efficiency and the editing window.
As a result, as shown in fig. 5, VP64 in TAF and HMGN1 in CAF were both fused at the nitrogen end of cda1Δ194-SpRY-BE3, and the editing window exhibited optimal C > T editing efficiency, without significant widening.
2. Further, the two DNA binding factors HMGN and VP64 found in FIG. 5 are combined and fused at the nitrogen end of CDA1Δ194-SpRY-BE3 in an arrangement mode, and test results show that the fusion proteins can remarkably improve the editing efficiency of C > T in a test site, the editing window is only 2-nt, and the highest editing efficiency point is still C-18 (FIG. 6).
As shown in FIG. 7, the efficiency improvement is carried out by carrying out universality test on the efficiency improvement by fusing HMGN1 and VP64 at the nitrogen end of CDA1Δ194-SpRY-BE3, and as a result, the efficiency improvement by single VP64 fusion is universal, and the editing efficiency can BE improved to a higher level by additional fusion HMGN1, wherein the average efficiency is about 2 times and can BE up to 15 times.
Example 5 application of base editor construction strategy in Rice
In higher eukaryote rice, the base editor provided by the invention is subjected to editing efficiency test, and is subjected to comparison analysis with the existing base editor version CDA1-SpRY-BE3 and the modified version CDA1Delta194-SpRY-BE 3 in the step (1), wherein the base editor in the rice is subjected to agrobacterium transformation and rice callus transformation, DNA is extracted, and high-throughput sequencing is performed on target fragments, and analysis and visualization are performed to obtain the editing efficiency. The specific method for agrobacterium transformation, rice callus transformation and DNA extraction is as follows:
1. Agrobacterium transformation:
(1) Placing the agrobacterium competent cells in a 37 ℃ water bath, and culturing for 5min;
(2) The vector plasmid DNA corresponding to FIG. 8 is added into the competent cells of Agrobacterium respectively, and the ice bath is carried out for 30min, liquid nitrogen for 5min, water bath at 37 ℃ for 5min and ice bath for 5min;
(3) Adding 700 mu L of LB liquid medium (without antibiotics), and shake culturing at 28 ℃ and 225rpm for 2-3 hours;
(4) Taking out the bacterial liquid, centrifuging at 6000rpm for 1min, collecting 100 μl of supernatant, and re-suspending bacterial blocks;
(5) And (3) coating the bacterial liquid on a resistance culture medium plate containing 50mg/L of carbaryl and 20mg/L of rifampicin, culturing for 2-3 days at 28 ℃ in an inverted mode, performing colony PCR on the colony, sequencing an amplification product, and obtaining agrobacterium with correct sequencing results, namely recombinant agrobacterium containing a corresponding vector.
2. Rice callus transformation:
(1) Soaking mature seeds of Ningjing No. 7 in NaCl for 2 hours, and washing with sterile water for 3-4 times;
(2) Placing the treated seeds on an induced callus culture medium, and culturing in darkness for 15-20 days;
(3) Shake culturing recombinant agrobacterium containing corresponding vectors in a liquid resistance culture medium containing 50mg/L of carbaryl and 20mg/L of rifampicin at 28 ℃ and 225rpm until the OD value is within the range of 0.6-1.0;
(4) Transformation into induced embryogenic callus;
(5) After 3 days of transformation, the calli were transferred to N6 medium containing 2, 4-dichlorophenoxyacetic acid, tyrosine, gelsolite, sucrose, carbenicillin and hygromycin for 30 days.
3. Extraction of rice callus DNA:
(1) Taking new-born rice callus, grinding with liquid nitrogen, and obtaining a grinding sample at 25 Hz for 90 s;
(2) The genomic DNA of the callus was extracted by CTAB method.
As shown in FIG. 8, compared with CDA1-SpRY-BE3 which shows lower editing efficiency in different PAM targets, CDA1Δ194-SpRY-BE3 has 1.4-4.5 times improvement on C-18 editing efficiency under a truncated strategy, and further, the editing efficiency in rice can BE improved to 1.3-12.6 times of that of a control by adding a DNA binding correlation factor.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.
Claims (10)
1. A base editor, characterized in that it contains one or more of HMGN1-VP64-CDA1Δ194-SpRY-BE3, VP64-HMGN1-CDA1Δ194-SpRY-BE3;
The HMGN-VP 64-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO 17, SEQ ID NO 13, SEQ ID NO 5 and SEQ ID NO 1 which are connected in sequence;
VP64-HMGN1-CDA1Δ194-SpRY-BE3 consists of SEQ ID NO.13, SEQ ID NO. 17, SEQ ID NO. 5 and SEQ ID NO.1 which are connected in sequence.
2. The base editor of claim 1 further comprising an sgRNA or an sgRNA expression vector.
3. A recombinant vector comprising the base editor of claim 1 or 2.
4. A recombinant microorganism comprising the base editor of claim 1 or 2.
5. Use of the base editor of claim 1 or 2 for the preparation of a gene edited product.
6. Use of the base editor of claim 1 or 2 to increase the range and/or efficiency of gene editing for non-disease therapeutic purposes.
7. Use of the recombinant vector of claim 3 or the recombinant microorganism of claim 4 for the preparation of a gene-edited product.
8. Use of the recombinant vector of claim 3 or the recombinant microorganism of claim 4 for improving the range and/or efficiency of gene editing for non-disease therapeutic purposes.
9. The method for preparing a base editor of claim 1, characterized in that it comprises the steps of:
(1) nCas9 (D10A) in CDA1-nCas (D10A) -UGI was replaced with SpRY;
(2) Shortening CDA1 to 194 amino acids to obtain CDA1Δ194, wherein the CDA1Δ194 sequence is shown as SEQ ID NO. 5;
(3) Fusing two DNA interaction factors to the nitrogen end of the base editor of step (2) to construct DBDs-CDA1Δ194-SpRY-BE3, wherein the DBDs-CDA1Δ194-SpRY-BE3 is HMGN-VP 64-CDA1Δ194-SpRY-BE3 and/or VP64-HMGN1-CDA1Δ194-SpRY-BE3;
The HMGN-VP 64-CDA1Delta194-SpRY-BE 3 consists of SEQ ID NO 17, SEQ ID NO 13, SEQ ID NO 5 and SEQ ID NO 1 which are connected in sequence;
VP64-HMGN1-CDA1Δ194-SpRY-BE3 consists of SEQ ID NO. 13, SEQ ID NO. 17, SEQ ID NO.5 and SEQ ID NO. 1 which are connected in sequence.
10. A method for improving the range or efficiency of gene editing for non-disease therapeutic purposes, characterized in that the method uses the base editor of claim 2 for in vitro editing.
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