CN116254265A - Design and assembly method of ultra-long flexible gRNA array and multi-target editing system composed of same - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a design and assembly method of an ultra-long flexible gRNA array and a multi-target editing system composed of the same. According to the invention, through the design of the gRNA array structure and the selection of non-repeated elements in the gRNA array structure, the overall repeated degree is reduced, so that the synthesis difficulty of the gRNA array is reduced, and the stability of the array is improved. The invention can realize the design and assembly of 168 gRNAs and even more arrays of gRNAs. Meanwhile, due to the addition of the vox sequence in the array, the array becomes flexible and variable, and random deletion and replication of the rearrangement unit in the ultra-long flexible gRNA array are realized, so that regional multi-target editing of the genome is finally realized.

Description

Design and assembly method of ultra-long flexible gRNA array and multi-target editing system composed of same

Technical Field

The invention relates to the technical field of biology, in particular to a design and assembly method of an ultra-long flexible gRNA array and a multi-target editing system composed of the same.

Background

With the development of genome coding technology, multiple genome engineering has been widely used in the fields of multiple genome editing, rapid metabolic pathway modification, and the like. The multi-target editing is used as a powerful molecular biology operation means, and can greatly improve the range and efficiency of gene editing and transcription regulation. Multiple automated genome engineering (Multiplex automated genome engineering, MAGE) and CRISPR-Cas9 based multi-target editing techniques are often used for multi-target editing. MAGE is an automated, high-throughput method that relies on annealing of oligonucleotide strands at target sites to accurately introduce mutations. But its efficiency is limited by the increased global mutation rate of the genome due to inhibition of DNA mismatch repair, which is a result of heterologous expression efficiency of single-stranded DNA binding proteins. Unlike MAGE systems, which have a high degree of host-specific selection, CRISPR-Cas9 systems are capable of efficient genome editing in a variety of organisms. The CRISPR-Cas 9-based multi-target editing technology has the characteristics of strong targeting and no host dependence, so that the technology is widely applied to the fields of genome modification, metabolic path regulation and the like. Furthermore, the CRISPR-Cas12a system is also used in multi-target editing due to the simplicity of the crRNA structure. However, the assembly of a large number of repeat structures in a gRNA array presents challenges for its application in the multi-target field.

The number of edits by CRISPR-Cas9 based multi-target editing techniques depends on the number of grnas in the system. The application of the ultra-long gRNA array can effectively improve the quantity and stability of editing a plurality of different targets in a single strain based on CRISPR-Cas 9. There are two approaches to expressing multiple gRNAs today, including (i) constructing multiple plasmids containing a single gRNA expression cassette; (ii) polycistronic expression of the gRNA. The design of the gRNA array based on the gRNA polycistronic expression can effectively improve the editing range and efficiency of the CRISPR-Cas9 multi-target editing technology. However, the number of type III promoters in yeast is limited, and the gRNA scaffold sequences cannot be altered, resulting in a high degree of repetition of the resulting gRNA array. Therefore, how to synthesize a stable ultralong gRNA array for multi-target editing of different sites in a single bacterium is a key factor.

Currently, researchers can use a single gRNA targeted to multiple copy sites to effect ten thousand edits to the genome. And editing of a plurality of specific sites can realize the replacement of 33 essential gene stop codons and the deletion of 25 porcine endogenous retroviruses (Porcine endogenous retroviruses, PERVs). By assembling and expressing different gRNA expression cassettes, 22 gRNAs can be assembled in vivo and 13 genes in the large intestine can be inhibited from being expressed.

However, the efficiency of existing multi-target editing techniques such as MAGE is limited by the efficiency of heterologous expression of single-stranded DNA binding proteins and the increased global mutation rate of the genome due to inhibition of DNA mismatch repair. Multi-target editing based on CRISPR systems is limited by the length of the gRNA array. Because of the high degree of element repetition in the gRNA array, assembly and problems with the array are challenged. Meanwhile, the genome is edited in a partitioning way, so that the genome editing area is diversified, and the research of multi-target editing in aspects of gene interaction, metabolic regulation and the like is promoted.

Disclosure of Invention

In view of the above, the present invention provides an ultra-long flexible gRNA array, a design method, an assembly method, and a multi-target editing system composed of the same. The invention reduces the overall repetition degree by adding vox sequences and selecting non-repeated elements in the gRNA array, thereby reducing the synthesis difficulty of the gRNA array, improving the stability of the array, realizing the design and assembly of 168 gRNAs and even more gRNAs, and realizing

In order to achieve the above object, the present invention provides the following technical solutions:

a gRNA transcription unit comprising a promoter, a tRNA, a gRNA of a target sequence, a VOX sequence, and a terminator;

The VOX sequence is shown as SEQ ID NO. 1.

In the invention, the number of the gRNAs of the target sequence is k, and k is more than or equal to 3 and less than or equal to 6; the gRNA of the target sequence comprises: a guide sequence complementary to the target sequence, a scaffold sequence and a termination sequence; in some embodiments, the number of grnas of the target gene is 3, specifically including gRNA1, gRNA2 and gRNA3, where the gRNA1, gRNA2 and gRNA3 comprise different scaffold sequences, and are randomly called from 3 synthetic non-repetitive gRNA scaffold libraries. Further, the bracket sequence is selected from the sequences shown in SEQ ID NO. 23-25, and the termination sequence is shown in SEQ ID NO. 26.

The number of tRNA in each gRNA transcription unit is 4-7, the tRNA can be randomly called from a sequence library consisting of 21 yeast tRNA, and specifically, the sequence of the tRNA is shown as SEQ ID NO. 2-22.

In the gRNA transcription unit, the promoter may be from a synthetic yeast type II promoter library ^[1] Any call in the above. The terminator can be randomly called from a sequence library consisting of 12 synthetic terminators (the sequences are shown as SEQ ID NOs: 27-38).

In some embodiments, when the number of gRNAs is 3 in each gRNA transcription unit, the number of tRNAs is 4, and specifically includes 4 different tRNAs, namely tRNA1, tRNA2, tRNA3 and tRNA4, which are respectively selected from different tRNAs shown in SEQ ID NOs 2-22; the gRNA transcription unit comprises the following components from the 5 'end to the 3' end: the structure of the promoter-tRNA 1-gRNA1-VOX sequence-tRNA 2-gRNA2-VOX sequence-tRNA 3-gRNA3-VOX sequence-tRNA 4-terminator is shown in FIG. 14. In the gRNA transcription unit, gRNA1, gRNA2 and gRNA3 represent only three different grnas, and do not represent the sequence of the grnas in the transcription unit, and the sequence can be arbitrary.

The invention also provides a gRNA array which consists of the gRNA transcription unit.

In some embodiments, the gRNA array of the invention comprises X mid-level fragments comprising a total of m first-level fragments, each first-level fragment comprising 3-5 gRNA transcription units, each transcription unit comprising k grnas, the m first-level fragments divided into X mid-level fragments, on average or non-average; wherein x is greater than or equal to 1, m is greater than or equal to 1, k is greater than or equal to 3, and x, m and k are integers.

The invention provides a design and assembly method of the gRNA array, which comprises the following steps:

step (1): obtaining n gRNAs according to target gene design, dividing the gRNAs into n/k gRNA transcription units according to any one of claims 1-5, wherein each transcription unit comprises k gRNAs, k is more than or equal to 3, n is more than or equal to 3, and n and k are integers;

step (2): every 3-5 gRNA transcription units form a first-stage fragment, and n/k gRNA transcription units are equally divided into m first-stage fragments;

step (3), the m primary fragments are grouped evenly or unevenly, and each group of primary fragments forms a middle stage fragment; the first-stage segment is designed as follows, and a first-stage assembly segment is obtained:

adding homologous arms and restriction enzyme cutting sites at two ends of each primary segment, and adding the same homologous arms at the 3 'end of the previous primary segment and the 5' segment of the next primary segment in two adjacent primary segments to integrate the primary segments through enzyme cutting and Gibson assembly; the homology arms added at the 5 'end of the first primary segment and the 3' end of the X primary segment are carrier homology arms, and the rest homology arms are random homology sequences;

Step (4): and respectively synthesizing m first-stage assembly fragments, and performing in vitro enzyme digestion and Gibson assembly to obtain the full-length sequence of the gRNA array.

In the step (1), the n/k gRNA transcription units may be started by more than one promoter, or may be started by different promoters, preferably different promoters are used, i.e., the n/k gRNA transcription units are started by n/k different promoters, respectively;

in the step (2), n/k gRNA transcription units are equally divided into m primary fragments; every 3-5 gRNA transcription units form a first-stage fragment, and the specific number can be 3, 4 or 5, and can be determined according to the total number and the total length of gRNAs.

In the step (3), dividing m primary fragments into x intermediate fragments on average or non-average; when m primary fragments cannot be equally divided into several intermediate fragments, for example, in the specific embodiment of the present invention, when the number of grnas is 168, the gRNA array includes 14 primary fragments, each primary fragment includes 4 gRNA transcription units, each transcription unit includes 3 grnas, that is, each primary fragment includes 12 grnas; the 14 primary segments are divided into four combinations, i.e., the first intermediate segment comprises: 1-4 primary segments, the second intermediate segment comprising: 5-8 primary segments; the third intermediate segment comprises: 9-12, the fourth intermediate segment comprising: 13-14 primary segment.

After the gRNAs are grouped according to the method, the primary fragments are also required to be designed as follows for synthesis and assembly of the primary fragments, and the grouping schemes with the number of the gRNAs of 30 and 168 are taken as an example for illustration respectively:

(1) The gRNA array comprises 30 gRNAs with the total length of 7821bp, the structure is shown in figure 15, the number of the gRNAs n=30 and k=3, namely each transcription unit comprises 3 gRNAs, the gRNA array comprises 10 gRNA transcription units, each 5 gRNA transcription units form a first-stage fragment, and the gRNA array comprises two first-stage fragments in total. The two primary segments were designed as follows, see fig. 19:

two homologous arms A and B are respectively added at two ends of a first primary segment, two homologous arms B and C are respectively added at two ends of another primary segment, wherein the homologous arms A and C are homologous arms at two ends of a carrier (such as a pCCI carrier), and meanwhile, a proper enzyme cutting site (such as EcoRI, xhoI, notI) is added at the tail end of the homologous arm, and the two primary assembly segments obtained through the design are respectively synthesized by a gene synthesis company.

The two primary assembled fragments synthesized were digested with restriction enzymes (after the synthesis of gene company, constructed into plasmid, i.e., the primary assembled fragments were actually in the form of plasmid), and assembled by Gibson, the two primary fragments were integrated with homology arm B, and the integrated fragments were ligated to pCCI-LEU plasmid in vitro using homology arms A and C at both ends, as shown in FIG. 16.

(2) When the number of the gRNAs is 168, the total length of the array is 50,530bp, and the array comprises 14 primary fragments, each primary fragment comprises 4 gRNA transcription units, each transcription unit comprises 3 gRNAs, namely each primary fragment comprises 12 gRNAs; the 14 primary segments are divided into four combinations P1, P2, P3 and P4, i.e. the P1 primary segment comprises: 1-4 primary fragments, P2 intermediate fragments comprising: 5-8 primary segments; the P3 mid-stage fragment includes: 9-12 grade fragments, and the P4 grade fragment comprises: 13-14 primary segment.

Homology arms are respectively added at two ends of the first-stage fragment, wherein the 5 'end of the first-stage fragment and the 3' end of the 14 th-stage fragment are respectively added with a carrier (such as pCCI carrier) homology arm A and L, and the rest positions are added with homology arms of random sequences, such as homology arms with the numbers of B-N. Meanwhile, appropriate enzyme cutting sites (such as EcoRI, xhoI, notI) are added at the tail ends of all homologous arms, and 14 first-stage assembly fragments obtained through the design are respectively marked as follows:

the P1 intermediate assembly segment includes: 1-4 first-stage assembly segments, wherein the P2 intermediate-stage assembly segments comprise: 5-8 first-stage assembly segments; the P3 mid-level assembly segment includes: 9-12 level assembly segments, the P4 level assembly segment comprising: 13-14 first-stage assembly segments. The 14 first-order assembly fragments were synthesized by Gene Synthesis company, respectively.

During assembly, the intermediate assembly segment is first assembled with the intermediate assembly segment as unit, and the intermediate assembly segment P1 is obtained through one enzyme cutting and one Gibson assembly of the 1-4 first assembly segments and connected to pUC57 plasmid. pUC57 recombinant plasmids containing P1, P2, P3 and P4 were obtained according to the method, respectively; the four pUC57 recombinant plasmids were then digested once and assembled once by Gibson, and finally the entire gRNA full-length sequence was ligated into the pCCI-LEU plasmid, completing the splicing of the entire gRNA array in vitro.

In one embodiment, the present invention demonstrates that all fragments are assembled correctly by verification of 13 ligation sites (14 primary fragments contain 13 ligation sites in total) (see FIG. 19). Then, the plasmid is completely extracted from the large intestine, the plasmid containing specific enzyme cleavage sites is cut, the plasmid is cut into a plurality of fragments with different lengths, and whether the length of the plasmid is correct or not is judged. The plasmid was cut into fragments of different lengths by cleavage with KpnI and XbaI, and analysis by agarose gel electrophoresis showed that the extracted plasmid length and the sequence of the corresponding cleavage site were identical to the design (FIG. 20). Meanwhile, the invention utilizes the double enzyme digestion experiments of XhoI and BamHI to completely cleave the gRNA array from the plasmid, and the specific recognition and cleavage of BamHI can linearize the complete plasmid. The analysis by PFGE verification showed that the gRNA array was the same as designed and was ligated intact to the pCCI vector to form plasmid pCCI-II-168gRNA containing 168 gRNAs (FIG. 21).

The middle fragments of P1-P4 are respectively obtained through once Gibson assembly and integration, meanwhile, the integrated middle fragments are respectively connected to pUC57 plasmid, two first-stage assembled fragments are subjected to enzyme digestion by restriction enzyme, and are integrated through Gibson assembly by using a homology arm B, and the integrated fragments are connected to pCCI-LEU plasmid in vitro by using two homology arms A and C, and the specific assembly process is shown in FIG. 18.

The invention also provides a recombinant vector comprising the gRNA transcription unit and the gRNA array. The skeleton carrier of the recombinant vector is not particularly required, and the types common in the field can be selected from the group consisting of PUC series carriers and pCCI series carriers. In some embodiments, the backbone vector of the recombinant vector is a PUC57 vector or a pCCI-LEU vector.

The invention also provides a multi-target editing system, which comprises a base editor, and the gRNA transcription unit, the gRNA array or the recombinant vector. In some embodiments, the multi-target editing system comprises a base editor and a gRNA array of the invention. The type of the base editor is not particularly limited, and any type common in the art may be used. The gRNA array developed by the invention can be adapted to a variety of Cas9 variants, applied in different scenarios, such as CRISPRa, CAISPRi, or for CRISPR/Cas9 based epigenetic modification, etc. In some embodiments, the base editor is nCDA1Δ198-BE3 from the base editor described in document "Engineering of high-precisionbase editors for site-specific single nucleotide replacement".

The invention also provides the gRNA transcription unit, the gRNA array, the recombinant vector and the multi-target editing system, and application of the gRNA transcription unit, the gRNA array, the recombinant vector and the multi-target editing system in multi-target gene editing. In some embodiments of the invention, the multi-target gene editing is performed in yeast, in particular Saccharomyces cerevisiae.

In the invention, the source of the carrier homology arm is not required to be special, and the carrier types common in the field can be used. For the specific sequence of the homology arm of the vector, a sequence of 20 to 60bp (preferably 60 bp) at both ends of the insertion site of the vector is generally selected depending on the kind of the vector selected. The sequences of the remaining homology arms except the vector homology arm are random sequences of 20bp that are not homologous to the genome.

The invention has the following beneficial effects:

1. the invention designs and assembles an ultra-long flexible array with the length of 50,530bp and 168 specific gRNAs by utilizing tRNA-gRNA structure, non-repetitive elements and vox sequence.

2. By adding vox sequences to the gRNA array, flexibility of the array is achieved.

3. And simultaneously editing at most 104 different sites in the saccharomyces cerevisiae by using an ultra-long flexible gRNA array and a base editor, and improving the editing sites in a single bacterium to 113 through two rounds of iteration.

4. And realizing regional multi-target editing of the genome by using rearrangement of the ultra-long flexible gRNA array.

Drawings

FIG. 1 shows a schematic diagram of a base editor editing glutamine and arginine codons to stop codons;

FIG. 2 shows a base editor open reading frame structure;

FIG. 3 shows a schematic representation of the base editor for ADE2 in Saccharomyces cerevisiae;

FIG. 4 shows editing efficiency of a base editor under different expression intensities and integration formats;

FIG. 5 shows the results of optimization of base editor expression conditions;

FIG. 6 shows a schematic structural diagram of construction of a non-repetitive gRNA plasmid using Gibson assembly;

FIG. 7 shows the efficiency of base editing of non-repetitive gRNAs in yeast;

FIG. 8 shows a two-dimensional block diagram of gRNA;

FIG. 9 shows the effect of gRNA structural changes on base editing efficiency;

FIG. 10 shows the structure of a gRNA array with 21 tRNA cleavage effects verified;

FIG. 11 shows the effect of three gRNA array driven base editing;

FIG. 12 shows the effect of different expression patterns of a base editor on the number of multi-target edits in a single bacterium;

FIG. 13 shows recognition and cleavage of tRNA by RNase P and RNase Z;

FIG. 14 shows a gRNA transcription unit in a gRNA array;

FIG. 15 shows the design of a gRNA array containing 30 gRNAs;

FIG. 16 shows the assembly of a gRNA array containing 30 gRNAs, wherein the green lines represent the gRNA array, A, B and C represent different homology arms, A and C represent homology arms at both ends of the vector, and B is a sequence-randomized homology arm;

FIG. 17 shows the validation of a gRNA array containing 30 gRNAs;

FIG. 18 shows an assembly process for a gRNA array containing 168 gRNAs, wherein the green line represents a first order segment, the same letter represents the same homology arm of the sequence, the different letters represent the homology arms of different sequences, A and L are homology arms at both ends of the vector, and the remaining homology arms are random homology sequences;

FIG. 19 shows PCR validation of a gRNA array plasmid containing 168 gRNAs;

FIG. 20 shows the cleavage verification of KpnI, xbaI of a gRNA array plasmid containing 168 gRNAs;

FIG. 21 shows the BamHI, xhoI cleavage verification of a gRNA array plasmid containing 168 gRNAs;

FIG. 22 shows Sanger sequencing validation of 12 sites in the plasmid;

FIG. 23 shows the sequencing depth of all sites in the plasmid;

FIG. 24 shows the sequencing length of DNA fragments in ONT sequencing;

FIG. 25 shows verification of the base sequence at position 39121 in a plasmid;

FIG. 26 shows the change in phenotype of the strain after multi-target editing based on a gRNA array containing 30 gRNAs;

FIG. 27 shows the multi-target editing effect of a gRNA array containing 30 gRNAs;

FIG. 28 shows base editing for all targets in yHX 0366;

FIG. 29 shows colony phenotype validation of 168 gRNAs mediated multi-target editing;

FIG. 30 shows an edit profile of 168 sites in 31 strains;

FIG. 31 shows a characterization of editing efficiency at different sites in 31 strains;

FIG. 32 shows a two-round, multi-target editing strain whole genome sequencing analysis;

FIG. 33 shows PCRtag verification of gRNA array rearrangement;

FIG. 34 shows whole genome sequencing validation of gRNA array rearrangements;

figure 35 shows whole genome sequencing validation of the induction editing strain.

Detailed Description

The invention provides an ultra-long flexible gRNA array and a design and assembly method thereof. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The sequences referred to herein are shown in table 1:

TABLE 1

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The invention is further illustrated by the following examples:

example 1

1.1 selection of gRNA array elements

1.1.1 construction of a RNA array element screening System

Evaluation of editing effects of a multi-target editing system based on an ultra-long flexible gRNA array requires effective visualization means for characterization. Previously, for CRISPR systems, researchers developed techniques for chromosome cleavage, gene silencing, base editing, gene driving, and the like. And for evaluation of multi-target editing and evaluation of screening of the gRNA array elements, a convenient and efficient detection means is provided for editing sites after editing events occur. The single base editor utilizes deaminase fused to Cas9 protein to effect editing of bases within a specific editing window. The change in bases in the DNA sequence can be detected by sequencing and does not change over time. In addition, cytosine (C) becomes thymine (T) under the action of cytidine deaminase, so that codons encoding glutamine (CAA; CAG) and arginine (CGA) can be converted into stop codons (TAA; TAG; TGA) under the action of a base editor, nonsense mutation (Nonsense mutation) is caused, synthesis of a peptide chain is terminated in advance, and the purpose of gene inactivation is achieved (figure 1). Thus, in this study, a base editor was used as a tool for multi-target editing, and the effectiveness of the gRNA array ligation elements was characterized and the number of edits in individual strains determined by counting the number of gene inactivation events versus base changes.

In CBE base editors, cas genes are typically included, along with cytidine deaminase and base excision repair inhibitors expressed in fusion therewith. For the content of the study, the required base editor should have the characteristics of high editing efficiency and wide editing range, so that the gRNA design can be performed for more targets. CDA1 is an ortholog from sea lamprey AID that has better C to T base editing activity in a specific DNA sequence than the previously reported cytidine deaminase apodec 1. Furthermore, dCas9 is more suitable for multi-target editing systems because it does not cause double strand breaks, but does not allow growth of multiple edited strains, compared to Cas9 and nCas9 versions. The nCDA1Δ198-BE3 was used for the base editor following the study.

The CRISPR base editing system construction process comprises the following steps: the nCDA1Δ198-BE3 optimized by saccharomyces cerevisiae codon is synthesized by general biological company, and the restriction enzyme of KpnI and NotI is reserved at both ends of the geneA site. The pRS413-P expression plasmid is obtained by linking the pRS413 vector obtained by PCR amplification after enzyme digestion _TEF1 -nCDA1Δ198-BE3-T _CYC1 . Deletion of ADE2 in s.cerevisiae results in accumulation of the intermediate ribosyl aminoimidazole in the cell. And the intermediate forms red pigment after oxidation, and the colony turns from white to red. Whereas CRISPR targeting sequences (5' -TCAACTTAAGGCGAAGTTGT) TGG-3') targeting the Glutamine (gin ) codon at position 233 of Saccharomyces cerevisiae ADE 2. Editing of the C to T bases can form a stop codon for TAA, and prematurely terminate the ADE2 translation process, resulting in a change in color of the yeast colony. The following screening of the gRNA array elements all characterized the effect of base editing by counting the efficiency of colony reddening.

Yeast harboring the CRISPR base editing system plasmid underwent base editing in vivo, and after 72 hours of culture, red colonies formed on the corresponding screening plates, indicating success of base editing (FIG. 3). Next, we tested the editing efficiency of the base editor at different expression intensities and integration formats. Including expression on low and high copy plasmids and inducible expression integrated on the genome. The experimental results showed that the base editor expression pattern was changed and the base editing efficiency was not significantly changed (FIG. 4). Considering the effect of plasmid instability and the different number of targets on the requirement of expression intensity, the base editor following this study was integrated between YOR072W and YOR073W of chrysxv and was started by GAL1 to induce expression with galactose in the absence of glucose.

1.1.2 optimization of the gRNA sequence

The sequence of the gRNA is relatively conserved in the gRNA array. CRISPR-Cas9 acts as a type II CRISPR system, in that the transactivated tracrRNA hybridizes to the crRNA, thereby guiding specific cleavage of a specific site by the Cas9 protein. In addition, crrnas comprising the target sequence are fused to tracrRNA, resulting in a complete gRNA sequence that is capable of achieving specific cleavage of the genome. In previous studies, non-repetitive libraries of grnas were developed by way of biochemical modeling, biochemical characterization, and machine learning of gRNA sequences. The repeated degree of the gRNA array is reduced, so that the array synthesis difficulty is reduced. However, no attempt was made to do this in yeast.

We amplified pRS42H vector using PCR reaction and assembled the newly synthesized gRNA scaffold (handle) sequence into vector using Gibson assembly, including the 20bp target sequence targeting ADE2 (5' -TCAACTTAAGGCGAAGTTGT)TGG-3') (fig. 6). The effectiveness of the editing effect and the effectiveness of the gRNA scaffold were determined by inducing a change in yeast phenotype after base editing. The newly constructed gRNA plasmid can be sequence confirmed by Sanger sequencing.

We constructed the 28 non-repetitive gRNA scaffolds previously reported into vectors and induced editing of ADE2 in yeast cells with integrated base editor. The effect of non-repetitive grnas was determined by counting the proportion of the number of phenotypically altered strains to the total number of all strains. For each non-repeated gRNA, three parallel experiments were performed, and the results indicate that most of the non-repeated grnas were not functional in yeast. Whereas for non-repetitive gRNAs numbered 1,5,8, 22, 34, 37, they were able to function in combination with base editors in yeast for targeting genes and base editing, but were generally less efficient (< 40%) (fig. 7).

For this reason, we compared the distinction between non-repeated grnas and laboratory use grnas. Unlike non-repetitive grnas, the grnas used in the laboratory have a 15bp additional DNA sequence that is considered a terminator herein (fig. 8). Therefore, we hypothesize that the addition of this sequence can lead to a corresponding increase in overall editing efficiency. Three highly efficient gRNAs numbered 1, 22, 37 (sequences shown as SEQ ID NOS: 23-25, respectively) were selected for subsequent study. The experimental result shows that after the 15bp terminator sequence is added, the editing effect on the target spot is improved, and the efficiency of single editing is improved to more than 75 percent (figure 9). The results indicate that the 76bp scaffold sequence is particularly important for binding and specific cleavage of gRNA to Cas9 in yeast. Thus, in subsequent studies, we will use the three non-repetitive gRNAs described above as scaffolds for expression to reduce the degree of repetition of the entire gRNA array.

Characterization of 1.1.421 tRNAs cleavage gRNA arrays

Based on the above research results, tRNA has been shown to be an effective gRNA flanking cleavage element, and the combination of tRNA-gRNA-tRNA's can effectively release mature gRNA for use in CRISPR systems. Utilizing tRNA endogenous to Saccharomyces cerevisiae ^Gly Cutting the gRNA flank can realize editing of eight different sites. However, larger scale synthesis of gRNA arrays can be difficult due to the large number of repeats of tRNA and gRNA scaffolds. The different types of tRNA in Saccharomyces cerevisiae, among which ten tRNA's have been shown to be effective in cleaving at the side of gRNA, are used for the transport of different amino acids ^[258] . To utilize more non-repeating tRNA elements and reduce the degree of repetition across the array, we need to characterize the ability of each tRNA to cleave in yeast. Similar to the array structure designed by the previous selection of cleavage elements, we divided the 21 tRNAs into three groups, distributed on either side of the gRNA. Four target sequences targeting the codon (5 '-TCAACTTAAGGCGAAGTTGT) of ADE2, gln 233, gln 374, gln 393 and Ile 244 were selected together (5' -TCAACTTAAGGCGAAGTTGT)TGG-3'；5'-CAAAGGCTGAACTACATTACAGG-3'；5'-TGTCGCTCAAAAGTTGGACTTGG-3'；5'-ATCAAATCTTTTCCCGGTTGTGG-3') was used for characterization of tRNA cleavage ability (FIG. 10).

Wherein, due to the limitation of tRNA quantity, the third gRNA array (tRNA (16-17)) targets only the first three sites and the tRNA is reused once therein ^Gly (FIG. 10).

All arrays were introduced into yHX0362 and induced in galactose medium for 24 hours. After 72 hours of incubation on the plates, the proportion of color change of the colonies was counted to characterize the efficiency of base editing. The results indicate that the efficiency of base editing induced by the gRNA array with three different trnas as the connecting elements can reach more than 50% (fig. 11 a). Meanwhile, we randomly picked 16 strains from three experiments to PCR amplify ADE2 and determined the editing of all sites using Sanger sequencing. As shown in fig. 11b, all arrays were edited for their targeting sites and the effect was no less than 50%. The effectiveness of the gRNA array structure was shown to be useful as a flanking cleavage element for all tRNA's, expanding the number of non-repeating elements in the gRNA array. Meanwhile, the influence of the expression of the base editor driven by two modes (low copy plasmid constitutive expression; chromosome integration induced expression) on multi-target editing is compared. As can be seen by comparison, the base editor was able to edit more sites in a single strain when galactose-induced expression was performed than when constitutively expressed in low copy plasmids (FIG. 12).

Thus, by characterization of the editing effect of the gRNA array composed of three different trnas, it was shown that 21 trnas were all able to cleave in the gRNA array transcript and be used in subsequent array designs.

the tRNA precursor is recognized and cleaved in vivo by RNAse P and RNAse Z, thereby removing the 5 'and 3' sequences. RNase Z will cleave exactly at the 3' end of the tRNA at the junction with the gRNA without disrupting the gRNA structure. At the 5 'end of the tRNA, cleavage by RNase P occurs within the 5' leader sequence. Meanwhile, previous studies have also demonstrated that the presence of a leader sequence can effectively enhance the processing ability of rnase P. Thus, the 5 'ends of tRNA's will each be added with their respective leader sequences during the design of the gRNA array (FIG. 13). And all arrays are composed of transcriptional units as shown in figure 14. Within this unit the grnas are linked at both ends by different types of trnas, and at the 3' end vox sequences are added for array rearrangement. Thus, during the subsequent design of the grnas, three grnas will be included in each transcription unit to ensure that all grnas can be transcribed efficiently. To this end, we have determined the basic structure of the gRNA array and the composition of the various types of elements, thereby reducing the degree of repetition of the entire array.

1.2 design and Assembly of gRNA arrays containing 30 gRNAs

First, we designed a proof of concept experiment to verify the feasibility of the array design. We selected 30 targets among the five genes of ADE2 and violacein metabolic pathways for the design of the gRNA array. These targets are in the range of-20 to-15 bases from the PAM site, with at least one cytosine being used to characterize the occurrence of an editing event. Since SpCas9 is used, PAM sequences for all targets are NGG. Wherein, each gene is designed with a gRNA target point which can cause nonsense mutation, and the translation of gene transcripts on ADE2 or violacein metabolic paths can be terminated in advance, so that colonies generate corresponding phenotypes. The design of the gRNA array was performed according to the principles described above, with a total of 10 transcription units, 10 synthetic promoters, 21 tRNA elements, 3 gRNA scaffolds and 10 synthetic terminators involved in the design of the entire gRNA array (FIG. 15).

The total length of the designed array is 7821bp, in order to reduce the difficulty of array synthesis, the whole array is divided into two parts, five transcription units are used as a group, and the five transcription units are delivered to a gene synthesis company for synthesis. Meanwhile, we add appropriate cleavage sites (EcoRI, xhoI, notI) at both ends of the synthesized fragment to facilitate the assembly of the fragment after completion of the synthesis. Two fragments were digested with restriction enzymes and assembled by Gibson and ligated in vitro to pCCI-LEU plasmid using 20bp homology arms at both ends of the fragments (FIG. 16). Although the degree of duplication of the gRNA array has been reduced, there is some duplication due to the limited number of elements. And then the synthesized gRNA array is longer in length, so eventually the gRNA array will be assembled onto the pCCI vector. The ligated plasmid was introduced into the large intestine for verification and amplification. By designing PCR primers at the junction, it was verified whether the plasmid was correctly ligated. The experimental results showed that the junctions between fragments, and the junctions between fragments and plasmid were all correctly ligated, indicating correct assembly of the gRNA array (fig. 17). Thus, based on the array design principle in section 5.2, we designed and successfully assembled a gRNA array containing 30 gRNAs targeting ADE2 and violacein metabolic pathway genes.

1.3 design and Assembly of ultra-long flexible gRNA arrays containing 168 gRNAs

Next, we tried to verify if the design and assembly method for a gRNA array containing hundreds of gRNAs was effective. The yeast genome contains a large number of nonessential genes, and the deletion of the nonessential genes independently does not cause death of the yeast strain. However, the combination of specific essential genes cannot be deleted due to the influence of gene interactions, which is a synthetic lethal phenomenon in yeast. Previous studies demonstrated that the 744,843613,184 region of chrII containing 38 non-essential genes was completely deleted by PCR-mediated chromosomal deletion (PCR-mediated chromosomal deletion, PCD) techniques without affecting the growth state of the strain. Thus, we selected this region as the targeting region for 168 gRNAs. By programming we searched for target sequences within this region, which have cytosine present in the interval from PAM site (NGG sequence) -20 to-15. And to disperse all target sequences as uniformly as possible throughout the chromosome. Meanwhile, for ADE2, we designed three gRNAs capable of prematurely terminating gene translation to characterize the occurrence of base editing events. Based on the previously set rules for the design of the gRNA array, we selected a combination of 56 non-replicated synthetic promoters, 3 gRNA scaffolds, 21 tRNA's, and 12 synthetic terminators for the design of the gRNA array. The overall length of the designed gRNA array reached 50,530bp, so we split the array into 14 primary fragments, each fragment containing 4 transcriptional units, for a total of 12 gRNAs. The 14 primary fragments were separated into four combinations, each of which could be integrated by one Gibson assembly. In this process, the fragments were assembled onto pUC57 plasmid, facilitating subsequent extraction and cleavage of plasmid. All the primary fragments have about 3600bp in length, and can be directly synthesized by a company due to lower repetition degree. Then, the obtained four plasmids containing the intermediate fragments are cut through a pre-reserved enzyme cutting site, and four linearized intermediate fragments are obtained. Similarly, the middle fragment can be assembled by Gibson using 60bp homology arms at both ends, and the entire gRNA array can be spliced in vitro by one-step assembly onto the pCCI vector (FIG. 18).

We reserved PCR sites at both the 5 'and 3' ends of the primary fragment for characterizing the correct ligation of the fragments. Verification of the 13 fragment ligation sites indicated that all fragments were assembled correctly (FIG. 19). Then, we extract the plasmid from E.coli completely, cut the plasmid with specific enzyme cutting site, cut the plasmid into several fragments with different length, and judge if the plasmid length is correct. The plasmid was cut into fragments of different lengths by cleavage with KpnI and XbaI, and analysis by agarose gel electrophoresis showed that the extracted plasmid length and the sequence of the corresponding cleavage site were identical to the design (FIG. 20). Meanwhile, we used the double cleavage experiments of XhoI and BamHI to cleave the gRNA array completely from the plasmid, whereas specific recognition and cleavage of BamHI linearized the complete plasmid. The analysis by PFGE verification showed that the gRNA array was the same as designed and was ligated intact to the pCCI vector to form plasmid pCCI-II-168gRNA containing 168 gRNAs (FIG. 21).

Next, to further verify if the sequence and design of the constructed plasmid were identical, we picked 12 sites on the plasmid for Sanger sequencing verification. By comparison with the control sequence, it was shown that all 12 sites were on the plasmid and that the bases were unchanged (FIG. 22). Meanwhile, we performed three-generation sequencing (ONT sequencing) validation of the extracted plasmid. The nanopore sequencing method can realize the head-to-head sequencing of all DNA molecules, so that the nanopore sequencing method is suitable for sequencing DNA fragments with complex structures. Sequencing results showed that the sequencing depth covered all sequences of the plasmid (fig. 23). And ONT sequencing quality remained at a higher level (fig. 24). By aligning the spliced sequences with the control, we found that adenine (a) at position 39,121 in the plasmid was replaced with guanine (G), and the site was examined in combination with Sanger sequencing, which showed no change in the base of the site (fig. 25).

To this end we designed and assembled correctly a complete ultra-long flexible gRNA array containing 168 gRNAs. Different from the previous array synthesis mode, the array with the reduced repetition degree can be synthesized by a company in a segmented way, so that the assembly difficulty of the array is reduced, a simple gRNA array design method is provided for multi-target editing, and the operating range of the multi-target editing is further improved.

2.1 characterization based on Multi-target editing of gRNA arrays containing 30 gRNAs

We used the designed gRNA array to edit the different loci of the genome and determined the efficiency of multi-target editing and the effect of different target editing. The pre-synthesized and assembled gRNA array targeting five genes of ADE2 and violacein metabolic pathways was introduced into yHX0365, the correctly introduced strain was picked up for 24 hours under galactose induction and plated onto the corresponding plate. The gRNA array was designed with gRNA capable of base editing to create nonsense mutations for genes on both ADE2 and violacein metabolic pathways, resulting in corresponding phenotypic changes in the strain (FIG. 26). During selection of strains, we used strain phenotype changes to determine sequenced strains. The colony with reddening colony color shows that nonsense mutation occurs in genes in ADE2 and violacein metabolic paths in the genome, and the occurrence of a multi-target editing event is proved. We picked 8 strains that appeared red in color and performed PCR amplification and Sanger sequencing validation on all their targets. The number of single-induction edits in a single strain, as well as the edit efficiency for different targets, were counted. The experimental results show that at least 19/30 sites were edited in each strain. And at least one editing event occurred at 29/30 sites in all strains (FIG. 27). Meanwhile, we note that for single bacteria, the average editing number accounts for more than 75% of the total target points, suggesting that more gRNA expression can realize multi-target editing in the strain. The experiment also shows that after single induction editing, the strain yHX0366 can realize editing of at most 27/30 different sites in the single bacterium, and lays a foundation for further improving the editing quantity of different sites in the single bacterium (figure 28).

2.2 characterization of Multi-target editing based on ultra-Long Flexible gRNA arrays

2.2.1 characterization of genome Multi-target editing Effect in Single bacteria

To evaluate how hundreds of different gRNAs were expressed in a single bacterium to edit different sites, we introduced correctly assembled plasmids containing gRNA arrays into yHX0362, resulting in strain yHX0463, and induced base editor expression to edit 168 targets. The inclusion of a gRNA in the gRNA array resulted in nonsense mutations in ADE2, and thus changes in strain color would be used to characterize the base editing event (fig. 29). By counting the number of color-changing strains, we found that the efficiency of nonsense mutations to ADE2 in this experiment (28.0%) was significantly less than the base editing efficiency (> 80%) of the same target guided by a single gRNA. We speculate that this is due to the large number of gRNAs expressed in a single bacterium, while the number of base editors that induce expression cannot mediate sufficient base editing of all gRNAs at the corresponding sites. In other studies, an increase in the expression level of dCas9 has also been demonstrated to increase the effect of gene transcription inhibition mediated by multiple gRNAs. Meanwhile, the strain can be subjected to iterative induction of base editing, the action time of the base editor is prolonged, the number of the point editing in single strain can be increased, and the part is described in detail in section 2.2.2.

We randomly picked 31 single colonies with reddish color for whole genome sequencing (FIG. 30). Whole genome sequencing was also performed on the same controls as on the starting strain yHX0362 and on the three strains yHX0463 obtained by induction in galactose medium for 24 hours (yHX 0392, yHX0550, yHX 0552).

Next, the data from whole genome sequencing of the test strains were analyzed and single nucleotide variations (Single nucleotide variation, SNV) in control yHX0362 were deleted as negative mutations from all experimental strains. All the changes of C to T bases in the target sequences of 20 bp-20 to-15 are recorded as the base edits of the sites, so that the number of site edits in a single strain after single editing and the editing efficiency of 168 sites in 31 strains are counted. The experimental results showed that multiple edits occurred in the genomes of all experimental strains, and the median of the number of edits was 77 and the average number of edits was 72. Of these, in strain yHX, base editing occurred at 104 different sites on the genome after a single induction editing (fig. 30). In all experimental strains, the average editing efficiency of all sites was 42.72% (fig. 31), and at least one editing of 145 sites occurred, indicating that most of the designed grnas were able to bind and edit at the corresponding targets.

2.2.2 iterative editing of a Multi-target editing System

The multi-target editing technique often fails to implement editing of all target sites in a single experiment. In the MAGE technique, the average number of bases changed in a single cell can be increased from 3.1 to 5.6 by increasing the number of MAGE cycle operations ^[2] . During experiments with codon substitutions in cells, researchers were editing by adding one round of transfection to cells, in a single roundEditing of 6/47 sites was added to the bacteria ^[15] . In this study, a single 102 and 104 target site editing was achieved in the induced strains yHX0455 and yHX456 by induced editing of strain yHX0463 containing the ultralong flexible gRNA array plasmid.

To characterize the change in edit number in single bacteria with increasing number of induction edits, we re-inoculated yHX0455 and yHX456 into galactose induction medium for induction for 24 hours. Since the strain color had changed in the previous round of editing, we randomly picked the colonies obtained in this round of editing and detected base changes in the strain genome using whole genome sequencing. By analysis of the base sequence of 168 sites, we found that by the second round of induction editing, editing of new target sites occurred in all single bacteria randomly selected (FIG. 30). Meanwhile, the newly added editing sites mostly occur in sites that have been edited before. Wherein, the positions corresponding to 89-gRNA and 138-gRNA which are not edited at the earlier stage have one editing event in the 9 strains. Furthermore, the genome of yHX0401 and yHX0402 was edited with 113 target sites in the strain obtained by the second round of editing on the basis of yHX0456, further increasing the number of different site edits in the single strain (fig. 32). Therefore, by increasing the number of induced edits, the number of edits in a single bacterium can be further increased.

2.2.3 rearrangement and Multi-target random editing of ultra-Long Flexible gRNA arrays

Because 34bp loxPSym sites are designed at the downstream of the non-essential genes of the Saccharomyces cerevisiae synthetic chromosome, the SCRaMble can be utilized to rearrange synV and synX synthesized in the early stage of a laboratory, so that the genes are duplicated, deleted, translocated, reversed and the like ^[3,4,5] . The diversity of the strain genotypes can be quickly constructed through SCRaMble, so that strains with different phenotypes can be obtained. In the research, the vox sequence is added into the designed and constructed ultra-long flexible gRNA array, so that site-specific recombination can occur under the action of Vika protein, and the diversity of the gRNA array can be rapidly formed. The abundant multi-target editing combination can be gene interaction in genomeBasic tools for excavation, strain transformation and the like ^[6,7,8] . Thus, in this section we will use the expression of the Vika protein to construct a gRNA array library and induce editing of it, characterizing the diversity of editing sites in a single bacterium.

First, a constitutively expressed plasmid pRS415-CYC1-Vika-EBD-tCYC1 was constructed in which Vika was expressed in fusion with an Estradiol Binding Domain (EBD) under the drive of a CYC1 weak promoter. The effect of Vika-EBD on vox is affected by estradiol, and the fusion protein is not folded in the absence of estradiol and remains in the cytoplasm and thus cannot come into contact with the nuclear DNA.

The constructed plasmid pCCI-II-168gRNA and pRS415-CYC1-Vika-EBD-tCYC1 were introduced into yHX0362 and correctly introduced strain yHX0549,0549 was obtained by selection. After overnight incubation of yHX0549,0549, 500uL of broth was transferred to 5mL of medium and 1uM of estradiol was added to induce gRNA array rearrangement. After 3 hours, the bacterial solution was diluted and spread on a plate medium, and after three days of culture, the strain was selected for analysis of rearrangement of the gRNA array therein.

First, the rearrangement of the array was characterized by PCR using 13 PCRtags designed on the gRNA array. Six strains (yHX 0465, yHX0466, yHX0467, yHX0468, yHX0469, yHX 0470) were selected for characterization of the change in the structure of the gRNA array they carry. The results showed that the gRNA array was deleted in different regions and the length of the deletion was varied to form a library of a certain size (fig. 33). To further characterize structural variations in different gRNA arrays, we performed whole genome sequencing on the six strains described above. The sequencing depth of the gRNA array is counted, and the result shows that the sequence of the gRNA array is deleted and duplicated after rearrangement. Wherein the gRNA array in yHX0468 replicates in the region yHX-22,305 and deletes in the region 15,853-18,528, resulting in complex structural changes of replication overlapping deletions. While the gRNA arrays in yHX469 and yHX470 have undergone at least three different region deletion events, resulting in a gRNA array with three different targeting point combinations (fig. 34). Subsequently, we performed induced editing on six strains, while rearrangement of the gRNA array in the strain may result in deletion of three ADE 2-grnas used to characterize multi-target editing. Thus, we will randomly pick the corresponding strains with and without color change, and analyze the editing sites in the picked strains using whole genome sequencing. The results show that different gRNA arrays produced after rearrangement resulted in a change in the multi-target editing preference, resulting in strains with different editing modes (fig. 35). Thus, rearrangement of the gRNA array can drive the base editor to edit in different regions, thereby creating a large library of strains of different genotypes.

Example 2 comparison of editing effects of different editing systems

Editing effects of the editing system reported in the prior art and the editing system of the invention are shown in table 2:

TABLE 2

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[2]Wang H H,Isaacs F J,Carr P A,et al.Programming cells by multiplex genome engineering and accelerated evolution[J].Nature,2009,460(7257):894-898；

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[4]Jia B,Wu Y,Li B Z,et al.Precise control of SCRaMbLE in synthetic haploid and diploidyeast[J].Nature Communications,2018,9(1):1933；

[5]Shen M J,WuY,Yang K,et al.Heterozygous diploid and interspecies SCRaMbLEing[J].Nature Communications,2018,9(1):1934；

[6]Roy K R,Smith J D,Vonesch S C,et al.Multiplexed precision genome editing with trackablegenomic barcodes in yeast[J].Nature Biotechnology,2018,36(6):512-520.

[7]Kuzmin E,VanderSluis B,Wang W,et al.Systematic analysis of complex genetic interactions[J].Science,2018,360(6386):eaao1729；

[8]Boone C,Bussey H,Andrews B J.Exploring genetic interactions and networks with yeast[J].Nature Reviews Genetics,2007,8(6):437-449；

[9]Liu B,Jing Z,Zhang X,et al.Large-scale multiplexed mosaic CRISPR perturbation in the whole organism[J].Cell,2022,185(16):3008-3024.e16；

[10]Reis A C,Halper S M,Vezeau G E,et al.Simultaneous repression ofmultiple bacterial genes using nonrepetitive extra-long sgRNA arrays[J].Nature Biotechnology,2019,37(11):1294-1301；

[11]Campa C C,Weisbach N R,Santinha A J,et al.Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts[J].Nature Methods,2019,16(9):887-893；

[12]Chen Y,Hysolli E,Chen A,et al.Multiplex base editing to convert TAG into TAA codons in the human genome[J].Nature communications,2022,13(1):1-13。

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (13)

1. A gRNA transcription unit comprising a promoter, a tRNA, a gRNA of a target sequence, a VOX sequence, and a terminator;

the VOX sequence is shown as SEQ ID NO. 1.

2. The gRNA transcription unit of claim 1, wherein the number of grnas of the target sequence is k, 3.ltoreq.k.ltoreq.6; the number of tRNA is 4-7.

3. The gRNA transcription unit of claim 1, wherein the gRNA of the target sequence comprises gRNA1, gRNA2, and gRNA3, the gRNA1, gRNA2, and gRNA3 comprising different scaffold sequences; the tRNA comprises tRNA1, tRNA2, tRNA3 and tRNA4 which have different sequences;

the gRNA transcription unit comprises the following components from the 5 'end to the 3' end: promoter-tRNA 1-gRNA1-VOX sequence-tRNA 2-gRNA2-VOX sequence-tRNA 3-gRNA3-VOX sequence-tRNA 4-terminator.

4. The gRNA transcription unit according to claim 1 to 3, wherein the sequence of the tRNA is shown in SEQ ID NO. 2 to 22.

5. The gRNA transcription unit of any one of claims 1-4, wherein the gRNA of the target sequence comprises: a guide sequence complementary to the target sequence, a scaffold sequence and a termination sequence;

the bracket sequences are respectively selected from sequences shown in SEQ ID NO. 23-25, and the termination sequence is shown in SEQ ID NO. 26.

6. A gRNA array, characterized by consisting of the gRNA transcription unit of any one of claims 1-5.

7. The gRNA array of claim 6, comprising X mid-level fragments comprising a total of m first-level fragments, each first-level fragment comprising 3-5 gRNA transcription units, each transcription unit comprising k grnas, the m first-level fragments being divided into X mid-level fragments, on average or non-average; wherein x is greater than or equal to 1, m is greater than or equal to 1, k is greater than or equal to 3, and x, m and k are integers.

8. The method for designing and assembling a gRNA array according to claim 6 or 7, comprising:

step (1): obtaining n gRNAs according to target sequence design, dividing the gRNAs into n/k gRNA transcription units according to any one of claims 1-5, wherein each transcription unit comprises k gRNAs, k is more than or equal to 3, n is more than or equal to 3, and n and k are integers;

Step (2): every 3-5 gRNA transcription units form a first-stage fragment, and n/k gRNA transcription units are equally divided into m first-stage fragments;

step (3), the m primary fragments are grouped evenly or unevenly, and each group of primary fragments forms a middle stage fragment; the first-stage segment is designed as follows, and a first-stage assembly segment is obtained:

adding homologous arms and restriction enzyme cutting sites at two ends of each primary segment, and adding the same homologous arms at the 3 'end of the previous primary segment and the 5' segment of the next primary segment in two adjacent primary segments to integrate the primary segments through enzyme cutting and Gibson assembly; the homology arms added at the 5 'end of the first primary segment and the 3' end of the X primary segment are carrier homology arms, and the rest homology arms are random homology sequences;

step (4): and respectively synthesizing m first-stage assembly fragments, and performing in vitro enzyme digestion and Gibson assembly to obtain the full-length sequence of the gRNA array.

9. The method of designing and assembling according to claim 7, wherein n=30, k=3, the gRNA array comprises 10 gRNA transcription units, each 5 gRNA transcription units forming a first-order segment comprising two first-order segments;

Or, n=168, k=3, the gRNA array comprises 56 gRNA transcription units, each 4 gRNA transcription units comprising one primary segment, comprising 14 primary segments in total.

10. A recombinant vector comprising the gRNA transcription unit of any one of claims 1-5, the gRNA array of claim 6 or 7.

11. A multi-target editing system, comprising:

a base editor;

and a gRNA transcription unit according to any one of claims 1 to 5, a gRNA array according to claim 6 or 7, a gRNA a array obtained by the design assembly method according to claim 8 or 9, or a recombinant vector according to claim 10.

12. The multi-target editing system of claim 11, wherein the base editor is nCDA1 a 198-BE3.

13. Use of the gRNA transcription unit of any one of claims 1-5, the gRNA array of claim 6 or 7, the gRNA array obtained by the design assembly method of claim 8 or 9, the recombinant vector of claim 10, the multi-target editing system of claim 11 or 12 for multi-target gene editing.

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