Multi-site dual base editor and use thereof

Abstract

Provided is a multi-site dual base editor and the use thereof, which belongs to the technical field of biology. Firstly, a constructed single plasmid editor framework is used to realize dual editing of multiple sites under the guidance of a single crRNA array by means of the composition and structural optimization of a fusion protein composed of cytosine deaminase, UGI, adenine deaminase and dCas12a. On this basis, the multi-site editing efficiency is further improved by means of promoter replacement, the introduction and optimization of artificial interval sequences in the crRNA array and the modification of a DR motif. Finally, the function of the multi-site dual base editor is verified by means of the generation of a multi-resistant strain, the de novo generation of a riboflavin synthetic strain, and the increased yield of a surfactin synthetic strain. The operable range of the dual base editor on a genome is expanded, the simultaneous dual editing of multiple sites on a genome is realized, and multiple sites on a genome can be edited simultaneously under the guidance of a single crRNA array.

Description

A multi-site dual base editor and its application Technical Field

[0001] The present invention relates to a multi-site dual base editor and applications thereof, and belongs to the field of biotechnology. Background Art

[0002] Gene editing is a technique that achieves gene knockout, insertion of foreign DNA fragments, or DNA base mutation by introducing sequence changes at specific DNA sites. In recent years, the CRISPR / Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) system, particularly the CRISPR / Cas9 system, has been widely used in gene editing. In this system, immature precursor CRISPR RNA (pre-crRNA) binds to tracrRNA (trans-activating crRNA) and, under the action of RNase III, forms a crRNA:tracrRNA complex, which guides the Cas9 protein to recognize and cleave specific sites in the genome, creating double-strand breaks. Sequence changes on the homologous template are then introduced to the target site via homologous recombination repair (HDR). Alternatively, the broken DNA fragments can be directly joined via non-homologous end joining (NHEJ), generating random insertions or deletions (Indels). Since unrepaired double-strand breaks are lethal, only cells that have successfully repaired and introduced mutations so that Cas9 no longer recognizes and cuts can survive. This is the basic principle of using CRISPR / Cas9 for gene editing.

[0003] In order to simplify the operation process and improve editing efficiency, crRNA and tracrRNA are often constructed into a chimera, namely sgRNA (small guide RNA), for expression. In this way, gene editing can be performed by expressing sgRNA and Cas9 protein. In addition to the CRISPR / Cas9 system, the CRISPR / Cas12a system (also known as CRISPR / Cpf1) is also often used in gene editing. Unlike the CRISPR / Cas9 system, CRISPR / Cas12a only requires crRNA to function; and Cas12a itself has RNase activity and can cut and process immature mRNA sequences containing multiple crRNAs; therefore, a crRNA array containing multiple crRNAs can be designed. When it is processed and matured by Cas12a, it can produce multiple crRNAs with independent functions and guide Cas12a to target the corresponding target sites in the genome, achieving simultaneous editing of multiple sites.

[0004] Many species lack the NHEJ pathway (even if it is active, it is weak), while the HDR pathway requires the participation of a homologous template to function, and there is also a competitive relationship between the two repair mechanisms, which leads to a high lethality and low editing efficiency of the gene editing process based on the above method. After inactivating the DNA enzyme of Cas9, nCas9, which can only cut one DNA chain, and dCas9, which cannot cut DNA, can be obtained. nCas9 and dCas9 can still bind to specific sites in the genome under the guidance of sgRNA, but will not produce lethal double-strand breaks. Similarly, after inactivating the DNA enzyme activity of Cas12a, dCas12a, which cannot cut DNA, can be obtained. dCas12a can also bind to specific sites in the genome under the guidance of crRNA and will not produce lethal double-strand breaks. In addition, because the RNA enzyme activity of dCas12a is retained, it can target multiple different sites in the genome under the guidance of a single crRNA array.

[0005] Cytosine deaminase can catalyze the deamination of cytosine (C) to uracil (U), and further convert U to thymine (T) through DNA repair or replication; adenine deaminase TadA and its mutants can deaminate adenine (A) to form inosine (I). At the DNA level, inosine will be read and replicated as guanine (G), ultimately achieving the conversion of A→G. As shown in Figure 1a, cytosine deaminase is fused to nCas9 or dCas9 to construct a cytosine base editor (CBE), which can achieve genomic base editing (C→T) independent of double-strand breaks under the guidance of sgRNA. Further fusion of a uracil glycosylase inhibitor (UGI) derived from bacteriophage PBS inhibits the activity of intracellular uracil DNA N-glycosylase (UNG), preventing the base mismatch repair pathway (BER) from restoring the edited U·G pairing to C·G, further improving the efficiency of base editing. As shown in Figure 1b, adenine deaminase is fused to nCas9 or dCas9 to construct an adenine base editor (ABE), which can achieve A→G conversion at specific genomic sites under the guidance of sgRNA. In addition, by simultaneously fusing cytosine deaminase and adenine deaminase with nCas9 or dCas9 to construct a dual editor (duBE), C→T and A→G conversions at specific sites on the genome can be simultaneously achieved under the guidance of sgRNA.

[0006] The sites recognized by Cas9 need to have a PAM sequence of NGG (N = A, T, C, G), which limits the operable range of Cas9-based dual base editors on the genome; in addition, each sgRNA guiding Cas9 requires a complete transcription unit, and multiple corresponding sgRNA expression frames need to be constructed when performing multi-site base editing (C→T and A→G). This not only increases the complexity of the construction process, but also reduces the stability of the DNA sequence due to the repeated use of elements such as promoters. The CRISPR / Cas9-based dual base editor has a limited operable range on the genome, and is inefficient and complex to operate during multi-site base editing (C→T and A→G). CBEs or ABEs based on dCas12a have been successfully constructed, and their PAM sequences are TTV (V = A, C, G), thus expanding the editable range on the genome. However, there are no reports on how to couple cytosine deaminase, adenine deaminase, and dCas12a to simultaneously maintain the activities of the three (C→T, A→G, and crRNA array processing and DNA targeting) to achieve CRISPR / Cas12a-mediated multi-site dual base editing (C→T and A→G).

[0007] Summary of the Invention

[0008] To solve the above problems, the present invention develops a multi-site dual base editor (MultiduBE) based on dCas12a, which can simultaneously perform efficient editing (C→T and A→G) on multiple sites on the genome under the guidance of a single crRNA array. First, by utilizing the constructed single plasmid editor skeleton, the composition and structure of the fusion protein composed of cytosine deaminase, UGI, adenine deaminase and dCas12a are optimized, and the dual editing (C→T and A→G) of multiple sites under the guidance of a single crRNA array is achieved; On this basis, the efficiency of multi-site editing is further improved by introducing and optimizing the artificial spacer sequence in the promoter replacement and crRNA array and the transformation of the DR motif; Finally, the function of the multi-site dual base editor (MultiduBE) is verified by generating multiple resistant strains, generating from scratch riboflavin synthesis strains and improving the yield of surfactant synthesis strains.

[0009] The first object of the present invention is to provide a multi-site dual base editor, which comprises a plasmid containing cytosine deaminase, uracil glycosidase inhibitor (UGI), adenine deaminase TadA, defective nuclease dcas12a and crRNA insertion region; wherein:

[0010] When the cytosine deaminase is hAPOBEC3A, the adenine deaminase TadA is located after the hAPOBEC3A;

[0011] When the cytosine deaminase is hAID, the adenine deaminase TadA is located before the hAID.

[0012] Furthermore, the order of the elements on the plasmid is any of the following: (1) from front to back: cytosine deaminase hAPOBEC3A, adenine deaminase TadA, defective nuclease dcas12a, uracil glycosidase inhibitor; (2) from front to back: adenine deaminase TadA, cytosine deaminase hAID, defective nuclease dcas12a, uracil glycosidase inhibitor.

[0013] Furthermore, the amino acid sequence of the defective nuclease dcas12a is shown in SEQ ID NO.1.

[0014] Furthermore, the GenBank number of cytosine deaminase hAPOBEC3A is KM266646.1; the GenBank number of cytosine deaminase hAID is AAM95402.1; and the amino acid sequence of the adenine deaminase TadA is shown in SEQ ID NO.2.

[0015] Furthermore, the crRNA insertion region is connected to a crRNA array. In practical applications, technicians can replace crRNA as needed to achieve multi-site dual editing of different sequences.

[0016] Furthermore, the crRNA array is constitutively expressed, and the cytosine deaminase, uracil glycosidase inhibitor, adenine deaminase TadA and defective nuclease dcas12a are inducibly expressed.

[0017] Furthermore, the cytosine deaminase, uracil glycosidase inhibitor, adenine deaminase TadA and defective nuclease dcas12a are regulated by repressor proteins LacI and P grac100 The expression is regulated by the promoter or by the repressor proteins TetR and P tet Promoter-regulated expression.

[0018] Furthermore, P with the repressor protein TetR tet The sequence is shown as SEQ ID NO.5.

[0019] Furthermore, the crRNA array is composed of P veg Promoter-regulated expression.

[0020] Furthermore, an artificial spacer sequence (Sp1-4) having a nucleotide sequence such as SEQ ID NO. 6-9 is inserted into the crRNA array, specifically, the artificial spacer sequence is inserted between the DR motif and the spacer.

[0021] Furthermore, the length of the spacer is greater than 17 bp, preferably 17-26 bp, and more preferably 23 bp.

[0022] Furthermore, the DR motif in the crRNA array is extended. Specifically, the extended DR motif nucleotide sequence is shown in SEQ ID NO.11.

[0023] Furthermore, the plasmid contains a temperature-sensitive replicon.

[0024] Furthermore, the temperature-sensitive replicon includes but is not limited to pE194ts and the like.

[0025] A second object of the present invention is to provide a fusion protein for multi-site dual-base editing, wherein the fusion protein comprises any of the following sequences:

[0026] (1) A sequence composed of a fusion of cytosine deaminase hAPOBEC3A, adenine deaminase TadA, defective nuclease dcas12a, and uracil glycosidase inhibitor;

[0027] (2) A sequence composed of adenine deaminase TadA, cytosine deaminase hAID, defective nuclease dcas12a and uracil glycosidase inhibitor.

[0028] Furthermore, the amino acid sequence of the fusion protein is shown in SEQ ID NO.3-4.

[0029] The third object of the present invention is to provide a recombinant strain containing the above-mentioned multi-site dual base editor or fusion protein.

[0030] Furthermore, the recombinant strain uses Bacillus subtilis or Escherichia coli as a starting strain.

[0031] The fourth object of the present invention is to provide the application of the above-mentioned multi-site dual base editor, fusion protein or recombinant strain in gene editing.

[0032] The fifth object of the present invention is to provide the use of the above-mentioned multi-site dual base editor, fusion protein or recombinant strain in constructing mutants.

[0033] The sixth object of the present invention is to provide the application of the above-mentioned multi-site dual base editor, fusion protein or recombinant strain in biosynthesis.

[0034] The seventh object of the present invention is to provide the application of the above-mentioned multi-site dual base editor, fusion protein or recombinant strain in metabolic regulation.

[0035] Beneficial effects of the present invention:

[0036] The present invention designed and constructed a single-plasmid multi-site dual base editor (MultiduBE) based on CRISPR / Cas12a, which achieved base editing (C→T and A→G) at five sites of the genome simultaneously under the guidance of a single crRNA array through optimization and transformation. According to the sequence alignment results, the relevant target genes were mutated, and the mutant strains simultaneously produced four types of resistance to tetracycline, rifampicin, spectinomycin and streptomycin. In addition, the four target genes related to riboflavin synthesis were mutated at the same time, and a mutant strain that could synthesize 344.8 mg / L riboflavin was obtained by mutation of the wild-type strain that could not synthesize riboflavin. This yield is 16 times that of the control strain that only strengthened the riboflavin synthesis gene cluster. Finally, the five relevant target genes of the surfactant synthesis strain were mutated to obtain a mutant strain with a 41.9% increase in yield. The multi-site dual base editor (MultiduBE) only requires the construction of a specific crRNA array. Compared with traditional genome editing methods based on homologous recombination, it eliminates the process of constructing a homologous repair template. Compared with dual base editors based on dCas9 or nCas9, it does not require the use of multiple promoters to express sgRNAs of different target genes. The editor consists of only one temperature-sensitive plasmid, which can be eliminated by heating and culturing after the genome editing is completed. The mutant strain finally obtained only undergoes base mutations at specific target sites on the genome (C→T and A→G, and the corresponding complementary chains are G→A and T→C), thereby achieving the effect of efficient multi-site targeted mutagenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 shows the working principle of base editors;

[0038] Figure 2 shows the design and analysis process of a CRISPR / Cas12a-based multi-site dual base editor (MultiduBE); (a) Schematic diagram of the base editor plasmid backbone; (b) Dual BsaI restriction sites for rapid assembly of crRNA arrays; (c) Coupling of cytosine deaminase, adenine deaminase, UGI, and dCas12a to construct a multi-site dual base editor (MultiduBE); (d) Rapid analysis of base editing based on Sanger sequencing;

[0039] Figure 3 shows the construction of a CRISPR / Cas12a-based multi-site dual base editor (MultiduBE); (a) Five base editing validation targets were selected on the aprE and nprE genes; (b) Schematic diagram of crRNA array-mediated multi-site dual base editing, where the array is composed of alternating stacks of direct repeat (DR) sequences and spacer sequences; (c) Composition and structural optimization of the multi-site dual base editor (MultiduBE);

[0040] Figure 4 shows promoter replacement by multi-site base editors;

[0041] Figure 5 shows the optimization and modification of the crRNA array; wherein, (a) adding an artificial spacer (synthetic separator, Sp) sequence to the crRNA array (b) extending the artificial spacer sequence and DR sequence (c) changing the spacer length in the crRNA array (d) optimizing the crRNA array construction method;

[0042] Figure 6 shows the effect of crRNA array optimization and modification on editing efficiency; (a) changes in editing efficiency after adding different Sp sequences and changing spacer (b) the effect of Sp sequence on dCas12a-mediated crRNA array processing;

[0043] Figures 7 and 8 show analysis of mutants generated by the dual base editor (MultiduBE); Figure 7 shows analysis of mutants generated by pWLT-duBE-1a, and Figure 8 shows analysis of mutants generated by pWLT-duBE-2b; the left and right data are the ratios before and after the addition of Sp4, respectively. Asterisks represent mutations in the complementary chain where crRNA is located, and ~ represents single-base deletions.

[0044] Figure 9 shows the effect of the dual base editor (MultiduBE) in Escherichia coli;

[0045] Figure 10 shows the application of MultiduBE in resistance generation; (a) related gene sequence alignment (b) editing targets for resistance generation (c) generation of dual-resistant mutants mediated by MultiduBE (d) generation of quadruple-resistant mutants mediated by MultiduBE;

[0046] Figure 11 shows the de novo generation of riboflavin synthesis strains mediated by the multi-site dual base editor (MultiduBE); (a) Riboflavin synthesis pathway in Bacillus subtilis (b) Targeted mutagenesis targets for riboflavin synthesis regulation (c) Screening of high-yielding riboflavin strains based on a microfluidic system (d) 96-well plate and fluorescence observation of riboflavin synthesis mutant strains (e) Shake flask fermentation of riboflavin synthesis mutant strains;

[0047] Figure 12 shows the increase in surfactin production mediated by multi-site dual base editor (MultiduBE); among them, (a) surfactin target regulation target (b) targeted mutagenesis and screening of surfactin mutant strains (c) shake flask fermentation of surfactin mutant strains. DETAILED DESCRIPTION

[0048] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0049] The materials and methods involved in the present invention are as follows:

[0050] PrimeSTAR high-fidelity DNA polymerase for genomic fragment amplification and T4 DNA ligase for crRNA array ligation were purchased from Takara. Phanta Max Master Mix (Dye Plus) polymerase for crRNA array fragment amplification and Taq DNA polymerase for colony PCR were purchased from Novartis. The restriction endonuclease BsaI for crRNA array construction was purchased from New England Biolabs. The plasmid extraction kit was purchased from Shanghai Biotech Co., Ltd., and the PCR product nucleic acid purification kit was purchased from Thermo Scientific. Riboflavin and surfactin standards were purchased from Yuanye Biotechnology.

[0051] Conventional cell culture was performed using LB medium containing 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl. The final concentrations of kanamycin, tetracycline, rifampicin, and spectinomycin were 50 μg / mL, 200 μg / mL, 50 μg / mL, and 200 μg / mL, respectively. IPTG and anhydrotetracycline (aTC) were 1 mM and 500 nM, respectively.

[0052] When performing shake flask fermentation of riboflavin and surfactin, a single colony after streak activation was picked and placed in a 250 mL flat-bottom shake flask containing 20 mL of LB medium and cultured at 37°C and 220 rpm for 12 h to prepare a seed solution. Then, the seed solution was transferred to a 250 mL baffled shake flask containing 50 mL of fermentation medium at a 5% inoculum and fermented at 37°C and 220 rpm. 1 mL of the sample was taken every 12 h to measure the OD value. 600 , glucose and product content. The fermentation medium contained: glucose 80 g / L, tryptone 6 g / L, yeast powder 12 g / L, urea 6 g / L, glycerol 5 g / L, K2HPO4·3H2O 12.5 g / L, KH2PO4 2.5 g / L, and Mg2SO4 3 g / L.

[0053] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, cell biology, recombinant DNA technology and related fields in the field of this technology, which have been fully described in existing literature.

[0054] Example 1 Design and construction of a multi-site dual base editor (MultiduBE) based on CRISPR / Cas12a

[0055] As shown in Figure 2a, a base editing plasmid backbone pWLBE-dCas12a-N (Molecular Cloud ID: MC_0101391) containing dCas12a (amino acid sequence shown in SEQ ID NO. 1) was constructed, wherein dCas12a was driven by the Bacillus subtilis IPTG-inducible promoter P grac100 The plasmid can be expressed in both Escherichia coli and Bacillus subtilis and can be selected by kanamycin. In addition, the backbone has the thermosensitive replicon pE194ts of Bacillus subtilis, which can be quickly eliminated from Bacillus subtilis by culturing at 50°C. In addition, the promoter P vegThe downstream of the crRNA array contains an insertion region, which contains two BsaI restriction sites. The multi-target crRNA array can be rapidly assembled using our previously optimized SOMACA (Synthetic Oligos Mediated Assembly of crRNA Array) method (Reference: Wu, Y., Liu, Y., Lv, X., Li, J., Du, G., Liu, L., 2020. CAMERS-B: CRISPR / Cpf1 assisted multiple-genes editing and regulation system for Bacillus subtilis. Biotechnology and Bioengineering 117, 1817-1825.) (Figure 2b). Specifically, when expressing a single crRNA, crRNA can be directly obtained by annealing a pair of primers with overlapping regions (primer concentration is 10uM, 10uL of upstream and downstream primers in a 20uL system; reaction conditions are: 98°C for 2min, 0.1°C / S cooling to 4°C and then keeping warm.), the product is diluted 10 times and 1uL is taken and connected to the vector after BsaI digestion. When multiple crRNAs are designed to form a crRNA array, multiple pairs of primers with overlapping regions are first PCR-treated (primer concentration is 10uM, 20uL system contains 10uL of DNA polymerase and 5uL of upstream and downstream primers; standard PCR program is used, extension time is 5 seconds, 10 cycles), and then diluted 10 times to obtain double-stranded DNA with BsaI at both ends. Take 1μL of each of the above double-stranded DNA and the plasmid for golden gate assembly. After the above product was transformed into Escherichia coli, a primer was selected on the plasmid backbone and crRNA for colony PCR, and a single colony with a band was picked for sequencing to screen for positive clones containing the required crRNA.

[0056] As shown in Figure 2c, in order to construct a multi-site dual base editor (MultiduBE), cytosine deaminase (hAPOBEC3A, hAID, LjCDA1L2_1, GenBank numbers KM266646.1, AAM95402.1, MG495262.1) that matches dCas12a well, adenine deaminase (TadA9, amino acid sequence as shown in SEQ ID NO.2) and UGI (GenBank: YP_009283008.1) were introduced into the plasmid backbone. Finally, the plasmid containing the specific crRNA array and different fusion proteins was transformed into Bacillus subtilis, and the base editing efficiency was rapidly analyzed using the operating steps shown in Figure 2d, and a multi-site dual base editor (MultiduBE) with strong activity was screened. The specific operation steps are as follows: First, the editing plasmid connected to the specific crRNA array was transformed into Bacillus subtilis, coated with a plate containing kanamycin, and cultured at 30°C for 12 hours; then, a single colony was picked and inoculated into a 14mL shake tube containing 2mL LB medium (containing kanamycin) and shake-cultured at 30°C for 12 hours; then, 5μL of the bacterial solution was transferred to three 14mL shake tubes containing 2mL LB medium (containing kanamycin and IPTG) and shake-cultured at 30°C for 36 hours to induce the expression of the base editing system; finally, 50μL was taken from each of the three parallel samples that had completed editing and mixed in equal volumes, and then 2μL was taken from the mixed bacterial solution to a 50μL PCR reaction system to amplify the target gene fragment. After purification, the DNA fragment was sequenced by Sanger sequencing and the sequencing results were analyzed using BEAT software (https: / / hanlab.cc / beat / ).

[0057] Here, aprE and nprE were selected as editing verification targets for the construction and optimization of multi-site dual base editors. As shown in Figure 3a, four targets were selected on the aprE gene and one target was selected on the nprE gene. The crRNA targeting the five targets was assembled by the SOMACA method described above to obtain a crRNA array, which can be cut and processed by dCas12a to complete multi-site guidance, and the dual base editing (C→T and A→G) of the non-targeted chain is completed under the action of the cytosine deaminase and adenine deaminase fused on dCas12a (Figure 3b).

[0058] As shown in Figure 3c, the composition and structure of the multi-site dual base editor (MultiduBE) were optimized. Three cytosine deaminases (hAPOBEC3A, hAID, LjCDA1L2_1), one adenine deaminase (TadA9), UGI, and dCas12a were combined and arranged on the pWLBE-dCas12a-N backbone to construct different fusion proteins. Subsequently, a crRNA array targeting five target sites of the aprE and nprE genes was constructed, and multi-site base editing analysis and verification were performed using the method described in Figure 2d (the primer sequences used for aprE and nprE amplification sequencing are shown in Table 1). It can be seen that pWLBE-duBE-1a and pWLBE-duBE-2b have strong multi-site dual editing effects, and can achieve dual base editing of four targets (C→T and A→G). The amino acid sequences of the fusion proteins duBE-1a and duBE-2b are shown in SEQ ID NO.3 and SEQ ID NO.4; although pWLBE-duBE-3b and pWLBE-duBE-3c can edit four sites, they only have editing activity for adenine (A→G), while the editing sites of the other fusion proteins are less than 4. Ultimately, duBE-1a with an amino acid sequence as shown in SEQ ID NO.3 and duBE-2b with an amino acid sequence as shown in SEQ ID NO.4 were selected as multi-site dual base editors (MultiduBE), and further optimization was performed to improve their editing efficiency.

[0059] Table 1 Primer sequences used for aprE and nprE amplification and sequencing

[0060] Example 2 Optimization and modification of multi-site dual base editor (MultiduBE)

[0061] pWLBE-duBE-1a and pWLBE-duBE-2b can only achieve dual base editing at 4 sites under the guidance of the crRNA array targeting 5 sites. Therefore, the multi-site dual base editor (MultiduBE) was further optimized and modified by promoter replacement and crRNA array structure modification to further improve the editing efficiency. As shown in Figure 4, using the tetracycline-inducible promoter P tet Replaced the original P of pWBE-duBE-1a and pWBE-duBE-2b grac100 The promoter (corresponding to the repressor protein LacI is also replaced by TetR, and the P with the repressor protein TetR tetThe sequence is shown in SEQ ID NO. 5), resulting in plasmids pWLT-duBE-1a and pWLT-duBE-2b. pWLT-duBE-1a enabled editing at five sites, extending the editing window compared to the pre-promoter modification, but the editing efficiency at the fifth site was only 3%. pWLT-duBE-2b, on the other hand, could only edit four sites, with the fourth site's editing efficiency below 10%.

[0062] In order to further enhance the editing efficiency of the multi-site dual base editor (MultiduBE), an attempt was made to add an artificial spacer (synthetic separator, Sp) sequence to the crRNA array. As shown in Figures 5a and 5b, we introduced different Sp sequences between DR and spacer (the nucleotide sequences of Sp1-5 are shown in SEQ ID NO.6-10) in order to further improve the editing efficiency; in addition, in order to optimize the construction process of the crRNA array, the original DR sequence was extended to increase its Tm value to about 55°C (the nucleotide sequence of DR+ is shown in SEQ ID NO.11). At the same time, an attempt was made to change the length of the spacer in the crRNA array to examine its effect on the editing efficiency (Figure 5c).

[0063] As shown in Figure 5d, the specific steps for optimizing the crRNA array construction method after adding the Sp sequence are as follows: (1) The crRNA at position 1 is directly amplified into a double strand by a round of PCR using a pair of primers. The amplification system contains 10 μL Phanta Max and 5 μL of upstream and downstream primers, and 30 cycles of amplification are performed according to the instructions. (2) The crRNA at the rear position was obtained by two rounds of PCR. In the first round, double-stranded DNA was obtained by PCR (the upstream primer was a single-stranded DR+, spacer and Sp sequence, and the downstream primer was a complementary single-stranded sequence of the universal Sp sequence) to obtain a double-stranded DNA fragment with repetitive sequences (DR+ and Sp sequence) at both ends. The amplification system contained 10 μL Phanta Max and 5 μL each of the upstream and downstream primers, and 15 cycles were amplified according to the instructions. In the second round of PCR, sticky ends were added to both ends of the double-stranded DNA fragment obtained in the first round for golden gate assembly. The 3′ ends of the upstream and downstream primers used would bind to the repetitive sequence and were therefore universal, while the 5′ ends were adjusted according to the enzyme cutting interface added as needed (refer to the SOMACA method). The amplification system contained 10 μL Phanta Max, 5 μL each of the upstream and downstream primers, and 1 μL of the PCR product from the first round, and 30 cycles were performed. (3) The crRNA double-stranded DNA fragments obtained by the above amplification were diluted ten-fold and mixed in equal volumes, and the golden gate assembly system was prepared (each 10 μL included 2 μL of T4 DNA ligase, 1 μL of BasI, 1 μL of T4 DNA ligase buffer, 1 μL of plasmid, 2 μL of crRNA mix, and 3 μL of ddH2O). The assembly was performed according to the program in Table 2. After completion, the fragments were transformed into Escherichia coli for sequencing. After confirming that the construction was successful, the fragments were transformed into Bacillus subtilis for genome editing verification.

[0064] Table 2 Assembly procedures of crRNA arrays

[0065] As shown in Figure 6a, Sp4 (nucleotide sequence is shown in SEQ ID NO.9) has a significant improvement effect on the editing efficiency of both pWLT-duBE-1a and pWLT-duBE-2b. Under the guidance of the crRNA array containing Sp4, dual base editing of 5 sites can be achieved, and Sp1-3 also has a certain degree of improvement in editing efficiency. In addition, changing the spacer length will also have a certain effect on the editing efficiency. After the length is shortened to 14bp, dCas12a cannot be guided to edit the genome, and spacers greater than 17bp have a guiding effect, but the spacer sequence of 23bp is still the most favorable for editing. In addition, we also performed sequencing analysis on the small RNA produced by the crRNA array expression before and after adding Sp4, and found that the addition of Sp4 did promote the processing and cutting of the crRNA array, thereby improving the targeting and editing efficiency of multiple sites (see Figure 6b).

[0066] Example 3 Analysis of mutants generated by multi-site dual base editor (MultiduBE)

[0067] During the above-mentioned transformation and optimization process, the base editing results were analyzed by Sanger sequencing. This analysis process is short and inexpensive, making it suitable for the initial construction and optimization of multi-site dual base editors (MultiduBE). However, the accuracy of Sanger sequencing is low, and low-frequency mutations cannot be detected. Moreover, it can only derive the approximate proportion of base mutations, and cannot derive the specific composition of the mutants, let alone determine whether dual base editing (C→T and A→G) occurs simultaneously in the same sequence. Therefore, we further analyzed the multi-site base editing of pWLT-duBE-1a and pWLT-duBE-2b under the guidance of crRNA arrays before and after the addition of Sp4 by high-throughput sequencing. The targets of aprE and nprE gene editing in the three parallel editings were amplified and sequenced, and the composition of the 9 most frequently occurring mutants was analyzed. As shown in Figures 7 and 8, there are significant differences in the composition of mutants generated by pWLT-duBE-1a and pWLT-duBE-2b. Moreover, for both, the introduction of Sp4 did not improve the editing efficiency at target 1. On the contrary, the editing efficiency decreased slightly after the addition of Sp4. This may be because the addition of Sp4 promoted the maturation of subsequent crRNAs, thereby reducing the relative proportion of dCas12a bound to the first crRNA. For target 5, where the editing efficiency has always been low, the addition of Sp4 can significantly improve the editing efficiency.

[0068] As shown in Figure 7, when using pWLT-duBE-1a for multi-site base editing, the A→G editing efficiency of target 1 was low. Among the 9 most frequently occurring mutants, 8 were single-point or multi-point C→T mutations, and no mutants with both C→T and A→G were found. In the following gene targets, mutants with both C→T and A→G were observed. A lower frequency (less than 2%) of bystander editing out-of-protospacer (BEOP) outside the crRNA target was also found at targets 1, 3, and 5. For example, a C→T mutation occurred in the complementary chain at position 40 of target 1 (0.31 and 0.32% before and after adding Sp4, respectively). As shown in Figure 8, when using pWLT-duBE-2b for multi-site base editing, the A→G editing efficiency of target 1 was improved compared with pWLT-duBE-1a, but no mutants with both C→T and A→G were observed. Unlike pWLT-duBE-1a, mutants with both C→T and A→G were only found at targets 2 and 3, and a lower frequency (less than 0.5%) of BEOP was found at targets 1, 4, and 5.

[0069] Example 4 Effect of MultiduBE in Escherichia coli

[0070] The promoter P used to express dCas12a in pWLT-duBE-1a and pWLT-duBE-2b tet and promoter P for crRNA expression veg It is active in both Escherichia coli and Bacillus subtilis, so we further verified whether the multi-site dual base editor (MultiduBE) can function in E. coli. As shown in Figure 9, five target sites were selected on the xylR and ykgH genes of E. coli, and the corresponding crRNA arrays were constructed. Sequencing analysis showed that pWLT-duBE-1a has multi-site dual base editing activity in E. coli, and the addition of Sp4 can increase the number of editing targets from 4 to 5.

[0071] Example 5 Application of Multi-site Dual Base Editor (MultiduBE) in Resistance Generation

[0072] First, the generation of multiple resistant mutants was selected as functional verification of the multi-site dual base editor (MultiduBE). As shown in Figure 10a, the antibiotic target gene rpsE (encoding ribosomal protein S5) in the spectinomycin-resistant mutants of Neisseria gonorrhoeae and Streptomyces roseosporus reported in the previous report was mutated. The RpsE proteins in the above two hosts were sequenced with the RpsE protein of Bacillus subtilis, and the potential mutation target that can cause spectinomycin resistance in Bacillus subtilis was identified; the rpsL gene (encoding ribosomal protein S12) of Borrelia burgdorferi, Mycobacterium tuberculosis, and Streptomyces coelicolor can cause streptomycin resistance after mutation. The RpsL proteins in the above hosts were sequenced with the RpsL protein of Bacillus subtilis, and the potential mutation target that can cause spectinomycin resistance in Bacillus subtilis was identified. According to previous reports on Bacillus subtilis, mutations in the promoter region of its tetL gene (tetracycline efflux protein leader peptide) can produce tetracycline resistance, mutations in its rpoB gene (encoding RNA polymerase β subunit) can produce rifampicin resistance, and mutations in its mthA gene (encoding methylthioadenosine / s-adenosine homocysteine ​​nucleosidase) can also produce streptomycin resistance.

[0073] Here, we first target P tetL The crRNAs targeting rpoB and rpsE were constructed into a binary array to investigate whether they could guide multi-site dual base editors (MultiduBE) to perform targeted mutagenesis on the genome and produce resistance to tetracycline and rifampicin. In addition, four different crRNAs were set up for the potential mutation sites of rpsE, which were consistent with the above-mentioned targeting P tetL crRNAs were combined to form four binary arrays to produce tetracycline and spectinomycin resistance; in addition, two different crRNAs were set for the rpsL gene and one crRNA was set for mthA, which were combined with the above-mentioned targeting P tetLcrRNA is combined to form three binary arrays to produce two resistances to tetracycline and streptomycin. Since the product spectra of the two multi-site dual base editors (MultiduBE) pWLT-duBE-1a and pWLT-duBE-2b are quite different, mixing them can produce more mutants. Therefore, the above-mentioned binary crRNA array library is simultaneously connected to a mixture of pWLT-duBE-1a and pWLT-duBE-2b, and then transformed into Bacillus subtilis BSZRG strain (preliminary construction based on Bacillus subtilis 168, reference Li, Y., Wu, Y., Liu, Y., Li, J., Du, G., Lv, X., Liu, L., 2022. A genetic toolkit for efficient production of secretory protein in Bacillus subtilis. Bioresource Technology 363, 127885.) for targeted induced mutation. As shown in Figure 10c, the binary crRNA array was transformed into Bacillus subtilis to induce mutation, and then the corresponding antibiotics were added for screening. The mixed bacterial solution obtained by screening was sequenced and analyzed for the target gene. Finally, three single colonies were picked for each resistance combination after streaking and separation for target gene sequencing. Before adding resistance screening, the crRNA array sequencing used to generate tetracycline + rifampicin showed a single peak, while the crRNA arrays used to generate tetracycline + spectinomycin and tetracycline + streptomycin showed superimposed peaks due to the presence of multiple combinations; after adding the corresponding dual antibodies for screening, the sequencing of the original mixed crRNA array also became a single peak, indicating that the targeted crRNA that can produce corresponding resistance was screened and enriched in this process; through sequencing analysis of the corresponding target genes on the genome of the mixed bacterial liquid after screening, it was found that the mutation site was consistent with the crRNA array finally enriched, indicating that the crRNA array can be used as a tracker of mutation targets on the genome; sequencing analysis of three single colonies of the three dual antibody combinations showed that the three single colonies of tetracycline + rifampicin and tetracycline + spectinomycin resistance mutants had the same mutation, while the three single colonies of tetracycline + streptomycin resistance mutants had different mutations.

[0074] On this basis, based on the enrichment process of the crRNA array during the screening of resistant mutants, the optimal crRNA corresponding to the four resistances was determined and constructed into a four-membered crRNA array (DNA sequence see sequence 12) for targeted mutagenesis of four mutants; using the above crRNA array, one tetracycline + rifampicin + spectinomycin + streptomycin quadruple-resistant mutant strain, two tetracycline + rifampicin + streptomycin triple-resistant mutant strains and three tetracycline + spectinomycin + streptomycin triple-resistant mutant strains were induced (Figure 10d).

[0075] Example 6 De novo generation of riboflavin synthesis strains mediated by multi-site dual base editor (MultiduBE)

[0076] Riboflavin (also known as vitamin B2) is a heat-resistant, water-soluble vitamin essential to the human body. It is a component of the cofactor of flavonoid enzymes in the body. Its neutral solution in water and ethanol is yellow and has strong fluorescence. As shown in Figure 11a, there are two riboswitches (guanine riboswitch, FMN riboswitch) in the riboflavin synthesis pathway in Bacillus subtilis, which will produce feedback inhibition on the synthesis pathway; in addition, the riboflavin kinase encoded by the essential gene ribC can convert riboflavin into FMN, which not only consumes riboflavin, but the generated FMN further strengthens the feedback inhibition of FMN riboswitch; in addition, the 6-phosphate glucose dehydrogenase encoded by zwf is a key step in the distribution of intracellular carbon flow in the pentose phosphate pathway and will also affect the synthesis of riboflavin. Therefore, we selected the two riboswitches, guanine riboswitch and FMN riboswitch, the ribC gene and the zwf gene as the targeted mutagenesis targets for riboflavin synthesis; we also selected a target P mentioned above. tetcrRNA was used to observe the base editing induced in the absence of the corresponding selection pressure (Figure 11b), and then the crRNA acting on the above target was constructed into a five-membered crRNA array (DNA sequence see sequence 13). As shown in Figure 11c, the base editing plasmid containing the crRNA array was transformed into a riboflavin-free Bacillus subtilis G600 strain (preliminary construction based on Bacillus subtilis 168, reference Li, Y., Wu, Y., Liu, Y., Li, J., Du, G., Lv, X., Liu, L., 2022. A genetic toolkit for efficient production of secretory protein in Bacillus subtilis. Bioresource Technology 363, 127885.), and then targeted mutagenesis of the five corresponding sites was performed to generate a mutant strain that can synthesize riboflavin from scratch; the mutant library after mutagenesis was diluted to OD 50 using a culture medium containing 30 g / L glucose and kanamycin. 600 =0.1, single-cell droplet encapsulation was performed and cultured at 30°C for 24 hours; then, a microfluidic system was used to sort the droplets with higher fluorescence (riboflavin itself is fluorescent, and the yield after secretion outside the cell is proportional to the fluorescence intensity). The cells in the collected droplets were spread on a plate containing kanamycin for culture, and single colonies were picked and screened in a 96-well plate for fluorescence measurement. A total of s1-s12 mutant strains were obtained (Figure 11d). Although tetracycline was not used for screening, P among these 12 strains was tet As shown in Figure 11e, five mutant strains, Rib-s4, Rib-s8, Rib-s11, Rib-s12, and Rib-s14, were selected and the base editing plasmids were eliminated before shake flask fermentation. The Rib-s0 strain was constructed as a control (using P veg The promoter replaced the promoter of the rib operon and the FMN riboswitch region), the shake flask yields of the five mutants were higher than that of the control strain Rib-s0 (21.5 mg / L), and the yield of the mutant strain with the highest yield, Rib-s12 (344.8 mg / L), was 16 times that of the control.

[0077] Example 7 Multi-site dual base editor (MultiduBE) mediated increased surfactin production

[0078] Surfactin is a lipopeptide biosurfactant synthesized by a non-ribosomal peptide synthetase (NRPS) from the genus Bacillus. Compared to chemical surfactants, it possesses advantages such as anti-adhesion, anti-biofilm, anti-bacterial and anti-inflammatory, anti-mycoplasma, and anti-viral properties. It has broad application prospects in biopharmaceuticals, environmental remediation, oilfield extraction, cosmetics, and daily necessities. As shown in Figure 12a, based on relevant reports in the literature, five genes—srfAA (encoding surfactin synthase), remA (encoding a regulatory protein of genes involved in biofilm formation), spoIVB (encoding a serine protease), lcfB (encoding), and ilvB (encoding)—were selected as editing targets for surfactin synthesis regulation. The five crRNAs acting on these targets were then constructed into a five-membered crRNA array (DNA sequence is shown in SEQ ID NO: 14). The wild-type Bacillus subtilis 168 strain, encoding the sfp gene for 4-phosphopantetheinyltransferase (which activates surfactin synthase), is inactive, making it unable to synthesize surfactin. Here, we first reversed the sfp gene in Bacillus subtilis G600 to restore its activity (the restored amino acid sequence is shown in SEQ ID NO: 15), generating strain Sur-S0. As shown in Figure 12b, the crRNA array containing genes targeting surfactin synthesis was connected to the base editing plasmid and transformed into the strain Sur-s0 for targeted mutagenesis of surfactin synthesis-related genes, and the mutant strains were analyzed and screened by colorimetric method (reference Yang, H., Yu, H., Shen, Z., 2015. A novel high-throughput and quantitative method based on visible color shifts for screening Bacillus subtilis THY-15 for surfactin production. Journal of Industrial Microbiology and Biotechnology 42, 1139-1147.), and mutant strains Sur-s1 to Sur-s12 were obtained (among which Sur-s6, Sur-s9 and Sur-s10 strains were wild type or repeated with other mutant strains).Finally, five strains, G600, Sur-s0, Sur-s1, Sur-s11 and Sur-s12, were selected for shake flask fermentation. No surfactin was detected in the fermentation broth of G600. The control strain Sur-s0 could synthesize 702.7 mg / L of surfactin, while the production of the mutant strain Sur-s1 increased by 42.0% compared with the control strain, reaching 997.5 mg / L (Figure 12c).

[0079] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A multi-site dual base editor, It is characterized in that The multi-site dual base editor comprises a plasmid containing cytosine deaminase, a uracil glycosidase inhibitor, an adenine deaminase TadA, a defective nuclease dcas12a and a crRNA insertion region; wherein: When the cytosine deaminase is hAPOBEC3A, the adenine deaminase TadA is located after the hAPOBEC3A; When the cytosine deaminase is hAID, the adenine deaminase TadA is located before the hAID.

2. The multi-site dual base editor according to claim 1, It is characterized in that The order of the elements on the plasmid is any of the following: (1) from front to back: cytosine deaminase hAPOBEC3A, adenine deaminase TadA, defective nuclease dcas12a, uracil glycosidase inhibitor; (2) from front to back: adenine deaminase TadA, cytosine deaminase hAID, defective nuclease dcas12a, uracil glycosidase inhibitor.

3. The multi-site dual base editor according to claim 1, Features: The crRNA insertion region is connected to a crRNA array.

4. The multi-site dual base editor according to claim 3, Features: The crRNA array is constitutively expressed, and the cytosine deaminase, uracil glycosidase inhibitor, adenine deaminase TadA and defective nuclease dcas12a are inducibly expressed.

5. The multi-site dual base editor according to claim 4, Features: The cytosine deaminase, uracil glycosidase inhibitor, adenine deaminase TadA and defective nuclease dcas12a are regulated by repressor proteins LacI and P grac100 The expression is regulated by the promoter or by the repressor proteins TetR and P tet Promoter regulates expression.

6. The multi-site dual base editor according to any one of claims 3 to 5, Features: The crRNA array is composed of P veg Promoter regulates expression.

7. The multi-site dual base editor according to any one of claims 3 to 5, Features: An artificial spacer sequence having a nucleotide sequence as shown in SEQ ID NO.6-9 is inserted into the crRNA array.

8. The multi-site dual base editor according to claim 7, Features: The artificial spacer sequence is inserted between the DR motif and the spacer region; the length of the spacer region is greater than 17 bp.

9. The multi-site dual base editor according to any one of claims 3 to 5, Features: The DR motif in the crRNA array is extended; the nucleotide sequence of the extended DR motif is shown in SEQ ID NO.

11.

10. The multi-site dual base editor according to claim 1, Features: The plasmid contains a temperature-sensitive replicon.

11. A fusion protein for multi-site dual-base editing, It is characterized in that The fusion protein comprises any of the following sequences: (1) A sequence composed of cytosine deaminase hAPOBEC3A, adenine deaminase TadA, defective nuclease dcas12a and uracil glycosidase inhibitor; (2) A sequence formed by the fusion of adenine deaminase TadA, cytosine deaminase hAID, defective nuclease dcas12a and uracil glycosidase inhibitor.

12. A recombinant strain containing the multi-site dual base editor according to any one of claims 1-10 or the fusion protein according to claim 11.

13. The recombinant strain according to claim 12, Features: The recombinant strain uses Bacillus subtilis or Escherichia coli as the starting strain.

14. Use of the multi-site dual base editor according to any one of claims 1 to 10, the fusion protein according to claim 11, or the recombinant strain according to claim 12 or 13 in gene editing.

15. Use of the multi-site dual base editor according to any one of claims 1 to 10, the fusion protein according to claim 11, or the recombinant strain according to claim 12 or 13 in constructing a mutant.

16. Use of the multi-site dual base editor according to any one of claims 1 to 10, the fusion protein according to claim 11, or the recombinant strain according to claim 12 or 13 in biosynthesis.

17. Use of the multi-site dual base editor according to any one of claims 1 to 10, the fusion protein according to claim 11, or the recombinant strain according to claim 12 or 13 in metabolic regulation.

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