CN108728468B

CN108728468B - Method and vector for cloning target DNA fragment

Info

Publication number: CN108728468B
Application number: CN201710264591.7A
Authority: CN
Inventors: 苗靳
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2017-04-21
Filing date: 2017-04-21
Publication date: 2022-02-01
Anticipated expiration: 2037-04-21
Also published as: CN108728468A

Abstract

The invention provides a method and a vector for cloning a target DNA fragment, wherein the method comprises the following steps: a recognition site for CRISPR-Cas9 located on one side of a target DNA fragment, and an integration site and a first recombination site located on both sides of the target DNA fragment, respectively, the first recombination site being located between the target DNA fragment and the CRISPR-Cas9 recognition site; cloning the integration site, the recognition site and the first recombination site into a vector, and integrating a spacer sequence into the 5' end of the sgRNA sequence on the vector; integrating the vector into the genetic material in which the DNA fragment of interest is located by homologous recombination; inducing expression of the Cas9 coding sequence of the positive clone, and carrying out homologous recombination between two first recombination sites at two ends of the fragment generated by cutting; and (4) screening plasmids carrying the target DNA fragments. The method has simple steps, low cost and high efficiency, and is particularly suitable for cloning large target DNA fragments from organisms.

Description

Method and vector for cloning target DNA fragment

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a method and a vector for cloning a target DNA fragment.

Background

The genome editing method based on the CRISPR (Clustered Regularly Interspaced Small Palindromic Repeats)/Cas9 system has deeply changed the research method and content in the field of life science. The CRISPR/Cas9 system is one of the widely existing adaptive immune systems in prokaryotes, and is used to cope with invasion of foreign nucleic acid molecules, such as degradation of phage-derived nucleic acid molecules, degradation of foreign plasmids transformed into cells, and the like. When the cell is first invaded, part of the exogenous DNA sequence is captured and regularly integrated in the form of a spacer (spacer) to become a CRISPR sequence. In order to accurately distinguish self from foreign DNA molecules, a PAM (promoter Adjacent Motif) motif 5 '-NGG-3' is present downstream (5 '-3' direction) of the spacer sequence in the foreign DNA molecule. When the cell is subjected to a secondary attack, this sequence is transcribed into the non-coding RNA crrna (crispr RNA). In CRISPR type II systems, there is also one non-coding RNA, tracrRNA, bound to the crRNA, which is processed and then directs the unique protease Cas9 to search for the spacer and PAM sequences, cleaving the twice-attacked DNA double-stranded molecule. For the convenience of vector construction, the crRNA tracrRNA binary molecule can be fused into a single RNA molecule, sgRNA (single guide RNA), 20nt of the 5' end of which is a spacer, which is responsible for recognizing the region of a specific gene. Thus, genome editing is possible only by expressing the Cas9 protein and the sgRNA simultaneously. Type II systems are more compact than other types of CRISPR systems. For the CRISPR/Cas9 system, only different RNA sequences need to be designed to realize the recognition and the cleavage aiming at specific sites.

In the current methods for directionally cloning large-fragment DNA, in vitro attempts to separate target fragments from genomic DNA using the CRISPR/Cas9 system, similar to the action of restriction enzymes, have been reported. The disadvantage of this approach is that it is overly dependent on high quality, library-building-grade genomic DNA prepared in vitro. Since the technology for preparing high-quality genomic DNA requires great experience, conventional laboratories often cannot be used conveniently. Therefore, cloning large-fragment DNA directly from an organism into a vector becomes an advantageous option. However, the methods for directly cloning large-fragment DNA from the body, which have been reported so far, are based on site-specific recombination systems, and require that recombinase recognition sites be pre-integrated on both sides of a target DNA fragment, and then recombinase expression is induced to loop out the target DNA fragment. The method has the disadvantages of repeated genetic operation and high time cost.

Disclosure of Invention

In the prior art, when large-fragment DNA is cloned, the method excessively depends on in vitro prepared high-quality genomic DNA with a library construction level, so the method has the advantages of high cost, complex technology and low efficiency. In addition, the method of in vivo cloning requires multiple genetic manipulations to introduce the recognition site of the site-specific recombinase, which is time-consuming and labor-intensive.

The present invention addresses the above-mentioned shortcomings by combining the CRISPR/Cas9 technology with the homologous recombination technology, developing a method for directly cloning a target DNA fragment from an organism onto a vector by simple genetic manipulation, the method comprising the steps of: (1) a CRISPR-Cas9 recognition site located at one side of the target DNA fragment, wherein the CRISPR-Cas9 recognition site is a 23nt sequence and comprises a 20nt spacer sequence and a 3nt PAM sequence located at the 3' end of the spacer sequence; (2) identifying an integration site and a first recombination site flanking the target DNA fragment, respectively, wherein the first recombination site is between the target DNA fragment and the CRISPR-Cas9 recognition site; (3) cloning the integration site, the CRISPR-Cas9 recognition site, and the first recombination site into the vector, wherein the CRISPR-Cas9 recognition site is located on the vector between the integration site and the first recombination site, the vector having a coding sequence for Cas9, a sgRNA sequence, and a coding sequence for a first resistance gene, wherein the Cas9 coding sequence is inducible expression, the resistance gene and sgRNA are constitutively expressed; (4) cloning the spacer sequence to the 5' end of the sgRNA sequence on the vector; (5) the integration site on the vector is subjected to homologous recombination with the integration site on the side of the target DNA fragment in the organism to integrate the vector into the genetic material in which the target DNA fragment is present, and then a positive clone in which vector integration occurs is selected by the first resistance gene; (6) inducing expression of the Cas9 coding sequence of the positive clone, followed by homologous recombination between two of the first recombination sites flanking the resulting fragment cleaved by the CRISPR-Cas9 system, respectively, to generate a plasmid comprising the DNA fragment of interest; (7) extracting plasmids, and screening the first resistance gene to obtain plasmids carrying the target DNA fragments. The method has simple steps, low cost and high efficiency. The method is particularly useful for cloning large target DNA fragments from organisms, particularly eukaryotes, while simultaneously knocking out target DNA fragments of said organisms.

According to one embodiment, the method further comprises: identifying a second recombination site flanking the target DNA fragment, wherein the CRISPR-Cas9 recognition site is located between the first recombination site and the second recombination site; and in step (3) also integrating said second recombination site into said vector, wherein said second recombination site is located on said vector between said integration site and said CRISPR-Cas9 recognition site; in step (6), homologous recombination occurs between the two second recombination sites located at the ends of the generated fragment cleaved by the CRISPR-Cas9 system. This method is particularly useful for cloning the target DNA fragment from a prokaryote, since prokaryotes may affect their metabolic function and cause death if they are unable to complete DNA repair by homologous recombination between the second recombination sites.

Preferably, the vector has a coding sequence for a second resistance gene and expression elements thereof, and the coding sequence for the second resistance gene is located on the vector between the second recombination site and the integration site. The selection of homologous recombination occurring through the second recombination site by the second resistance gene can be used to facilitate the selection of the organism that simultaneously knocks out the DNA segment of interest.

According to one embodiment, the vector comprises any one or more of the pMB1, p15A, pSC101 replicons. The replicon is a low copy number replicon, and can make large-fragment DNA relatively stable.

According to one embodiment, the vector comprises a single cleavage site for one or more restriction enzymes. The exogenous DNA fragment can be integrated on the carrier through the enzyme cutting sites.

According to one embodiment, the vector is a SG5 replicon deleted pCRISPR-Cas9 vector and the screening in steps (5) and (7) is performed with Apramycin (Apramycin) and in step (6) expression of the Cas9 coding sequence is induced with thiostrepton. The vector may also be the SG5 replicon deleted pKCCas9do vector.

According to one embodiment, the integration site 1 is 2-3 kb in length, and the first recombination site and the second recombination site are 300-700 bp in length. This ensures that homologous recombination-mediated plasmid integration occurs predominantly through the integration site and not at the first recombination site.

The target DNA fragment includes a large fragment regulatory sequence, a gene containing a large fragment intron and a regulatory sequence, or a gene cluster composed of functionally related genes.

The invention also provides a vector for cloning a target DNA fragment from an organism, wherein the vector comprises a coding sequence of Cas9, a sgRNA sequence and a coding sequence of a first resistance gene, wherein the Cas9 coding sequence is inducible expression and the resistance gene and sgRNA are constitutively expressed; and the vector has an integration site, a CRISPR-Cas9 recognition site and a first recombination site, the position of the CRISPR-Cas9 recognition site on the vector is between the integration site and the first recombination site, wherein the CRISPR-Cas9 recognition site is a 23nt sequence comprising a 20nt spacer sequence and a 3nt PAM sequence at the 3' end of the spacer sequence; and in the organism, the CRISPR-Cas9 recognition site is located on one side of the target DNA fragment, the integration site and first recombination site are located on both sides of the target DNA fragment, respectively, and the first recombination site is located between the DNA fragment and the CRISPR-Cas9 recognition site.

According to one embodiment, the vector further has integrated thereon a second recombination site located between the CRISPR-Cas9 recognition site and the integration site, and in the organism the CRISPR-Cas9 recognition site is located between the first recombination site and the second recombination site.

Preferably, the vector has a coding sequence for a second resistance gene and expression elements thereof, and the coding sequence for the second resistance gene is between the second recombination site and the integration site.

According to one embodiment, the vector is a SG5 replicon deleted pCRISPR-Cas9 vector. The vector may also be the SG5 replicon deleted pKCCas9do vector.

Drawings

Other features, objects and advantages of the present application will become more apparent upon reading of the following detailed description of non-limiting embodiments thereof, made with reference to the accompanying drawings in which:

fig. 1 is a technical principle schematic diagram of a method according to an embodiment of the present invention;

FIG. 2 is a schematic technical view of a method according to another embodiment of the present invention;

FIG. 3 is a map of the pCRISPR-Cas9 vector;

FIG. 4 is a map of pNTU33101 vector;

FIG. 5 is a schematic representation of the act synthesis gene cluster, integration site and recombination site of S.coelicolor M145 strain;

FIG. 6 is a map of pNTU33102 vector;

FIG. 7 is a restriction map of a plasmid carrying a target DNA fragment;

FIG. 8 shows the sequencing results of the plasmid carrying the target DNA fragment at both ends of the target DNA fragment.

Detailed Description

The present application will be described in further detail with reference to the following drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the relevant invention and not restrictive of the invention. It should be noted that, for convenience of description, only the portions related to the present invention are shown in the drawings.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments with reference to the attached drawings.

The invention combines the characteristics of accurate and efficient CRISPR/Cas9 technology and the characteristics of simple and feasible homologous recombination technology, invents a method for directly cloning large-fragment target DNA from an organism by using the CRISPR/Cas9 technology, applies the method to cloning a gene cluster of streptomyces, and creates a novel method for cloning large-fragment target DNA.

The organism can be various eucaryon and prokaryote such as animals, plants or microorganisms capable of genetic manipulation, for example, an organism of which annotation genes contain large segments of introns and regulatory sequences, or an organism of a gene cluster containing functional genes such as insect-resistant genes and disease-resistant genes. Methods of genetic manipulation include transformation, conjugal transfer, transduction, and transfection, among others.

The organism may be a streptomycete, which is an important source of nearly half of antibiotics. Antibiotic synthetic genes in streptomyces often exist in clusters, so that cloning large-fragment DNA is an important technical support for excavating natural product resources. Streptomyces as important sources of antibiotics include, for example, Streptomyces griseus (streptomycin-producing bacterium), Streptomyces avermitilis (avermectin-producing bacterium), Streptomyces lividans (thiostrepton-producing bacterium), Streptomyces cinnamomi (monensin-producing bacterium), Streptomyces albus (salinomycin-producing bacterium), and Streptomyces aureofaciens (tetracycline-producing bacterium).

The target DNA fragment is a piece of DNA on the genome of the organism of interest. The DNA segment of interest can also be derived from organelles such as mitochondria (mitochondrial DNA), chloroplasts (chloroplast DNA), and bacterial and fungal plasmids. The target DNA fragment may be a large fragment of regulatory sequence, may be a gene containing a large fragment of intron and regulatory sequence, or may be a gene cluster, for example, a gene cluster composed of functionally related genes. These functionally related genes may be synthetic or regulatory genes in the same metabolite synthesis pathway. These metabolites include secondary metabolites with activity against insects, antibiotics, etc.

Specifically, the method comprises the following steps:

(1) a CRISPR-Cas9 recognition site located at one side of the target DNA fragment, wherein the CRISPR-Cas9 recognition site is a 23nt sequence comprising a 20nt spacer and a 3nt PAM sequence located at the 3' end of the spacer sequence; the CRISPR-Cas9 recognition site can be located in the flanking sequence of the target DNA fragment.

The steps of screening for CRISPR-Cas9 recognition sites are well known in the art, and specifically, identifying CRISPR-Cas9 comprises the steps of:

i) identifying a 23nt sequence ending in 5 '-NGG-3' located on one side of the DNA fragment (the N is A, T, C or G), a 20nt comprising spacer and a 3nt comprising PAM, excluding sequences comprising five consecutive Ts;

ii) BLAST aligning 15 bases at the 3' end, including 12nt of spacer and 3nt of PAM, with the genome sequence of the organism, excluding non-specific sequences;

iii) fusing the selected 20nt spacer sequence with the sgRNA sequence, and predicting the RNA secondary structure by using an RNAfold server.

(2) Identifying an integration site and a first recombination site flanking the DNA fragment, respectively, wherein the first recombination site is between the DNA fragment and the CRISPR-Cas9 recognition site. The full length sequence of the integration site and the full length sequence of the first recombination site are not more than 30% identical to other sequences on the genome, thereby avoiding disturbance of the genome structure, and BLAST alignment of the full length sequence of the integration site and the full length sequence of the first recombination site with the genome sequence of the organism is performed, thereby excluding non-specific sequences.

(3) Cloning the integration site, the CRISPR-Cas9 recognition site, and the first recombination site into a vector, the position of the CRISPR-Cas9 recognition site on the vector being between the integration site and the first recombination site.

Homologous fragment-based cloning methods can be used, such as the In-fusion kit from Clontech, and also the yeast assembly method TAR. Cloning is preferably carried out using the Gibson Assembly method, which has the advantage of being simple and inexpensive.

The vector has a coding sequence for Cas9, a sgRNA sequence, and a coding sequence for a first resistance gene, wherein the Cas9 coding sequence is inducible expression and the resistance gene and sgRNA are constitutively expressed. The vector may comprise any one or more of the replicons pMB1, p15A, pSC101, which are low copy number replicons and may render large fragments of DNA relatively stable. The vector may also include a single cleavage site for one or more restriction enzymes to facilitate integration of the foreign DNA into the vector via these cleavage sites. Such vectors include the SG5 replicon-deleted pCRISPR-Cas9 vector or the SG5 replicon-deleted pKCas 9do vector (published in Acta Biochim Biophys Sin,2015,47(4), 231-243, commercially available from the addge website (http:// www.addgene.org /), numbered No. 62552).

(4) Integrating the spacer sequence into the vector at the 5' end of the sgRNA sequence; the spacer sequence can be integrated into the vector at the 5' end of the sgRNA sequence by the site of cleavage by a specific restriction endonuclease.

(5) The integration site on the vector is subjected to homologous recombination with the integration site on the side of the target DNA fragment in the organism to integrate the vector into the genetic material in which the target DNA fragment is present, and then a positive clone in which vector integration occurs is selected by the first resistance gene. The vector may be integrated into the genome of the DNA fragment by homologous recombination mediated plasmid integration.

The length of the integration site is 2-3 kb, and the length of the first recombination site is 300-700 bp. This ensures that homologous recombination-mediated plasmid integration occurs primarily through the integration site and not at the first recombination site.

For the method of plasmid integration, conjugative transfer is appropriate for Streptomyces; for other organisms, for example animals or fungi, corresponding genetic manipulation methods, such as transfection and transformation, are used.

When the organism is Streptomyces, a SG5 replicon-deleted pCRISPR-Cas9 vector can be used; the vector contains oriT, which is responsible for DNA shuttling between cells during conjugal transfer. If the vector is a SG5 replicon-deleted pCRISPR-Cas9 vector, screening is performed with apramycin.

(6) Inducing expression of the Cas9 coding sequence of the positive clone, and then allowing homologous recombination to occur between the two first recombination sites located at both ends of the same fragment generated by cleavage of the CRISPR-Cas9 system, respectively, to generate a plasmid comprising the DNA fragment of interest. If the vector is a SG5 replicon-deleted pCRISPR-Cas9 vector, thiostrepton (Tsr) is used for inducing the expression of the CRISPR-Cas9 system.

(7) Extracting plasmids, and screening the first resistance gene to obtain plasmids carrying the target DNA fragments. If the vector is a SG5 replicon-deleted pCRISPR-Cas9 vector, screening is performed with apramycin.

As shown in fig. 1, the method according to the above embodiment includes the steps of:

(1) identifying a CRISPR-Cas9 recognition site (, integration site (1), first recombination site (2) located on one side of the target DNA fragment. The DNA fragment and the sites are in the order of the integration site (1), the target DNA fragment, the first recombination site (2) and the CRISPR-Cas9 recognition site on the chromosome, and the order can also be reversed.

(2) The CRISPR-Cas9 recognition site (, the integration site (1) and the first recombination site (2) are cloned and integrated into a vector in the order integration site (1), CRISPR-Cas9 recognition site and first recombination site (2), which can also be reversed.

(3) The sequences of the vector are integrated into the chromosome by homologous recombination;

(4) inducing expression of CRISPR-Cas9 system, and cutting two CRISPR-Cas9 recognition sites;

(5) the CRISPR-Cas9 system cleavage produces a fragment carrying the target DNA, which generates a vector carrying the target DNA by homologous recombination occurring between the first recombination sites (2).

According to one embodiment, the method may further comprise identifying a second recombination site located on one side of the DNA fragment, wherein the CRISPR-Cas9 recognition site is located between the first recombination site and the second recombination site, and in step (3) the second recombination site is also integrated into the vector, wherein the second recombination site is located on the vector between the integration site and the CRISPR-Cas9 recognition site; in step (6), homologous recombination occurs between the two said second recombination sites located at the ends of the different fragments generated by the CRISPR-Cas9 system cleavage. Thus, an organism in which the target DNA fragment is knocked out can be obtained while cloning the DNA fragment. The full-length sequence of the second recombination site does not have more than 30% identity to other sequences on the genome, thereby avoiding disturbance of the genome structure, and the sequence of the second recombination site is BLAST aligned with the genome sequence of the organism, thereby excluding non-specific sequences.

Preferably, the vector has a coding sequence for a second resistance gene and expression elements thereof, and the coding sequence for the second resistance gene is located on the vector between the second recombination site and the integration site, so that homologous recombination occurring through the second recombination site can be screened for by the second resistance gene.

As shown in fig. 2, the method according to an embodiment of the present invention includes the steps of:

(1) a CRISPR-Cas9 recognition site (4), an integration site (1), a first recombination site (2), and a second recombination site (3) located on one side of the target DNA fragment. The DNA fragment and the sites are in the order of the integration site (1), the target DNA fragment, the first recombination site (2), the CRISPR-Cas9 recognition site and the second recombination site (3) on the chromosome, and the order can also be reversed.

(2) The CRISPR-Cas9 recognition site (4), the integration site (1), the first recombination site (2) and the second recombination site (3) are cloned and integrated into a vector, and the sequence of the sites on the vector is the integration site (1), the second recombination site (3), the CRISPR-Cas9 recognition site and the first recombination site (2), and the sequence can also be reversed.

(4) inducing expression of CRISPR-Cas9 system, and cutting CRISPR-Cas9 recognition site;

(5) and (3) cutting the CRISPR-Cas9 system to generate a fragment carrying the target DNA and a broken chromosome sequence, and carrying out homologous recombination on the fragment carrying the target DNA and the broken chromosome sequence through a first recombination site (2) and a second recombination site (3) respectively to generate a vector carrying the target DNA and a chromosome with the knocked-out target DNA.

It should be noted that while the operations of the method of the present invention are depicted in the drawings in a particular order, this does not require or imply that the operations must be performed in this particular order, or that all of the illustrated operations must be performed, to achieve desirable results. Rather, the steps depicted in the flowcharts may change the order of execution. Additionally or alternatively, multiple steps may be combined into one step execution and/or one step may be broken down into multiple step executions.

The invention also provides a vector for cloning a target DNA fragment from an organism, wherein the vector comprises a coding sequence of Cas9, a sgRNA sequence and a coding sequence of a first resistance gene, wherein the Cas9 coding sequence is inducible expression and the resistance gene and sgRNA are constitutively expressed; and the vector has an integration site, a CRISPR-Cas9 recognition site and a first recombination site, the position of the CRISPR-Cas9 recognition site on the vector is between the integration site and the first recombination site, wherein the CRISPR-Cas9 recognition site is a 23nt sequence comprising a 20nt spacer sequence and a 3nt PAM sequence at the 3' end of the spacer sequence; and in the organism, the CRISPR-Cas9 recognition site is located on one side of the target DNA fragment, the integration site and first recombination site are located on both sides of the target DNA fragment, respectively, and the first recombination site is located between the DNA fragment and the CRISPR-Cas9 recognition site. Also integrated on the vector is a second recombination site located between the CRISPR-Cas9 recognition site and the target DNA fragment, and in the organism the CRISPR-Cas9 recognition site is located between the first recombination site and the second recombination site. Preferably, the vector has a coding sequence for a second resistance gene and expression elements thereof, and the coding sequence for the second resistance gene is between the second recombination site and the integration site.

FIG. 6 shows a schematic representation of a vector according to one embodiment of the present invention, which is designated pNTU 33102. The vector has a coding sequence of Cas9, a sequence of sgRNA, a coding sequence of a first resistance gene apramycin resistance protein site acc (3) IV and a coding sequence of a second resistance gene Tsr resistance gene, wherein the Cas9 coding sequence has a tipA promoter responding to Tsr. The vector also has enzyme cleavage sites for Stu I, Nco I and SnaB I. The spacer sequence was inserted into the 5' end of the sgRNA sequence via the restriction sites of Nco I and SnaB I. The integration site (1), the first recombination site (2), the CRISPR-Cas9 recognition site (4), and the second recombination site (3) are integrated into the Stu I site of the vector by the Gibson Assembly DNA Assembly method in that order. The vector can also be obtained by genetic manipulation of the pKCas 9do vector deleted of the SG5 replicon.

Example 1 method for direct cloning of act gene cluster from streptomyces coelicolor using CRISPR-Cas9 technology.

Reagents and carriers therefor

pCRISPR-Cas9(ACS Synth. biol.,2015,4(9), 1020-1029; Genbank accession number KR011749, FIG. 3) vectors were from the University of Denmark technology (Technical University of Denmark) professor Tilmann Weber laboratory. The vector can also be synthesized by itself based on the vector sequences published by Genbank.

The Streptomyces coelicolor M145 strain is a widely used model strain for Streptomyces research. The Streptomyces coelicolor M145 strain of the invention is derived from ATCC species depository (accession number ATCC BAA-471).

This example directly clones the act synthetic gene cluster from S.coelicolor M145 strain. The method comprises the following steps:

1. the pCRISPR-Cas9 vector is modified, and an SG5 replicon (located at 9627-11069 bp of the vector) in the vector is deleted, so that the vector cannot autonomously replicate in streptomyces. The new vector was named pNTU33101 (FIG. 4). The resistance screening marker of the vector is apramycin, and the expression of the CRISPR-Cas9 system of the vector is induced by thiostrepton. The method comprises the following specific steps:

PCR was performed using pCRISPR-Cas9 vector as a template, primer 1 and primer 2.

Primer 1(5 '-3'): gcgttcaagggccgaaagccgagggtctgcctgccgtgaggt

Primer 2(5 '-3'): tcggctttcggcccttgaacgcctcgttcagcgacaccgtct

KOD plus (cat. KOD-211) from Toyobo (TOYOBO) was used for PCR.

The PCR reaction system is 50 μ L:

the PCR conditions were as follows:

pre-denaturation: 94 ℃ for 2 minutes

30 cycles: denaturation at 98 ℃ for 10 seconds

Extension at 68 ℃ for 10 min

After the PCR product is recovered, the pCRISPR-Cas9 vector with the SG5 replicon deleted can be obtained by transforming competent Escherichia coli. Coli can be any commercially available E.coli for cloning vectors, such as Top10, DH5 α, JM109, and the like.

2. And (3) recognizing CRISPR-Cas9 recognition sites (4) in the flanking sequences of the target DNA fragment, i.e. the streptomyces coelicolor act gene cluster, and selecting proper integration sites (1), first recombination sites (2) and second recombination sites (3). The positions and sizes of the 4 sites on the streptomyces coelicolor genome (Genebank accession number: NC-003888) are respectively as follows:

integration site (1): 5509957-5512067, size: 2111bp

First recombination site (2): 5534737-5535274, size: 541bp of

Second recombination site (3): 5535361-5535887, size: 527bp

CRISPR-Cas9 recognition site (4): 5535275-5535297, size: 23nt

The CRISPR-Cas9 recognition site (4) is located between

recombination sites

2 and 3 and comprises a spacer sequence of 20nt and a PAM sequence (CGG) of 3 nt.

Fig. 5 shows a schematic diagram of the act synthesis gene cluster of M145 strain, and the positions of CRISPR-Cas9 recognition site (4), integration site (1), first recombination site (2), and second recombination site (3) on chromosome, wherein the grey background indicates the spacer sequence and the PAM sequence is underlined. Each arrowed box represents a gene, with genes SCO5072 and SCO5092 shown in black.

3. The spacer was cloned between Nco I and SnaB I cleavage sites as published for the original vector (ACS Synth. biol.,2015,4(9), 1020-1029).

Primers used for cloning the spacer were:

primer 3(5 '-3'):

CATGCCATGGcacgcggttcgcccgttcgaGTTTTAGAGCTAGAAATAGC

(restriction sites for Nco I are underlined, the lower case bases are spacer sequences)

Primer 4(5 '-3'):

ACGCCTACGTAAAAAAAGCACCGACTCGGTGCC

(the restriction sites of SnaB I are underlined)

KOD plus (cat. KOD-211) from Toyobo (TOYOBO) was used for PCR.

The PCR reaction system is 50 μ L:

the PCR conditions were as follows:

pre-denaturation: 94 ℃ for 2 minutes

30 cycles: denaturation at 98 ℃ for 10 seconds

Annealing at 58 ℃ for 30 seconds

The extension was carried out at 68 ℃ for 10 seconds,

then, 1. mu.L of the PCR product was directly digested, and 10. mu.L of the digestion system (Fermentas):

pNTU33101 was digested (Fermentas), 50. mu.L system:

the cleavage product of pNTU33101 was subjected to electrophoresis, and a plasmid fragment (about 10Kb) was recovered. mu.L of PCR digestion products of the

primers

3 and 4 and 1. mu.L of pNTU33101 digestion recovery products are taken for ligation, 3. mu.L of TAKARA ligation kit Solution I (cat No. 6022) is used, the final system is 6. mu.L, reaction is carried out for 5 minutes, Escherichia coli is transformed, and then positive clones are screened.

4. The CRISPR-Cas9 recognition site (4), integration site (1), first recombination site (2), and second recombination site (3) were integrated onto the vector by the Gibson Assembly DNA method. Wherein the template is the genomic DNA of the Streptomyces coelicolor M145 strain. The three PCR products were integrated into the Stu I site in the spacer-ligated pNTU33101 vector using the widely used Gibson Assembly DNA Assembly method, and the resulting vector was named pNTU33102 (FIG. 5).

The primers used for cloning the integration site (1) were:

primer 5(5 '-3'): tctcgtcgaaggcactagaagggaagagggcaacctctacctggtc

Primer 6(5 '-3'):

tgcgaagctggcGAGGGGTTGTTGCTGAACATCTTGAC

the primers used for cloning the second recombination site (3) were:

primer 9(5 '-3'):CAACAACCCCTCgccagcttcgcacctcctccccga

primer 10(5 '-3'):

gcgggcggctgctggtcgtc

the primers used for cloning the first recombination site (2) were:

primer 7(5 '-3'):

cccggtgttcgacagttgcggcgag

primer 8(5 '-3'):

ggtcgatccccgcatataggGTGCTCGACGCCTGCACCGACCT

cas9 recognition site 4 is introduced during PCR by primer synthesis, specifically, primer 7 is underlined in bold and is a partial sequence of spacer, a partial sequence of PAM is indicated by a box, the underlined in bold and is a reverse complement sequence of spacer in primer 10, and a partial sequence of PAM reverse complement is indicated by a box.

KOD plus (cat. KOD-211) from Toyobo (TOYOBO) was used for PCR.

The PCR reaction system is 50 μ L:

the PCR conditions were as follows:

pre-denaturation: 94 ℃ for 2 minutes

30 cycles: denaturation at 98 ℃ for 10 seconds

Annealing at 58 ℃ for 30 seconds

Extension at 68 ℃ for 2 min

The spacer-ligated pNTU33101 vector was digested with Stu I (Fermentas cat # FD0424) in a 10. mu.L system:

gibson Assembly used a 2 Xpremix reaction system of NEB (cat. No. E2611S), 20. mu.L, at 50 ℃ for 1 hour:

the reaction product was transformed into E.coli and positive clones were selected.

5. The vector pNTU33102 was integrated into the S.coelicolor genome by means of conjugative transfer (see experimental procedures in Tobias Kieser, Mervyn J. Bibb, Mark J. Buttner, Keith F. Chater, David A. Hopwood, Practical Streptomyces Genetics, John Innes Foundation (2000), pp. 249-250), i.e.the pNTU33102 plasmid was introduced into the competent S.coelicolor M145 strain.

The bonding transfer experiment procedure was:

(1) fresh spores of Streptomyces coelicolor were suspended in 1mL of sterile water.

(2) After heat shock in a water bath at 50 ℃ for 10 minutes, the mixture was left at room temperature.

(3) An overnight culture of E.coli ET12567 strain (widely used by Streptomyces laboratories) containing the pNTU33102 plasmid was added to an equal volume of spore suspension.

(4) MS medium plates were plated and incubated at 28 ℃.

(5) After culturing for 14 hours, covering the culture medium with 1mL of sterile water containing 0.5mg of nalidixic acid and 1mg of apramycin, standing, airing, culturing at 28 ℃ for 4-5 days, and screening the apramycin-resistant zygotes after the zygotes grow out. Since the plasmid must recombine with the chromosome because it cannot replicate autonomously, apramycin can be used to screen for conjugative transfer of the zygote.

MS medium (1L): 20 g of Mannitol (Mannitol), 20 g of soybean cake powder and 20 g of agar powder.

6. Culturing an ampomycin-resistant zygote in a YEME liquid medium, adding 1 mu M thiostrepton to induce a CRISPR-Cas9 system after culturing for 48 hours at 28 ℃, cutting recognition sites 4 of Cas9 on both sides of a target DNA fragment to generate a fragment carrying the target DNA and a broken chromosome sequence, and carrying out homologous recombination through a first recombination site (2) and a second recombination site (3) respectively to generate a vector carrying the target DNA and a chromosome with the knocked-out target DNA.

YEME Medium (1L): 3 g of Yeast Extract powder (Yeast Extract), 5 g of Bacto-peptone (Bacto-peptone), 3 g of Malt Extract (Malt Extract), 10 g of glucose and 100 g of sucrose.

7. Extracting plasmid, transferring the plasmid into escherichia coli again, coating the escherichia coli on a flat plate containing apramycin, and screening the obtained clone to obtain the clone carrying the target DNA fragment. Enzyme digestion and sequencing validation of Positive clones

Extracting positive cloning plasmid, electrophoresis, identifying the obtained cloning according to the size of plasmid band, and screening the cloning containing the capture large fragment. Plasmids were extracted from positive clones and further digested (FIG. 7) and sequence verified (FIG. 8). In FIG. 7, the left graph shows the result of BamHI cleavage assay, and the right graph shows the result of Bgl II and StuI double cleavage assay. FIG. 8 shows the sequencing results of both ends of the target DNA fragment of the plasmid carrying the target DNA fragment. As can be seen from FIGS. 7 and 8, the DNA fragment of interest has been cloned into the vector.

The invention combines the characteristics of accurate and efficient CRISPR/Cas9 technology and the characteristics of simple and feasible homologous recombination technology, and initiates a method for directly cloning large-fragment target DNA from an organism by using the CRISPR/Cas9 technology. The method has simple steps, low cost and high efficiency, and is particularly suitable for cloning large target DNA fragments from organisms.

The above description is only a preferred embodiment of the application and is illustrative of the principles of the technology employed. It will be appreciated by a person skilled in the art that the scope of the invention as referred to in the present application is not limited to the embodiments with a specific combination of the above-mentioned features, but also covers other embodiments with any combination of the above-mentioned features or their equivalents without departing from the inventive concept. For example, the above features may be replaced with (but not limited to) features having similar functions disclosed in the present application.

Claims

1. A method for cloning a DNA fragment of interest from an organism into a vector, comprising the steps of:

(1) identifying a CRISPR-Cas9 recognition site located at one side of the target DNA fragment, wherein the CRISPR-Cas9 recognition site is a 23nt sequence and comprises a 20nt spacer sequence and a 3nt PAM sequence located at the 3' end of the spacer sequence;

(2) identifying an integration site and a first recombination site flanking the target DNA fragment, respectively, wherein the first recombination site is between the target DNA fragment and the CRISPR-Cas9 recognition site;

(3) cloning the integration site, the CRISPR-Cas9 recognition site, and the first recombination site into the vector, wherein the CRISPR-Cas9 recognition site is located on the vector between the integration site and the first recombination site, and the CRISPR-Cas9 recognition site is linked at one end to the integration site and at the other end to the first recombination site, wherein the vector has a coding sequence of Cas9, a sgRNA sequence, and a coding sequence of a first resistance gene, wherein the Cas9 coding sequence is inducible expression, and the coding sequence of the first resistance gene and the sgRNA sequence are constitutive expression;

(4) cloning the spacer sequence to the 5' end of the sgRNA sequence on the vector;

(5) the integration site on the vector is subjected to homologous recombination with the integration site on the side of the target DNA fragment in the organism to integrate the vector into the genetic material in which the target DNA fragment is present, and then a positive clone in which vector integration occurs is selected by the first resistance gene;

(6) inducing the expression of the Cas9 coding sequence of the positive clone, cutting two CRISPR-Cas9 recognition sites which are respectively positioned at two sides of the target DNA fragment, and then carrying out homologous recombination between two first recombination sites which are respectively positioned at two ends of the same DNA fragment generated by the cutting of a CRISPR-Cas9 system to generate a plasmid containing the target DNA fragment;

(7) extracting the plasmid, and screening the first resistance gene to obtain the plasmid carrying the target DNA fragment.

2. The method of claim 1, further comprising:

identifying a second recombination site flanking the target DNA fragment, wherein the CRISPR-Cas9 recognition site is located between the first recombination site and the second recombination site; and is

Also integrating the second recombination site into the vector in step (3), wherein the second recombination site is located on the vector between the integration site and the CRISPR-Cas9 recognition site, and the integration site, the second recombination site, the CRISPR-Cas9 recognition site, and the first recombination site are linked in sequence;

in step (6), homologous recombination occurs between the two second recombination sites located at the ends of the fragment resulting from cleavage by the CRISPR-Cas9 system.

3. The method of claim 1 or 2, wherein the vector comprises any one or more of the pMB1, p15A, pSC101 replicons, and a single cleavage site for one or more restriction endonucleases.

4. The method according to claim 1 or 2, wherein the vector is a SG5 replicon deleted pCRISPR-Cas9 vector and the screening with apramycin in step (5) and step (7) and thiostrepton in step (6) induce expression of the Cas9 coding sequence.

5. The method according to claim 1 or 2, wherein the target DNA fragment comprises a large fragment of regulatory sequences, a gene comprising a large fragment of introns and regulatory sequences, or a gene cluster consisting of functionally related genes.

6. The method of claim 2, wherein the integration site is 2-3 kb in length and the first and second recombination sites are 300-700 bp in length.

7. A vector for cloning a target DNA fragment from within an organism, wherein the vector comprises a coding sequence for Cas9, a sgRNA sequence, and a coding sequence for a first resistance gene, wherein the Cas9 coding sequence is inducible expression and the resistance gene and sgRNA are constitutively expressed;

the vector is provided with an integration site, a CRISPR-Cas9 recognition site and a first recombination site, the CRISPR-Cas9 recognition site is positioned between the integration site and the first recombination site on the vector, one end of the CRISPR-Cas9 recognition site is connected with the integration site, and the other end is connected with the first recombination site, wherein the CRISPR-Cas9 recognition site is connected with the integration site and the first recombination site on the vector

The CRISPR-Cas9 recognition site is a 23nt sequence and comprises a 20nt spacer sequence and a 3nt PAM sequence positioned at the 3' end of the spacer sequence; and is

In the organism, the CRISPR-Cas9 recognition site is located on one side of the target DNA fragment, the integration site and first recombination site are located on both sides of the target DNA fragment, respectively, and the first recombination site is located between the DNA fragment and the CRISPR-Cas9 recognition site;

the integration site on the vector is subjected to homologous recombination with the integration site on the side of the target DNA fragment in the organism, thereby integrating the vector into the genetic material in which the target DNA fragment is present; homologous recombination occurs between two of the first recombination sites located at both ends of the same DNA fragment resulting from the cleavage of the CRISPR-Cas9 system, respectively, to generate a plasmid comprising the DNA fragment of interest.

8. The vector of claim 7, wherein said vector further has a second recombination site integrated thereon, wherein said second recombination site is located on said vector between said integration site and said CRISPR-Cas9 recognition site, and said integration site, said second recombination site, said CRISPR-Cas9 recognition site, and said first recombination site are linked in sequence;

and in said organism, said CRISPR-Cas9 recognition site is located between said first recombination site and said second recombination site;

wherein homologous recombination occurs between the two second recombination sites at the ends of the fragment resulting from the cleavage by the CRISPR-Cas9 system to generate a plasmid comprising the DNA fragment of interest.

9. The vector of claim 7 or 8, wherein the vector comprises any one or more replicons of pMB1, p15A, pSC101, and a single cleavage site for one or more restriction endonucleases.

10. The vector of claim 7 or 8, wherein the vector is a SG5 replicon deleted pCRISPR-Cas9 vector.