CN106480083B

CN106480083B - CRISPR/Cas 9-mediated large-fragment DNA splicing method

Info

Publication number: CN106480083B
Application number: CN201510531714.XA
Authority: CN
Inventors: 覃重军; 周见庭; 吴荣海; 薛小莉
Original assignee: Center for Excellence in Molecular Plant Sciences of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2015-08-26
Filing date: 2015-08-26
Publication date: 2021-12-14
Anticipated expiration: 2035-08-26
Also published as: CN106480083A

Abstract

The invention discloses a CRISPR/Cas 9-mediated large-fragment DNA splicing method. Specifically, the present invention provides a nucleic acid construct capable of constitutively expressing Cas9 in yeast, comprising the yeast Tef1 promoter, the replication initiation site from pBR322 and its selection marker, and the replication region from CEN6ARS4 and its selection marker, operably linked to Cas9 gene, said nucleic acid construct being replicated in yeast in single copy and in high copy in e. The invention also provides a nucleic acid construct capable of constitutively expressing the sgRNA in yeast, a nucleic acid construct used as a receptor vector, and a DNA splicing method. The invention can be used for directly and successfully splicing more than two large DNA fragments to be spliced into large plasmids in yeast at one time, saves the low-efficiency operations of in vitro enzyme digestion recovery, genetic transformation and the like of the large DNA fragments, and is more convenient and efficient than the traditional method.

Description

CRISPR/Cas 9-mediated large-fragment DNA splicing method

Technical Field

The invention relates to the fields of microbial synthetic biology, genome engineering and molecular biology, in particular to a CRISPR/Cas 9-mediated large-fragment DNA splicing method.

Background

Synthetic biology is an emerging field of research. In 2010, the first artificial life in the world was reported by the American scientist Venter and its research team [ Gibson, D.G et al, Creation of a bacterial cell controlled by a chemical synthesized gene, Science, 2010, 329(5987): p.52-6 ], which made synthetic biology a research hotspot in modern life sciences. The development of DNA synthesis, large fragment splicing and genome transplantation technologies is an important technological base in synthetic biology research. The in vitro splicing of DNA developed by the Venter and its research team [ Gibson, D.G., et al, Complete chemical synthesis, assembly, and cloning of a Mycoplasma genetic genome, Science, 2008, 319(5867): p.1215-20 ] and in vivo splicing [ Gibson, D.G et al, 2010, supra ] techniques are widely used in synthetic biology research. The principle of DNA splicing in vitro is that DNA with a homologous sequence at the end is treated by 3 '-5' exonuclease, a plurality of DNA fragments can be spliced together after a generated single-stranded overhang is annealed and renatured, and the nick of a single strand can be repaired by DNA polymerase and DNA ligase, so that a complete spliced fragment is formed. The DNA in vivo splicing system is characterized in that a DNA fragment with a homologous sequence at the end and a linearized vector are transferred into a yeast body for splicing by utilizing an efficient homologous recombination system in the yeast body. By these two techniques, the 580kb Mycoplasma genitalium genome has been successfully assembled by Venter et al [ Gibson, D.G. et al, 2008, supra ]. Furthermore, they have synthesized a 1.08Mbp genome JCVI-syn1.0 of mycoplasma filiformis (m.mycoides) and transplanted and introduced into mycoplasma capricolum (m.capricolum) cells to obtain cells controlled by the synthesized mycoplasma filiformis genome, and successfully realized artificial life for the first time [ Gibson, D.G et al, 2010, supra ]. Subsequently, the research team led by Boeke et al in 2014 artificially synthesized a functional Saccharomyces cerevisiae III chromosome [ Annaluru, N. et al, Totalsynthesis of a functional designer eutetic chromosome, Science, 2014, 344(6179): p.55-8 ], which is the chromosome of the first artificially synthesized eukaryote, indicating that humans are moving further towards artificially synthesized life. Although the cost of DNA synthesis is decreasing with advances in technology, the efficiency of assembly of very large pieces of DNA, even complete genomes, is still relatively low due to the difficulties in handling large pieces of DNA.

Disclosure of Invention

In a first aspect, the invention provides a nucleic acid construct comprising a yeast Tef1 promoter operably linked to a Cas9 gene, and which is capable of constitutively expressing Cas9 in yeast.

In one embodiment, the nucleic acid construct is a plasmid.

In one embodiment, the nucleic acid construct further comprises a replication origin from pBR322 and a selectable marker therefor, such as an ampicillin resistance gene.

In one embodiment, the nucleic acid construct further comprises a replication region derived from CEN6ARS4 and a selectable marker thereof, such as MET 14.

In one embodiment, the nucleic acid construct is single copy replication in yeast and high copy replication in E.coli.

In one embodiment, the nucleic acid construct comprises, in operative association, in order, an origin of replication from pBR322, the promoter Tef1, the cas9 gene, the termination sequence CYC1, lacZ α, the origin of f1, the selectable marker Met14, the replication region from CEN6ARS4, and the selectable marker ampicillin resistance gene.

In one embodiment, the nucleic acid construct comprises the replication region from CEN6ARS4 as shown at bases 65-319 of SEQ ID NO. 1 and the cas9 gene as shown at bases 3610-7749 of SEQ ID NO. 1.

In one embodiment, the nucleic acid construct is set forth in SEQ ID NO 1.

In a second aspect, the invention provides a nucleic acid construct that constitutively expresses a sgRNA comprising, operably linked, a yeast promoter SNR52, a 20bp recognition site, and a Handle Cas9 sequence in yeast.

In one embodiment, the nucleic acid construct is a plasmid.

In one embodiment, the nucleic acid construct further comprises a vector replication region derived from 2 μ and a selectable marker therefor, such as TRP 1.

In one embodiment, the nucleic acid construct further comprises a replication initiation site from pBR322 and a selectable marker therefor, such as a ampicillin resistance gene.

In one embodiment, the nucleic acid construct comprises, in operative association in sequence, an origin of replication from pBR322, the SNR52 promoter, the 20bp recognition site 1, the Handle Cas9 sequence, the SNR52 promoter, the 20bp recognition site 2, the Handle Cas9 sequence, the CYC1 primer, the CYC1 termination sequence, lacZ α, the f1 origin, the TRP1, the vector replication region from 2 μ, and the ampicillin resistance gene.

In one embodiment, the nucleic acid construct comprises the sequences shown at positions 4624-4998 and 5012-5386 of SEQ ID NO 2.

In one embodiment, the nucleic acid construct is set forth in SEQ ID NO 2.

In a third aspect, the invention provides a nucleic acid construct comprising a vector replication and selectable marker, e.g., ADE2, from CEN6ARS4, and a vector replication origin and partitioning and selectable marker, e.g., a chloramphenicol resistance gene, from pBeloBAC 11.

In one embodiment, the nucleic acid construct is a plasmid.

In one embodiment, the nucleic acid construct comprises the vector replication region derived from CEN6ARS4, selection marker ADE2, the chloramphenicol resistance gene, the assignment control gene rctB, Vibrio chromosome II replication region oriCII, and the assignment control genes inc, rctA, parA, and parB, operably linked.

In one embodiment, the nucleic acid construct is set forth in SEQ ID NO 3.

In a fourth aspect, the invention provides a cell comprising any one, two or all three of the nucleic acid constructs of the invention.

In a particular embodiment, the cell is an escherichia coli or yeast cell.

In one embodiment, the nucleic acid construct is present in the cell in the form of an episomal plasmid.

In a specific embodiment, the nucleic acid construct is constitutively expressed in the cell.

In a fifth aspect, the present invention provides a method for splicing DNA, the method comprising:

(1) transferring a donor plasmid containing a DNA sequence to be spliced, a nucleic acid construct capable of constitutively expressing Cas9 in yeast, a linearized acceptor vector for splicing, and a sgRNA-containing nucleic acid construct capable of directing Cas9 to cleave the donor plasmid into the same yeast cell; and

(2) incubating the yeast cells of step (1);

thereby realizing DNA splicing.

In one embodiment, a donor plasmid containing the DNA sequence to be spliced and a nucleic acid construct capable of constitutively expressing Cas9 in yeast are transferred into the same yeast cell by yeast protoplast fusion technology.

In one specific example, linearized acceptor vectors for splicing and sgRNA-containing nucleic acid constructs capable of directing Cas9 to cleave the donor plasmid are transferred into yeast cells using lithium acetate.

In one embodiment, a donor plasmid containing the DNA sequence to be spliced and a nucleic acid construct capable of constitutively expressing Cas9 in yeast are transferred into yeast cells using protoplast fusion technology, and then a linearized acceptor vector for splicing and a sgRNA-containing nucleic acid construct capable of directing Cas9 to cleave the donor plasmid are transferred into the yeast cells using the LiAc method.

In a specific embodiment, the donor plasmid comprises a single copy replication region in yeast, a selection marker, a sequence recognized by the sgRNA, and a sequence recognized for cleavage by Cas 9.

In one embodiment, both ends of the DNA to be spliced contain sequences homologous to the ends of the DNA to be spliced. In one embodiment, the homologous sequence is 300-800 bp in length, such as about 500 bp. In a particular embodiment, there is no sequence homologous to the homologous sequence in other regions of the DNA, except at both ends of the DNA.

In one embodiment, both ends of the recipient vector contain sequences homologous to both ends of the DNA to be spliced. In one embodiment, the homologous sequences are 60-500 bp in length, such as about 60-100 bp.

In a particular embodiment, the plasmid capable of constitutively expressing Cas9 in yeast is a nucleic acid construct according to the first aspect of the invention.

In a particular embodiment, the receptor vector is a nucleic acid construct according to the third aspect of the invention.

In a specific embodiment, the sgRNA-containing plasmid is a nucleic acid construct according to the second aspect of the invention.

In one embodiment, after step (2), the desired transformants are selected by a marker on the receptor vector, the plasmid capable of expressing Cas9, and the plasmid containing the sgRNA.

In one embodiment, the method of the present invention comprises:

(i) transferring the nucleic acid construct of the first, second and third aspects of the invention and a donor plasmid containing a DNA sequence to be spliced into the same yeast cell; and (ii) incubating the yeast cells of step (i); thereby realizing DNA splicing.

The present invention also provides a kit comprising any one, any two or all three of the nucleic acid constructs of the first, second and third aspects of the invention described above; and optionally a donor plasmid according to the invention. The kit may optionally contain various reagents necessary for transferring the nucleic acid construct or donor plasmid into a yeast cell by the yeast protoplast fusion technique or the LiAc method.

Drawings

FIG. 1 shows a principle diagram of a CRISPR/Cas 9-mediated large-fragment DNA splicing method. Three yeasts containing different donor plasmids (one of the yeasts contains a Cas9 expression plasmid) are fused through protoplasts, so that the three plasmids are fused into the same yeast cell; the LiAc transformed linearized receptor vector and psgRNA. After expression of sgRNA in yeast, the sgRNA binds to Cas9, which is positioned at a specific recognition site so that it can cleave sites near both ends of the large-fragment DNA on the donor plasmid (positions indicated by arrows), and the cleaved fragments are spliced with the linear acceptor vector into a circular plasmid by homologous recombination in yeast.

Fig. 2 shows a flow chart of a CRISPR/Cas 9-mediated large-fragment DNA splicing method. The Cas9 expression plasmid and all donor plasmids can be fused into the same yeast cell by protoplast fusion method, which takes 7 days. During this time, a linearized receptor vector can be prepared and the construction of the psgRNA can be performed. The linearized acceptor vector and the psgRNA were transformed by the LiAc method into yeast cells that already contained the Cas9 expression plasmid and all donor plasmids. The expression of CRISPR/Cas9 in yeast can cut donor plasmid release large fragment DNA, and by using a homologous recombination system of yeast, the large fragment DNA containing homologous arms and a linear acceptor vector can be correctly spliced into a circular plasmid, and the step needs 5 days.

Figure 3 shows a schematic representation of Cas9 expression plasmid pMetcas 9. Plasmid pMetcas9 contains the promoter Tef1 that allows Cas9 to be constitutively expressed in yeast. The plasmid contains a yeast replication region (CEN 6ARS 4) and a selection marker (MET14), and is replicated in yeast in a single copy; it also contains the E.coli replication region (from pBR322) and the selection marker Ampicillin (Ampicillin), and is high copy replication in E.coli. The sequence is shown in SEG ID NO: 1.

Fig. 4 shows a schematic diagram of sgRNA expression plasmid psgRNA. The plasmid psgRNA contains the promoter SNR52 that allows the sgRNA Target1 and Target2 to be constitutively expressed in yeast. The plasmid contains a yeast replication region (derived from 2 mu) and a selection marker (TRP1), and is replicated in yeast in multiple copies; it also contains the E.coli replication region (from pBR322) and the selection marker Ampicillin (Ampicillin), and is high copy replication in E.coli. The sequence is shown in SEG ID NO: 2.

FIG. 5 shows a schematic diagram of the linearized vector template pcriv0 plasmid. Plasmid pcriv0 contains a yeast replication region (CEN 6ARS 4) and a selection marker (ADE2), and is replicated in yeast in single copy; meanwhile, the vibrio chromosome II replication region oriCII and distribution regulation related genes rctA, rctB, inc, parA, parB and chloramphenicol screening markers are contained, and single copy replication is realized in escherichia coli. The sequence is shown in SEG ID NO 3.

FIG. 6 shows the restriction map and schematic representation of pCriv 6. A shows the restriction enzyme verification diagram of pCriv6, wherein M is Lambda Ladder PFG Marker; 1 is pCriv6NotI enzyme cutting pCriv 6; SpeI was used as SpeI for pCriv 6. pCriv6 was digested with NotI to give a band of 311kb in size, and digested with SpeI to give two bands of 98kb and 213kb in size. PFGE conditions: 1% agarose gel, 6V/cm, 14 ℃, 16 hours, and the conversion time is 1-25 seconds; b shows a schematic diagram of pCriv 6.

FIG. 7 shows the cleavage result of pCriv7 and a schematic diagram. A is pCriv7 restriction enzyme verification diagram, wherein M is Lambda Ladder PFG Marker; SpeI enzyme pCriv7 is shown as 1. The fragment of pCriv7 was digested with SpeI to obtain five bands of 200kb, 182kb, 157kb, 98kb and 32 kb. The 32kb band was similar in size to the yeast chromosome fragment and therefore could not be separated on the gel. PFGE conditions: 1% agarose gel, 6V/cm, 14 ℃, 16 hours, and the conversion time is 1-25 seconds; b is a schematic diagram of pCriv 7.

Detailed Description

The invention aims to develop a method for efficiently splicing large-fragment DNA. The invention reduces the difficult and low-efficiency in vitro operation of large-fragment DNA, and ensures that the splicing of the large-fragment DNA becomes simple and easy.

The invention provides a method for efficiently splicing DNA in yeast by using a CRISPR/Cas9 system (the principle is shown in figure 1). Transferring donor plasmids containing large-fragment DNA with homologous sequences at two ends to be spliced into the same yeast cell by a yeast protoplast fusion technology, wherein the yeast cell contains a plasmid for constitutive expression of Cas 9; jointly transforming a linearized acceptor vector for splicing and a sgRNA plasmid capable of cutting a donor vector by a LiAc method; after the donor vector was cut, the large fragment DNA was spliced to the acceptor vector by in vivo homologous recombination in yeast, and the desired transformants were selected by markers on the acceptor vector, Cas9 vector, and sgRNA vector (see fig. 2). Compared with the splicing method established by the predecessor, the method has the advantages that the complicated and low-efficiency steps of in-vitro extraction, enzyme digestion, recovery and transformation of large-fragment DNA are avoided in the experimental operation process, so that the experimental process becomes simple and easy to operate. The invention has no strict limitation on the size of DNA to be spliced and has the potential of being applied to splicing of oversized fragment DNA, such as assembly and splicing of genome for splicing several megabases. For example, DNA fragments of at least 100kbp, e.g., at least 500kbp, and up to several megabases, are suitable for splicing by the method of the present invention.

Herein, high copy replication refers to copy numbers greater than 1, typically 5 or more, such as 10 or more.

The invention has the following detailed features:

construction of Cas9 expression vector: plasmid pMetcas9 was constructed which is high copy replication in E.coli (origin of replication origin pBR322, selection marker Ampicillin (Ampicillin) resistance gene), single copy replication in yeast (origin of vector replication region CEN6ARS4, selection marker MET14) and constitutive expression of Cas 9. CEN6ARS4 was available from pSH47(Guldener, U. et al, A new effective gene deletion cassette for predicted use in filing year, Nucleic Acids Research 24(13): 2519-. The sequence of the replication region can be shown as 65 th-319 th bases of SEQ ID NO. 1. The sequence of Cas9 can be shown as bases 3610-7749 of SEQ ID NO. 1. HpaI, SpeI double digestion of plasmid p415-GalL-hCAS9 [ DiCarlo, J.E., et al, Genome engineering in Saccharomyces cerevisiae use CRISPR-Cas systems, Nucleic Acids Research, 2013, 41(7): p.4336-4343 ], two 5.4kbp fragments were recovered. MET14 marker (using Saccharomyces cerevisiae S.288c genome as template) and Tef1 promoter (using plasmid pAG36 (Goldstein, A.L., etc., and Three new dominant drug resistance cassettes for gene delivery in Saccharomyces cerevisiae, Yeast. Yeast.1999 Oct; 15(14): 1541-53) as template) were amplified by PCR, 40bp homologous sequence of adjacent fragment to be spliced was carried on 3' end band of primer, PCR product was purified and transferred into yeast for splicing, spliced plasmid was verified by PCR and sequencing, and E.coli 10B was transformed, and plasmid DH was extracted for use.

FIG. 3 shows a plasmid map of pMetcas9 of the present invention, the nucleotide sequence of which is shown in SEQ ID NO: 1.

Construction of sgRNA expression vector: plasmid psgRNA was constructed which replicated in E.coli in high copy (origin of replication origin from pBR322, selection marker ampicillin resistance gene), replicated in yeast in multiple copies (origin of vector replication region from 2. mu., selection marker TRP1) and was able to constitutively express sgRNA. The sgRNA included the SNR52 promoter, a 20bp recognition site, and a Handle Cas9 sequence. The 20bp recognition site is responsible for recognizing the corresponding recognition site on the donor plasmid, while the Handle Cas9 is responsible for recruiting Cas9 to the cleavage site, thereby effecting cleavage of the donor plasmid. The 20bp recognition site can be designed at will according to the framework sequence of the donor plasmid vector. In a specific example, bases 4624-4998 of SEQ ID NO. 2 show the sequence of the sgRNA and bases 5012-5386 show the sequence of another sgRNA, including the SNR5 promoter, the 20bp recognition site and the Handle Cas9 sequence. In the present example, since the backbone used for each donor plasmid is the same, only two recognition sites are required to achieve cleavage of all donor plasmids. The sequence of the 20bp recognition site can be designed according to techniques conventional in the art. The sequence of the two 20bp recognition sites used in the specific examples of the present invention is shown in Table I.

In one embodiment, two DNA fragments, Target1(SEQ ID NO:4) and Target2(SEQ ID NO:5), were synthesized, wherein Target1 was digested with BglII and SpeI, Target2 was digested with SpeI and NcoI, 400bp sized fragments were recovered, ligated to BglII and NcoI digested vector pTRPgRNA (plasmid p426-SNR52 p-SNR. Y-SUP4t [ DiCarlo, J.E., et al, supra ] plasmid engineering, URA3 selection marker was changed to TRP1 selection marker), E.coli DH10B was transformed, resulting in a plasmid containing two sgRNAs targeting recognition sites. The spliced plasmid is verified to be correct by PCR and sequencing.

Table one: sgRNA recognition sequence

FIG. 4 shows a plasmid map of the psgRNA of the invention, whose nucleotide sequence is shown in SEQ ID NO 2.

Construction and linearization of the receptor vector pcriv 0: the Replication region of pcriv0 vector (Vibrio second chromosomal Replication region [ Egan, E.S. and M.K.Waldor, discrimination Replication for the Two Vibrio cholerae Chomomosomes, Cell, 2003, 114(4): p.521-530 ]) and chloramphenicol screening marker (using the commercial plasmid pBeloBAC11(NEB Co., Ltd.) as a template), as well as the yeast Replication region (derived from CEN6ARS 4) and ADE2 screening marker (derived from Saccharomyces cerevisiae genome) were PCR-amplified with 40bp of homologous sequence to the adjacent fragment to be spliced on the 3' end of the primer. And purifying the PCR product, and transferring the purified PCR product into a yeast body for splicing. Positive clones were PCR verified and sequenced correctly and transformed into E.coli DH 10B. After the plasmid digestion verification is correct, the sequence correctness of the donor vector pcriv0 is confirmed by sequencing. The linearized vector can be obtained by PCR amplification, and has a sequence of 60-500 bp, preferably 60-200 bp (e.g. about 60bp) homologous to the large fragment DNA to be spliced on the primer band. FIG. 5 shows a plasmid map of the receptor vector pcriv0 of the present invention, the nucleotide sequence of which is shown in SEQ ID NO. 3.

Preparation of donor plasmid containing large fragment DNA: the donor plasmid containing the large fragment DNA to be spliced needs to contain a single copy replication region in yeast (derived from CEN6ARS 4) and a different selectable marker. In addition, the two ends of the large fragment DNA need to be added with sequences which are homologous with the adjacent large fragment DNA to be spliced and are 300-800 bp (for example, about 500 bp). All homologous sequences have no significant homologous sequence in other regions of these large fragments of DNA. Furthermore, it is understood that the donor plasmid should contain sequences that can be recognized by the sgRNA. Typically, this sequence is located outside the insertion site of the large fragment of DNA on the donor vector backbone and is identical to the 20bp recognition site in the sgRNA. The donor plasmid should also contain a site that can be cleaved by Cas9, such as a PAM site. Such sequences can be designed according to techniques known in the art. As an example, the following recognition/cleavage sites can be designed on the donor plasmid: ATAGTGTCACCTAAATAGCTTGG and CGTAGCAACCAGGCGTTTAAGGG (i.e., 20bp sequence plus PAM site in Table one). It is understood that the method of the present invention can splice more than 2 large fragments of DNA. Thus, the present invention can use more than two donor plasmids, e.g., 3, 4 or even more, as long as the structures of these donor plasmids satisfy the above requirements.

Transformation/protoplast fusion: for plasmids smaller than 30kb, transformation into yeast can be carried out by the LiAc method [ Gietz, R.D. and R.H.Schiestl, High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method, Nature Protocols, 2007, 2(1): p.31-34 ]. Plasmids larger than 30kb, which are difficult to transform into yeast by the LiAc method, require the use of the yeast Protoplast fusion method [ Curran, B.P. and V.C.Bugeja, protocol fusion in Saccharomyces cerevisiae, Methods in molecular biology (Clifton, N J), 1996, 53: p.45-9 ]. Two or three yeast cells containing different donor plasmids can be fused at one time, and the marker of all plasmids is used for screening, so that the yeast with all plasmids in the same cell can be obtained.

Splicing of large fragment DNA in yeast: the psgRNA and linearized receptor vector are transformed into yeast cells that already contain all of the large fragment DNA plasmids to be spliced. Under the combined action of sgRNA and Cas9, all donor plasmids are cleaved, releasing all large fragments of DNA to be spliced. Through a high-efficiency homologous recombination system in yeast, the ends of the homologous sequence containing large-fragment DNA and a linearized acceptor vector can be spliced together. The desired transformed recombinants were obtained by co-screening pMetcas9, psgRNA and the receptor vector for auxotrophic markers. Positive clones need to be verified by PCR, sequencing and pulsed field gel electrophoresis.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specifying specific conditions in the following examples are generally carried out according to conventional conditions such as those described in Molecular Cloning, A Laboratory Manual (third edition of Molecular Cloning, Laboratory Manual), by Sambrook & Russell, or according to the manufacturer's recommendations. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1: splicing of two large fragments of DNA

In this example, plasmids pSP5 and pTP3-U, which had been constructed in Saccharomyces cerevisiae VL6-48 in the laboratory, respectively, were used (Table II). pSP5 contains 116643bp of donor DNA, designated SP 5; pTP3-U contained 184475bp of the donor DNA and was designated TP 3. SP5 and TP3 sequences are both from Escherichia coli, and are larger plasmids obtained by PCR amplification of certain essential genes of Escherichia coli and two-stage or three-stage splicing in yeast, and 491bp homologous sequences are arranged at the right end of SP5 and the left end of TP 3.

Table two: information on plasmids pSP5 and pTP3-U

Donor plasmid	Screening markers	Name of Donor DNA	Insert size (bp)
				pSP5	HIS3	SP5	116643

pTP3-U

URA3

TP3

184475

Cas9 expression plasmid pMetcas9 (FIG. 3, SEQ ID NO:1) was transferred into yeast cells VL6-48 already containing the pSP5 plasmid. VL6-48/pSP5/pMetcas9 and VL6-48/pTP3-U were subjected to Protoplast fusion, yeast Protoplast fusion Methods and reagent formulations references [ Curran, B.P. and V.C. Bugeja, protocol fusion in Saccharomyces cerevisiae, Methods in molecular biology (Clifton, N J), 1996.53: p.45-9 ] and [ Koprina, N.and V.Lariovov, Selective isolation of genomic from complex genes by transformation-assisted cloning in the yeast Saccharomyces cerevisiae, Nature, 2008, 3(3): p.371-377, modified by the steps:

VL6-48/pSP5/pMetcas9 and VL6-48/pTP3-U were streaked onto SC-Met, Ura and SC-His plates, respectively, and cultured at 30 ℃ for 3 days.

2. Inoculating single colony of selected yeast into 5ml of corresponding SC liquid culture medium, and culturing at 30 ℃ overnight at 200 rpm;

3. overnight yeast liquid dilution 10 times for OD determination₆₀₀At this point, the OD should be 0.3-0.5 (indicating good growth). 1:20 to 1:25 to 50ml of the corresponding SC broth to initiate OD₆₀₀Less than or equal to 0.2. Culturing at 30 deg.C and 200rpm to OD₆₀₀From 1.0 to 1.5, about 5 to 6 hours.

Centrifuging at 4.20 deg.C and 5000rpm for 5min, resuspending with 30ml sterile water, centrifuging, and collecting bacteria; the cells were resuspended in 20ml of 1M sterile sorbitol solution and centrifuged at 5000rpm for 5 minutes to harvest the cells.

5. Using 20ml of SPE solution (1M sorbitol, 0.01M sodium phosphate, 0.01M Na)₂EDTA, pH 7.5), adding 20. mu.l of yeast lyase (Zymolyase) solution (20mg/ml Zymolyase-20T, 50% glycerol, 2.5% glucose, 50mM Tris-HCl, pH8.0) and 40. mu.l of beta-mercaptoethanol, mixing, and treating at 30 ℃ for 30 min.

Centrifuging at 6.5 deg.C for 5min at 570g, washing with 50ml 1M sorbitol once, and centrifuging at 600g for 5min at 300-.

7. The cells were resuspended in 20ml of MP buffer (1M sorbitol, 0.1M NaCl, 0.01M HAc, pH 5.5 adjusted with 10M sodium hydroxide solution), and centrifuged at 600g for 5min to harvest the cells.

8. The pellet was resuspended in 3ml of MP buffer, diluted 1/80-fold and counted in a platelet counter. Three strains were taken 1x 10 respectively⁷Mixing the cells, centrifuging at 600g for 5min and precipitating with 2ml of 60% PEG4000 and 0.2ml of 1M CaCl₂The solution was resuspended.

9. Standing at room temperature for 3min, adding 6ml MP buffer solution, mixing, and standing at room temperature for 6 min.

10. The bacteria were harvested by centrifugation as described above, washed once with 10ml of MP buffer, centrifuged and resuspended in 1ml of MP buffer.

11. Mu.l and 900. mu.l were added to the previously thawed supernatant medium (SC-Met, Ura, His), plated, and cultured at 30 ℃ for 5 to 7 days, respectively.

After protoplast fusion, 30 fusions were grown in SC triple-deleted medium, which were yeast containing three plasmids, pMetcas9, pSP5 and pTP3-U, and designated ZJ 1.

Two primers pCriv6-VR and pCriv6-VF (see Table III) having about 60bp homologous sequences with SP55 'end and TP 3-U3' end respectively were designed, pCriv0 was used as a template, KOD-plus-neo (purchased from Toyobo Co.) was used for PCR amplification of a linear donor vector under the conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 98 ℃ for 10s, annealing at 52 ℃ for 20s, and extension at 68 ℃ for 10min, for 30 cycles.

Table three: primers used in examples one and two

Lithium acetate (LiAc) transformed 1ug of the linear receptor vector (FIG. 5, SEQ ID NO:3) and 1ug of psgRNA (FIG. 4, SEQ ID NO:2) into strain ZJ 1. The formulation of the transformation method of LiAc and the reagents and culture medium are described in the reference [ Gietz, R.D. and R.H. schiestl, 2007, supra ], on the basis of which some improvements are made, the basic steps of which are:

1. the yeast ZJ1 was streaked on SC-Met, Ura, His plates and cultured at 30 ℃ for 2 days;

2. inoculating single colony of picked yeast into 5ml YPAD liquid culture medium, and culturing at 30 deg.C and 200rpm overnight;

3. overnight yeast liquid dilution 10 times for OD determination₆₀₀At this point, the OD should be 0.4-0.5 (indicating good growth). 1:20 to 1:25 transfer to 2 × YPAD liquid Medium to initiate OD₆₀₀Less than or equal to 0.2. Culturing at 30 deg.C and 200rpm to OD₆₀₀From 0.8 to 1.0 for about 4 to 5 hours;

centrifuging at 4.20 deg.C and 5000rpm for 5min, and resuspending with 1/2 volume of sterile water (such as 50ml bacterial solution resuspended with 25ml sterile water) and centrifuging to collect bacteria;

washing with 5.1/2 volume of sterile water again, centrifuging to collect bacteria, resuspending with 1ml of sterile water, transferring to a 1.5ml EP tube, centrifuging to collect bacteria at 13000g for 30 seconds;

6. resuspend with 1/50 volumes of sterile water (e.g., 50ml of bacterial suspension finally resuspended with 1ml of sterile water), 100 μ L per tube for use;

7. the ssDNA (salmon sperm DNA) frozen at-20 ℃ is taken out and denatured at 100 ℃ for 5 minutes, and then immediately inserted into ice;

8. 13000g of yeast was centrifuged for 30 seconds and the supernatant was discarded. Sequentially adding (a mixture except the DNA fragments can be prepared, and then the DNA fragments are added respectively):

note: positive control: circular yeast-E.coli shuttle plasmid pXX 11. Negative control: only add ddH₂O 34μL。

9. Resuspending yeast with the above mixture (vortex);

heat shock at 10.42 ℃ for 25 minutes (mix every 5 minutes);

11.13000 g centrifuged for 1 min to remove supernatant, and 200uL ddH was added₂And O, re-suspending the yeast, and coating SC-Met, Trp and Ade three-lacking plates.

And (3) screening by using SC-Met, Trp and Ade three-plate deletion, and culturing for 3-4 days at 30 ℃ to obtain 28 transformants, wherein the colony PCR verified positive rate is 5/7, and the transformants contain the correct splicing plasmid pCriv 6.

The colony PCR verification steps are as follows: designing three pairs of verification primers (shown in table III) at joints among fragments, wherein the size of amplified fragments is between 0.5 and 1.5kbp, selecting 7 yeast transformants to be suspended in 20uL sterile water, uniformly mixing, taking 0.2uL as a template, and carrying out PCR verification by using KOD-FX enzyme under the PCR amplification conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 98 ℃ for 10s, annealing at 52 ℃ for 20s, extension at 68 ℃ for 1.5min, 35 cycles.

Selecting a yeast to prepare a gel block, and carrying out pulse field gel electrophoresis detection. The method for preparing the rubber block by the yeast comprises the following steps:

1. the clones were inoculated into 50ml of SC-Met, Trp, Ade medium and incubated overnight at 30 ℃.

2.OD₆₀₀When the strain is 1.0-2.0, the strain is collected by centrifugation at 4000rpm for 5 min.

3. With 50ml ddH₂O, resuspending the thalli, centrifuging at 4000rpm for 5min to collect the thalli,

4. step 3 was repeated with 10ml 50mM EDTA pH8.0 and the cells washed once.

5. The cell pellet was resuspended in 750. mu.l of cell resuspension buffer (10mM Tris-HCl, pH 7.2), centrifuged at 4000rpm for 5min, resuspended in 150. mu.l of cell resuspension buffer, and equilibrated at 50 ℃.

6. Mu.l of Zymolyase-20T solution (20mg/ml Zymolyase-20T, 50% glycerol, 2.5% glucose, 50mM Tris-HCl, pH8.0) and 225. mu.l of 2% TE25S solubilized low melting agarose (prepared beforehand, equilibrated at 50 ℃) were added. Mixing, pouring into a mold, and cooling on ice or in a refrigerator at 4 deg.C.

7. After about 30min, the gel mass was removed and 5ml of yeast lyase (lyticase) buffer (10mM Tris-HCl pH7.5, 50mM EDTA pH8.0) and 500. mu.l Zymolyase-20T were added and incubated at 37 ℃ for 2h.

8. The gel cake was washed once with 25ml water and then once with wash buffer (20mM Tris-HCl pH8.0, 50mM EDTA).

9. 5ml of proteinase K reaction solution (100mM EDTA pH8.0, 0.2% sodium deoxycholate, 1% sodium lauroyl sarcosinate, 1mg/ml proteinase K) was added to each 1ml of gel block and digested at 50 ℃ overnight (cell characteristics were different, some samples required an extended digestion time of 4 days, but this did not damage the DNA).

10. The gel block was washed 4 times with 50ml of wash buffer and gently shaken at room temperature for 30-60min each time.

11. The linear genome of the yeast is run out of the gel block by pulsed field gel electrophoresis, the circular plasmid is still remained in the gel block, the electrophoresis conditions are 0.5 XTBE buffer solution, the voltage is 6v/cm, the conversion time is 60-120 seconds, and 12 hours are carried out.

12. The next day the gel block was removed, as in step 10, and the gel block was washed 4 times with 50ml of wash buffer, gently shaken at room temperature for 30-60min each time. In the 2 nd and 3 rd washing, 1mM PMSF was added to inactivate proteinase K.

13. Prior to restriction enzyme digestion, the gel pieces were washed with 10-fold diluted wash buffer or TE for 30min and then soaked in 1 Xcutmark buffer (from NEB).

14. NotI and SpeI (from NEB) were used to cleave 1/3 gel masses. And (3) detecting by pulsed field electrophoresis under the following electrophoresis conditions: 0.5 XTBE buffer, voltage 6v/cm, conversion time 1-25 seconds, 24 h.

The results of pulsed field gel electrophoresis showed that pCriv6 was digested with NotI to give a 311kb band, and digested with SpeI to give 98kb and 213kb bands, which were the same size as the predicted band (see FIG. 6).

Example 2: splicing of three large fragments of DNA

In this example, the plasmids pTP1, pTP2 and pTP3-L (Table IV), which had been constructed in Saccharomyces cerevisiae VL6-48 in this laboratory, were used.

Table four: information on plasmids pTP1, pTP2 and pTP3-L

	Screening markers	Insert name	Insert size (bp)
				pTP1	HIS3	TP1	177147
pTP2	URA3	TP2	297952
				pTP3-L	LYS2	TP3	184475

pTP1 contains 177147bp of donor DNA and is named TP 1; pTP2 contains 297952bp of donor DNA and is named TP 2; pTP3-L contained 184475bp of donor DNA and was designated TP 3. The TP1, TP2 and TP3 sequences were derived from E.coli and were constructed in a manner similar to that of pSP5 and pTP3-U in example I. The right end of TP1 has 385bp homologous sequence with the left end of TP2, and the right end of TP2 and the left end of TP3 have 491bp homologous sequence. The Cas9 expression plasmid pMetcas9 (see FIG. 3 and SEQ ID NO:1) was transferred into yeast cells already containing the pTP2 plasmid. The yeast VL6-48/pTP1, VL6-48/pTP2/pMetcas9 and VL6-48/pTP3-L were fused into the same yeast cell using the protoplast fusion method described in example one to obtain strain ZJ 2.

Designing two primers pCriv7-VR and pCriv7-VF (shown in Table II) which are respectively homologous to about 60bp at the TP15 'end and the TP 33' end, using pCriv0 as a template, and amplifying a linear donor vector by KOD-plus-neo PCR under the conditions that: pre-denaturation at 95 ℃ for 5 min; denaturation at 98 ℃ for 10s, annealing at 52 ℃ for 20s, and extension at 68 ℃ for 10min, for 30 cycles.

Mu.g donor linear vector (FIG. 5, SEQ ID NO:3) and 1. mu.g psgRNA (FIG. 4, SEQ ID NO:2) were co-transformed into ZJ2 by LiAc method, screened by SC-Met, Trp, Ade triple deletion plate, cultured at 30 ℃ for 3-4 days to obtain 7 transformants, the colony PCR verified positive rate was 2/3, and the correct spliced plasmid was named pCriv 7. The primers verified by PCR are shown in Table three.

One yeast is selected to prepare a gel block, and the spliced plasmid pCriv7 is detected by pulse field gel electrophoresis. The fragment size of pCriv7 was 5 bands of 200kb, 182kb, 157kb, 98kb and 32kb (covered by the yeast chromosome genome) by SpeI digestion, and the results of PFGE are shown in FIG. 7.

Each of the relevant documents listed above is incorporated by reference into this application in its entirety. The various aspects of the invention are addressed above. Nevertheless, it will be understood that various modifications may be made to the above teachings without departing from the spirit and scope of the invention. Also, similar aspects are included in the claims.

Claims

1. A method of splicing DNA, comprising:

(2) incubating the yeast cells of step (1);

thereby realizing DNA splicing;

wherein the DNA sequence obtained by splicing is at least 100 kbp;

the donor plasmid comprises a single copy replication region in yeast, a screening marker, a sequence capable of being recognized by sgRNA and a sequence capable of being cut by Cas9, wherein two ends of the DNA to be spliced contain sequences homologous with the tail ends of the spliced DNA, and the length of the homologous sequences is 300-800 bp;

the nucleic acid construct capable of constitutively expressing Cas9 in yeast comprises a nucleic acid construct having a sequence of nucleotides corresponding tocas9The yeast Tef1 promoter is connected with gene operation, the replication initiation site from pBR322 and a screening marker thereof, the replication region from CEN6ARS4 and a screening marker thereof, and the nucleic acid construct is in single copy replication in yeast and high copy replication in Escherichia coli;

two ends of a linearized receptor vector for splicing contain sequences homologous with two ends of DNA to be spliced, and the length of the homologous sequences is 60-500 bp;

the sgRNA-containing nucleic acid construct capable of guiding Cas9 to cut the donor plasmid is a nucleic acid construct capable of constitutively expressing sgRNA in yeast, and comprises a vector replication region and a selection marker thereof which are derived from 2 mu m, a replication initiation site and a selection marker thereof from pBR322 and sgRNA, wherein the sgRNA comprises an operably linked yeast promoter SNR52, a 20bp recognition site and a Handle Cas9 sequence; and

the linearized receptor vector for splicing contains a vector replication region derived from CEN6ARS4 and a screening marker thereof, is single-copy replication in yeast, and simultaneously contains vibrio chromosome II replication region oriCII and distribution regulation related genesrctA、rctB、inc、parA、parBAnd a selection marker, in Escherichia coli for single copy replication.

2. The method of claim 1,

(a) transferring the nucleic acid construct, the recipient vector and the donor plasmid into the same yeast cell by yeast protoplast fusion technology or the LiAc method; and/or

(b) The length of the homologous sequence in the donor plasmid is 500 bp; and/or

(c) The length of the homologous sequence in the receptor vector is 60-100 bp; and/or

(d) After step (2), the desired transformants are selected by the recipient vector, a nucleic acid construct capable of constitutively expressing Cas9 in yeast, and a marker on the sgRNA-containing nucleic acid construct capable of directing Cas9 to cleave the donor plasmid.

3. The method according to claim 1, characterized in that a donor plasmid containing the DNA sequence to be spliced and a nucleic acid construct capable of constitutively expressing Cas9 in yeast are transferred into the yeast cells using protoplast fusion technology, and then a linearized acceptor vector for splicing and a sgRNA-containing nucleic acid construct capable of directing Cas9 to cleave the donor plasmid are transferred into the yeast cells using the LiAc method.

4. The method of claim 1, wherein the nucleic acid construct capable of constitutively expressing Cas9 in yeast comprises, in operative association with each other, an origin of replication from pBR322, the promoter Tef1, and,cas9Genes, a termination sequence CYC1, lacZ α, the f1 origin, the selectable marker Met14, the replication region from CEN6ARS4 and the selectable marker ampicillin resistance gene.

5. The method of claim 1, wherein the nucleic acid construct constitutively expressing Cas9 in yeast comprises the replication region from CEN6ARS4 at bases 65-319 of SEQ ID NO:1 and the Cas9 gene at bases 3610-7749 of SEQ ID NO: 1.

6. The method of claim 1, wherein the nucleic acid construct capable of constitutively expressing Cas9 in yeast has the sequence shown in SEQ ID NO 1.

7. The method of claim 1, wherein the sgRNA-containing nucleic acid construct capable of directing Cas9 cleavage of the donor plasmid comprises, in operative association in sequence, an origin of replication from pBR322, an SNR52 promoter, a 20bp recognition site 1, a Handle Cas9 sequence, an SNR52 promoter, a 20bp recognition site 2, a Handle Cas9 sequence, a CYC1 primer, a CYC1 termination sequence, lacZ α, a f1 origin, TRP1, a vector replication region derived from 2 μ and an ampicillin resistance gene.

8. The method of claim 1, wherein the sgRNA-containing nucleic acid construct capable of directing Cas9 to cleave the donor plasmid comprises the sequences as shown at positions 4624-4998 and 5012-5386 of SEQ ID NO 2.

9. The method of claim 1, wherein the sequence of the sgRNA-containing nucleic acid construct capable of directing Cas9 to cleave the donor plasmid is set forth in SEQ ID No. 2.

10. The method of claim 1, wherein the linearized acceptor vector for splicing comprises an operably linked vector replication region derived from CEN6ARS4, selection marker ADE2, chloramphenicol resistance gene, partition regulator generctBVibrio II chromosomal replication region oriCII, and distribution regulatory geneinc、rctA、parAAndparB。

11. the method of claim 1, wherein the linearized acceptor vector for splicing has the sequence shown in SEQ ID No. 3.

12. The method of claim 1, wherein the DNA sequence obtained by splicing is at least 500 kbp.