CN108728468A - Clone the method and carrier of target DNA fragments - Google Patents
Clone the method and carrier of target DNA fragments Download PDFInfo
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- CN108728468A CN108728468A CN201710264591.7A CN201710264591A CN108728468A CN 108728468 A CN108728468 A CN 108728468A CN 201710264591 A CN201710264591 A CN 201710264591A CN 108728468 A CN108728468 A CN 108728468A
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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Abstract
The present invention provides clone target DNA fragments method and carrier, the method includes：The CRISPR-Cas9 recognition sites that identification is located at the side of target DNA fragments and the integration site and the first recombination site for being located at the target DNA fragments both sides, first recombination site is between the target DNA fragments and the CRISPR-Cas9 recognition sites；Integration site, recognition site and the first recombination site are cloned into carrier, spacer sequences are integrated into 5 ' ends of sgRNA sequences on the carrier；By homologous recombination by will be on the inhereditary material where the vector integration to the target DNA fragments；The expression for inducing the Cas9 coded sequences of the positive colony cuts and homologous recombination occurs between two first recombination sites at the segment both ends of generation；Screening carries the plasmid of the target DNA fragments.The method and step of the present invention is simple, at low cost, efficient, particularly suitable that big target DNA fragments are cloned from organism.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of method and carrier of clone's target DNA fragments.
Background technology
Based on CRISPR (the short palindrome repetitive sequence in Regularity interval, Clustered Regularly Interspaced
Small Palindromic Repeats)/Cas9 systems genome edit methods it is deep change life science neck
The research method and research contents in domain.CRISPR/Cas9 systems be the adaptive immune system that is widely present in prokaryotes it
One, for coping with exogenous nucleic acid molecule invasion, such as the nucleic acid molecules in degradation bacteriophage source, Degradation and Transformation enters outer in cell
Source plasmid etc..After cell is invaded for the first time, partial exogenous DNA sequence dna is captured, has rule in the form of introns (spacer)
The integration of rule becomes CRISPR sequences.In order to accurately distinguish the DNA molecular of itself and external source, spacer in the DNA molecular of external source
There are 5 '-NGG-3 ' of PAM (Protospacer Adjacent Motif) motif for sequence downstream (5 ' -3 ' direction).When cell meets with
When meeting secondary attack, this section of sequence can be transcribed into non-coding RNA --- crRNA (CRISPR RNA).In CRISPR II type systems
In, there is also a non-coding RNAs --- and tracrRNA is combined with crRNA, is processed, is then instructed unique protease
Cas9 finds spacer sequences and PAM sequences, cuts the DNA double chain molecule of secondary attack.For the ease of vector construction, crRNA:
TracrRNA double bases molecule can be fused to single RNA molecule --- sgRNA (single guide RNA), 5 ' ends
20nt is spacer, is responsible for the region of identification specific gene.As long as expressing Cas9 albumen simultaneously in this way and sgRNA being possible to reality
Existing genome editor.Compared with other kinds of CRISPR systems, II type system structures are more compact.For CRISPR/Cas9 systems
System, it is only necessary to design different RNA sequences and identification and cutting for specific site can be realized.
At present in the method for directed cloning large fragment DNA, has been reported that and attempted in vitro using CRISPR/Cas9 systems
System is similar to the effect of restriction enzyme, target fragment is separated from genomic DNA.The shortcomings that this method, exists
In the genomic DNA of the excessively high quality of dependence external preparation, library construction rank.Due to preparing high quality genomic DNA
Technology needs rich experiences, Routine Test Lab often can not easily use.Therefore, directly from biological vivo clone large fragment
Just become a kind of advantageous selection on DNA to carrier.However, at present it has been reported that it is direct from vivo clone large fragment
The method of DNA is all based on site-specific recombination system, and enzyme recognition site will be recombinated by needing is integrated into target DNA fragments in advance
Both sides, then induction recombination expression of enzymes so that target DNA fragments ring is gone out.The shortcomings that this method is the heredity needed by repeatedly
Operation, time cost are larger.
Invention content
When cloning large fragment DNA in the prior art, the high quality of external preparation, the gene of library construction rank are excessively relied on
Group DNA, so cost is higher, technical sophistication, less efficient.In addition, the method for vivo clone needs multistep genetic manipulation to import
The recognition site of locus specificity recombinase, it is time-consuming and laborious.
The present invention develops one for disadvantages mentioned above by the way that CRISPR/Cas9 technologies to be combined with homologous recombination technique
Kind only can be directly from the method on biological vivo clone target DNA fragments to carrier, the method by simple inheritance operation
Include the following steps：(1) identification is located at the CRISPR-Cas9 recognition sites of the side of the target DNA fragments, described
CRISPR-Cas9 recognition sites are the sequence of 23nt, comprising the spacer sequences of 20nt and positioned at the spacer sequences 3 '
The PAM sequences of the 3nt at end；(2) identification is located at the integration site and the first recombination site of the target DNA fragments both sides,
Wherein, first recombination site is between the target DNA fragments and the CRISPR-Cas9 recognition sites；(3) will
The integration site, the CRISPR-Cas9 recognition sites and first recombination site are cloned into the carrier,
In, the position of the CRISPR-Cas9 recognition sites on the carrier is located at the integration site and first recombination position
Between point, coded sequence of the carrier with the coded sequence of Cas9, sgRNA sequences and the first resistant gene, wherein institute
It is inducible expression to state Cas9 coded sequences, and the resistant gene and sgRNA are constitutive expressions；(4) by the spacer sequences
Row are cloned into 5 ' ends of sgRNA sequences on the carrier；(5) integration site on the carrier in the organism with it is described
Homologous recombination occurs for the integration site of target DNA fragments side, to will the vector integration to target DNA fragments place
Inhereditary material on, then pass through first resistant gene screening occur vector integration positive colony；(6) sun is induced
Property clone Cas9 coded sequences expression, be then located at the segment both sides of the generation of CRISPR-Cas9 systems cutting
Homologous recombination occurs between two first recombination sites, generates the plasmid for including the target DNA fragments；(7) matter is extracted
Grain is screened by first resistant gene and obtains the plasmid for carrying the target DNA fragments.This method step is simple, cost
It is low, it is efficient.This method is particularly suitable to clone big target DNA fragments from organism, especially eucaryote, while can be with
Knock out the target DNA fragments of the organism.
According to one embodiment, the method further includes：Identification is located at the second of the target DNA fragments side
Recombination site, wherein the CRISPR-Cas9 recognition sites are located at first recombination site and are located at and second recombination position
Between point；And second recombination site is also integral in the carrier in step (3), wherein second recombination position
Point is on the carrier between the integration site and the CRISPR-Cas9 recognition sites；In step (6), it is located at
Occur between two second recombination sites of the fragment ends of the generation of the CRISPR-Cas9 systems cutting homologous heavy
Group.This method is particularly suitable to clone the target DNA fragments from prokaryotes, if because prokaryotes cannot pass through second
The homologous recombination occurred between recombination site is completed DNA and is repaired, and may influence its metabolic function, lead to death.
Preferably, the carrier has the coded sequence and its Expression element of the second resistant gene, and described second is anti-
The coded sequence of property gene is on the carrier between second recombination site and the integration site.Can be passed through
The homologous recombination that two resistance genescreens are occurred by the second recombination site helps to screen while knocking out the target dna piece
The organism of section.
According to one embodiment, the carrier includes any one or more of pMB1, p15A, pSC101 replicon.On
The replicon that replicon is low copy number is stated, the DNA of large fragment can be made more stable.
According to one embodiment, the carrier includes the single restriction enzyme site of one or more restriction enzymes.Outside
Source DNA segment can be by carrier that these restriction enzyme sites are integrated.
According to one embodiment, the pCRISPR-Cas9 carriers that the carrier is deleted for SG5 replicons, and step
(5) and in step (7) it is screened with apramycin (Apramycin), the Cas9 is induced with thiostrepton in step (6)
The expression of coded sequence.The carrier can also be the deleted pKCCas9do carriers of SG5 replicons.
According to one embodiment, the length of the integration site 1 is 2~3kb, first recombination site and described the
The length of two recombination sites is 300~700bp.It can ensure that the plasmid integration of homologous recombination mediation mainly by described whole in this way
It closes site to carry out, without carrying out in first recombination site.
The target DNA fragments include the regulating and controlling sequence of large fragment, the gene containing large fragment introne and regulating and controlling sequence,
Or the gene cluster being made of function related gene.
The present invention also provides for the carrier from biological vivo clone target DNA fragments, wherein the carrier includes
The coded sequence of Cas9, the coded sequence of sgRNA sequences and the first resistant gene, wherein the Cas9 coded sequences are inductions
Type is expressed, and the resistant gene and sgRNA are constitutive expressions；And there is integration site, CRISPR-Cas9 on the carrier
Recognition site and the first recombination site, the position of the CRISPR-Cas9 recognition sites on the carrier are located at described whole
It closes between site and first recombination site, wherein the CRISPR-Cas9 recognition sites are the sequence of 23nt, including 20nt
Spacer sequences and positioned at the spacer sequences 3 ' end 3nt PAM sequences；And it is described in the organism
CRISPR-Cas9 recognition sites are located at the side of the target DNA fragments, and the integration site and the first recombination site distinguish position
In the both sides of the target DNA fragments, also, first recombination site is located at the DNA fragmentation and the CRISPR-Cas9
Between recognition site.
According to one embodiment, the second recombination site is also integrated on the carrier, second recombination site is located at
Between the CRISPR-Cas9 recognition sites and integration site, and in the organism, the CRISPR-Cas9 identifications
Site is located at first recombination site and is located between second recombination site.
Preferably, the carrier has the coded sequence and its Expression element of the second resistant gene, and described second is anti-
The coded sequence of property gene is between second recombination site and the integration site.
According to one embodiment, the carrier includes any one or more of pMB1, p15A, pSC101 replicon.On
The replicon that replicon is low copy number is stated, the DNA of large fragment can be made more stable.
According to one embodiment, the carrier includes the single restriction enzyme site of one or more restriction enzymes.Outside
Source DNA segment can be by carrier that these restriction enzyme sites are integrated.
According to one embodiment, the carrier is the pCRISPR-Cas9 carriers that SG5 replicons are deleted.The carrier
It can also be the deleted pKCCas9do carriers of SG5 replicons.
Description of the drawings
By reading a detailed description of non-restrictive embodiments in the light of the attached drawings below, the application's is other
Feature, objects and advantages will become more apparent upon：
Fig. 1 is the technical principle schematic diagram according to the method for one embodiment of the present invention；
Fig. 2 is the technical principle schematic diagram according to the method for another embodiment of the present invention；
Fig. 3 is the collection of illustrative plates of pCRISPR-Cas9 carriers；
Fig. 4 is the collection of illustrative plates of pNTU33101 carriers；
Fig. 5 is the signal of act the synthetic genes cluster, integration site and recombination site of Streptomyces coelicolor M145 bacterial strain
Figure；
Fig. 6 is the collection of illustrative plates of pNTU33102 carriers；
Fig. 7 is the restriction enzyme mapping for the plasmid for carrying target DNA fragments；
Fig. 8 is the sequencing result at target DNA fragments both ends for the plasmid for carrying target DNA fragments.
Specific implementation mode
The application is described in further detail with reference to the accompanying drawings and examples.It is understood that this place is retouched
The specific embodiment stated is only used for explaining related invention, rather than the restriction to the invention.It also should be noted that in order to just
It is illustrated only in description, attached drawing and invents relevant part.
It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase
Mutually combination.The application is described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.
The simple and practicable feature of present invention combination CRISPR/Cas9 technologies accurate, efficient feature and homologous recombination technique,
Invented it is a kind of using CRISPR/Cas9 technologies out of organism Direct Cloning large fragment target dna method, and applied
In the gene cluster of clone streptomycete, a kind of method of new clone's large fragment target dna has been started.
The organism can be various eukaryons, the prokaryotes that animal, plant or microorganism etc. can carry out genetic manipulation,
Such as it annotates the organism that gene contains large fragment introne and regulating and controlling sequence, or contain anti insect gene, disease-resistant gene
The organism of the gene cluster of equal functional genes.The method of genetic manipulation includes conversion, engagement transfer, transduction and transfection etc..
The organism can be streptomycete, and streptomycete is the important sources of nearly half antibiotic.Antibiotic closes in streptomycete
At gene, often cluster exists, therefore it is the important technology support for excavating natural products resource to clone large fragment DNA.As antibiosis
The streptomycete of the important sources of element includes, such as (avermectin generates for streptomyces griseus (streptomysin producing strains), Avid kyowamycin
Bacterium), remote green streptomycete (thiostrepton producing strains), Chinese cassia tree streptomycete (coban producing strains), streptomyces albus (salinomycin
Producing strains) and streptomyces aureus (tetracycline producing strains) etc..
The target DNA fragments are the section of DNA on biological genome of interest.The target DNA fragments can also source
In organelle, such as the plasmid of mitochondria (mitochondrial DNA), chloroplaset (chloroplast DNA) and bacterium and fungi.The mesh
The regulating and controlling sequence that DNA fragmentation can be large fragment is marked, can be gene or base containing large fragment introne and regulating and controlling sequence
Because of cluster, such as the gene cluster that is made of function related gene.The relevant gene of these functions can be synthesized in same metabolin
The synthesis of approach or controlling gene.These metabolins include having pest-resistant, antibiotic activity secondary metabolites etc..
Specifically, described method includes following steps：
(1) identification is positioned at the CRISPR-Cas9 recognition sites of the side of the target DNA fragments, the CRISPR-Cas9
Recognition site is the sequence of 23nt, including the PAM sequences of the spacer of 20nt and the 3nt positioned at 3 ' ends of the spacer sequences
Row；The CRISPR-Cas9 recognition sites can be located in the flanking sequence of target DNA fragments.
The step of screening CRISPR-Cas9 recognition sites known in the art, specifically, identification CRISPR-Cas9 includes
Following steps：
I) identification positioned at the DNA fragmentation side with the 23nt sequences of 5 '-NGG-3 ' endings (N be A, T, C or
G), the 3nt for including the 20nt and PAM of spacer will be excluded comprising the sequence of continuous five T；
Ii) by 15 bases at 3 ' ends, include the 3nt of the 12nt and PAM of spacer, the genome sequence with the organism
Row carry out BLAST comparisons, exclude not specified sequence；
Iii the 20nt spacer sequences chosen are merged with sgRNA sequences), RNA two levels are carried out with RNAfold servers
The prediction of structure.
(2) identification is located at the integration site and the first recombination site of the DNA fragmentation both sides, wherein described first
Recombination site is between the DNA fragmentation and the CRISPR-Cas9 recognition sites.Integration site full length sequence and first
Recombination site full length sequence and the consistency of the other sequences on genome are no more than 30%, to avoid genome structure
Disorder, integration site full length sequence and the first recombination site full length sequence and the genome sequence of the organism carry out BLAST
It compares, to exclude not specified sequence.
(3) integration site, the CRISPR-Cas9 recognition sites and first recombination site are cloned into load
In body, the position of the CRISPR-Cas9 recognition sites on the carrier is located at the integration site and first recombination
Between site.
The cloning process based on homologous fragment, such as the In-fusion kits of Clontech companies can be used, also
Yeast assemble method TAR.The advantages of it is preferable to use Gibson Assembly methods to be cloned, Gibson Assembly methods
Be it is simple, inexpensively.
Coded sequence of the carrier with the coded sequence of Cas9, sgRNA sequences and the first resistant gene, wherein institute
It is inducible expression to state Cas9 coded sequences, and the resistant gene and sgRNA are constitutive expressions.The carrier may include
PMB1, p15A, pSC101 replicon it is any one or more, above-mentioned replicon is the replicon of low copy number, can be made big
The DNA of segment is more stable.The carrier may also include the single restriction enzyme site of one or more restriction enzymes, in order to
On the carrier that exogenous DNA is integrated by these restriction enzyme sites.Such carrier includes the deleted pCRISPR- of SG5 replicons
Cas9 carriers, or delete the pKCCas9do carriers of SG5 replicons (carrier is published in Acta Biochim Biophys
Sin, 2015,47 (4), 231~243.It can be from the websites addgene (http://www.addgene.org/) it buys, number is
No.62552)。
(4) the spacer sequences are integrated into 5 ' ends of sgRNA sequences on the carrier；It can be limited by specificity
Property restriction endonuclease restriction enzyme site by the spacer sequences be integrated into sgRNA sequences on the carrier 5 ' end.
(5) integration site of the integration site on the carrier in the organism with the target DNA fragments side
Homologous recombination occurs, to by the inhereditary material where the vector integration to the target DNA fragments, then pass through described
The positive colony of vector integration occurs for the screening of the first resistant gene.The carrier can be arrived by the plasmid integration that homologous recombination mediates
In genome where the DNA fragmentation.
The length of the integration site is 2~3kb, and the length of first recombination site is 300~700bp.It is true in this way
The plasmid integration for having protected homologous recombination mediation is mainly carried out by integration site, without being carried out in the first recombination site.
About the method for plasmid integration, engagement transfer is proper for streptomycete；For other biologies, for example, it is dynamic
Object or fungi need to use corresponding genetic manipulation method, such as transfection and conversion.
When the organism is streptomycete, the deleted pCRISPR-Cas9 carriers of SG5 replicons can be used；The load
Body contains oriT, and DNA shuttles between cell when being responsible for engagement transfer.If the carrier is what SG5 replicons were deleted
PCRISPR-Cas9 carriers, then screened with apramycin.
(6) expression for inducing the Cas9 coded sequences of the positive colony, then so that being located at CRISPR-Cas9 systems
Homologous recombination occurs between two first recombination sites at the same segment both ends of the generation of system cutting, generates comprising described
The plasmid of target DNA fragments.If the carrier is the pCRISPR-Cas9 carriers that SG5 replicons are deleted, sulphur chain silk is used
Rhzomorph (Tsr) induces the expression of CRISPR-Cas9 systems.
(7) plasmid is extracted, is screened by first resistant gene and obtains the plasmid for carrying the target DNA fragments.Such as
Carrier described in fruit is the pCRISPR-Cas9 carriers that SG5 replicons are deleted, then is screened with apramycin.
As shown in Figure 1, the method according to the above embodiment includes the following steps：
(1) identification positioned at the CRISPR-Cas9 recognition sites (*) of side of the target DNA fragments, integration site (1),
First recombination site (2).The DNA fragmentation is integration site (1), target dna piece with the sequence of these sites on chromosome
Section, the first recombination site (2) and CRISPR-Cas9 recognition sites, their sequence can also be opposite.
(2) CRISPR-Cas9 recognition sites (*), integration site (1) and the first recombination site (2) are cloned and is integrated into
On carrier, sequence of these sites on carrier is integration site (1), CRISPR-Cas9 recognition sites and the first recombination site
(2), their sequence can also be opposite.
(3) sequence of the carrier is integrated by homologous recombination on chromosome；
(4) expression for inducing CRISPR-Cas9 systems, cuts two CRISPR-Cas9 recognition sites；
(5) CRISPR-Cas9 systems cutting generates the segment for carrying target dna, by between the first recombination site (2)
The homologous recombination of generation generates the carrier for carrying target dna.
According to one embodiment, the method can also include second recombination of the identification positioned at the DNA fragmentation side
Site, wherein the CRISPR-Cas9 recognition sites be located at first recombination site be located at and second recombination site it
Between, and second recombination site is also integral in the carrier in step (3), wherein second recombination site exists
On the carrier between the integration site and the CRISPR-Cas9 recognition sites；In step (6) so that be located at
Occur between two second recombination sites of the different fragments end of the generation of CRISPR-Cas9 systems cutting homologous heavy
Group.In this way, the organism that target DNA fragments are knocked can be obtained while cloning the DNA fragmentation.Second recombination position
The consistency of other sequences on the full length sequence and genome of point is no more than 30%, to avoid the disorder of genome structure,
The sequence of second recombination site carries out the comparison of BLAST with the genome sequence of the organism, to exclude not specified sequence
Row.
Preferably, the carrier has the coded sequence and its Expression element of the second resistant gene, and described second is anti-
The coded sequence of property gene between second recombination site and the integration site, can lead to so on the carrier
Cross the homologous recombination that the screening of the second resistant gene is occurred by the second recombination site.
As shown in Fig. 2, method according to one embodiment of the present invention includes the following steps：
(1) identification positioned at the CRISPR-Cas9 recognition sites (4) of side of the target DNA fragments, integration site (1),
First recombination site (2) and the second recombination site (3).The DNA fragmentation is to integrate with the sequence of these sites on chromosome
Site (1), target DNA fragments, the first recombination site (2), CRISPR-Cas9 recognition sites and the second recombination site (3), they
Sequence can also be opposite.
(2) by CRISPR-Cas9 recognition sites (4), integration site (1), the first recombination site (2) and the second recombination site
(3) clone and be integrated on carrier, sequence of these sites on carrier be integration site (1), the second recombination site (3),
CRISPR-Cas9 recognition sites and the first recombination site (2), their sequence can also be opposite.
(3) sequence of the carrier is integrated by homologous recombination on chromosome；
(4) expression for inducing CRISPR-Cas9 systems, cuts CRISPR-Cas9 recognition sites；
(5) chromosome sequence that the cutting of CRISPR-Cas9 systems generates the segment of carrying target dna and is interrupted, two
By the first recombination site (2) and the second recombination site (3) site homologous recombination occurs for person respectively, generates and carries target dna
The chromosome that carrier and target dna are knocked.
It should be noted that although describing the operation of the method for the present invention with particular order in the accompanying drawings, this is not required that
Or imply and must execute these operations according to the particular order, it could the realization phase or have to carry out operation shown in whole
The result of prestige.On the contrary, the step of describing in flow chart, which can change, executes sequence.It additionally or alternatively, can be by multiple steps
It is rapid to merge into a step execution, and/or a step is decomposed into execution of multiple steps.
The present invention also provides for the carrier from biological vivo clone target DNA fragments, wherein the carrier includes
The coded sequence of Cas9, the coded sequence of sgRNA sequences and the first resistant gene, wherein the Cas9 coded sequences are inductions
Type is expressed, and the resistant gene and sgRNA are constitutive expressions；And there is integration site, CRISPR-Cas9 on the carrier
Recognition site and the first recombination site, the position of the CRISPR-Cas9 recognition sites on the carrier are located at described whole
It closes between site and first recombination site, wherein the CRISPR-Cas9 recognition sites are the sequence of 23nt, including 20nt
Spacer sequences and positioned at the spacer sequences 3 ' end 3nt PAM sequences；And it is described in the organism
CRISPR-Cas9 recognition sites are located at the side of the target DNA fragments, and the integration site and the first recombination site distinguish position
In the both sides of the target DNA fragments, also, first recombination site is located at the DNA fragmentation and the CRISPR-Cas9
Between recognition site.The second recombination site is also integrated on the carrier, second recombination site is located at the CRISPR-
Between Cas9 recognition sites and the target DNA fragments, and in the organism, the CRISPR-Cas9 recognition sites
It is located between second recombination site positioned at first recombination site.Preferably, the carrier has the second resistance base
The coded sequence and its Expression element of cause, and the coded sequence of second resistant gene is in second recombination site and institute
It states between integration site.
Fig. 6 shows that the schematic diagram of carrier according to one embodiment of the present invention, the carrier are named as
pNTU33102.The carrier is with the coded sequence of Cas9, sgRNA sequences, the first resistant gene apramycin resistance protein position
The coded sequence of the coded sequence of point acc (3) IV and the second resistant gene tsr resistant genes, wherein the Cas9 code sequences
Arranging has the tipA promoters of response Tsr.The carrier also restriction enzyme site with Stu I, Nco I and SnaB I.spacer
Sequence is inserted into 5 ' ends of sgRNA sequences by the restriction enzyme site of Nco I and SnaB I.Integration site (1), the first recombination position
Point (2), CRISPR-Cas9 recognition sites (4) and the second recombination site (3) pass through Gibson Assembly with the sequence
DNA assemble methods are integrated into the Stu I sites of carrier.The carrier can also be carried by the pKCCas9do for deleting SG5 replicons
Body obtains after genetic manipulation.
Embodiment 1 utilizes the method for CRISPR-Cas9 technologies Direct Cloning act gene clusters from streptomyces coelicolor.
Reagent and carrier for using etc.
PCRISPR-Cas9 (ACS Synth.Biol., 2015,4 (9), page 1020-1029；The Genbank numbers of logging in
KR011749, Fig. 3) carrier derive from Technical University Of Denmark (Technical University of Denmark) Tilmann
Weber teaches laboratory.The carrier can also be voluntarily synthesized according to the Genbank carrier sequences announced.
Streptomyces coelicolor M145 bacterial strain is the type strain of widely used streptomycete research.The sky blue chain of the present invention
Mould M145 bacterium sources are in ATCC culture presevation library (deposit number ATCC BAA-471).
The present embodiment is from Streptomyces coelicolor M145 bacterial strain Direct Cloning act synthetic gene clusters.Include the following steps：
1, pCRISPR-Cas9 carriers are transformed, delete SG5 replicons therein (9627~11069bp for being located at carrier),
Make the carrier can not autonomous replication in streptomycete.New support is named as pNTU33101 (Fig. 4).The resistance screening mark of the carrier
It is denoted as apramycin, the expression of the CRISPR-Cas9 systems of the carrier is induced by thiostrepton.It is as follows：
PCRISPR-Cas9 carriers are used to carry out PCR as template, primer 1 and primer 2.
Primer 1 (5 ' -3 ')：gcgttcaagggccgaaagccgagggtctgcctgccgtgaggt
Primer 2 (5 ' -3 ')：tcggctttcggcccttgaacgcctcgttcagcgacaccgtct
PCR spins the KOD plus (article No. KOD-211) of (TOYOBO) using Japan.
PCR reaction systems are 50 μ L：
The condition of PCR is as follows：
Pre-degeneration：94 DEG C 2 minutes
30 cycles：It is denaturalized 98 DEG C 10 seconds
Extend 68 DEG C 10 minutes
The Escherichia coli of transformed competence colibacillus can obtain deleting the pCRISPR- of SG5 replicons after PCR product recycling
Cas9 carriers.Escherichia coli can be any commercialized carrier cloning with Escherichia coli, such as Top10, DH5 α, JM109 etc..
2, identification is in target DNA fragments -- and the CRISPR-Cas9 in the flanking sequence of streptomyces coelicolor act gene clusters knows
Other site (4), and choose suitable integration site (1), the first recombination site (2) and the second recombination site (3).This 4 positions
Point is in streptomyces coelicolor genome (the Genebank numbers of logging in：NC_003888 position and size on) are respectively：
Integration site (1)：5509957~5512067, size：2111bp
First recombination site (2)：5534737~5535274, size：541bp
Second recombination site (3)：5535361~5535887, size：527bp
CRISPR-Cas9 recognition sites (4)：5535275~5535297, size：23nt
CRISPR-Cas9 recognition sites (4) include the spacer sequences and 3nt of 20nt between recombination site 2 and 3
PAM sequences (CGG).
Fig. 5 shows the act synthetic genes cluster and CRISPR-Cas9 recognition sites (4), integration site of M145 bacterial strains
(1), the position view of the first recombination site (2) and the second recombination site (3) on chromosome, wherein gray background indicates
Spacer sequences, PAM sequences add underscore.Each box with the arrow represents a gene, wherein gene SCO5072 and
SCO5092 blackings are shown.
3, the method (ACS Synth.Biol., 2015,4 (9), page 1020-1029) delivered according to initial carrier,
Spacer is cloned between Nco I and SnaB I restriction enzyme sites.
Cloning the primer used in spacer is：
Primer 3 (5 ' -3 ')：
CATGCCATGGcacgcggttcgcccgttcgaGTTTTAGAGCTAGAAATAGC
(underscore is the restriction enzyme site of Nco I, and the base of small letter is spacer sequences)
Primer 4 (5 ' -3 ')：
ACGCCTACGTAAAAAAAGCACCGACTCGGTGCC
(underscore is the restriction enzyme site of SnaB I)
PCR spins the KOD plus (article No. KOD-211) of (TOYOBO) using Japan.
PCR reaction systems are 50 μ L:
The condition of PCR is as follows：
Pre-degeneration：94 DEG C 2 minutes
30 cycles：It is denaturalized 98 DEG C 10 seconds
Annealing 58 DEG C 30 seconds
Extend 68 DEG C 10 seconds,
Then the PCR product of 1 μ L is directly taken to carry out digestion, the digestion system (Fermentas) of 10 μ L：
PNTU33101 is subjected to digestion (Fermentas), 50 μ L systems：
PNTU33101 digestion products are subjected to electrophoresis, and recycle plasmid fragments (about 10Kb).Take the PCR of 2 μ L primers 3 and 4
Digestion products take 1 μ L pNTU33101 digestion recovery products to be attached, use TAKARA connection kit Solution I
(article No. 6022) 3 μ L, final system are 6 μ L, react 5 minutes, convert Escherichia coli, then screening positive clone.
4, by Gibson Assembly DNA methods, by CRISPR-Cas9 recognition sites (4), integration site (1),
One recombination site (2) and the second recombination site (3) are integrated on carrier.Wherein, template is Streptomyces coelicolor M145 bacterial strain
Genomic DNA.Three PCR products are integrated into company using the Gibson Assembly DNA assemble methods being widely used
The Stu I sites being connected in the pNTU33101 carriers of spacer, obtained carrier are named as pNTU33102 (Fig. 5).
Primer used in Clonal integration site (1) is：
Primer 5 (5 ' -3 ')：tctcgtcgaaggcactagaagggaagagggcaacctctacctggtc
Primer 6 (5 ' -3 ')：
tgcgaagctggcGAGGGGTTGTTGCTGAACATCTTGAC
Cloning the primer used in the second recombination site (3) is：
Primer 9 (5 ' -3 ')：CAACAACCCCTCgccagcttcgcacctcctccccga
Primer 10 (5 ' -3 ')：gcgggcggctgctggtcgtc
Cloning the primer used in the first recombination site (2) is：
Primer 7 (5 ' -3 ')：cccggtgttcgacagttgcggcgag
Primer 8 (5 ' -3 ')：
ggtcgatccccgcatataggGTGCTCGACGCCTGCACCGACCT
Cas9 recognition sites 4 are synthesized by primer and are introduced during PCR, specifically, 7 black matrix underscore portion of primer
It is divided into the partial sequence of spacer, the partial sequence of PAM is marked with box, and black matrix underscore part is spacer's in primer 10
The partial sequence of reverse complementary sequence, PAM reverse complementals is marked with box.
PCR spins the KOD plus (article No. KOD-211) of (TOYOBO) using Japan.
PCR reaction systems are 50 μ L：
The condition of PCR is as follows：
Pre-degeneration：94 DEG C 2 minutes
30 cycles：It is denaturalized 98 DEG C 10 seconds
Annealing 58 DEG C 30 seconds
Extend 68 DEG C 2 minutes
It is connected in the pNTU33101 carriers of spacer and carries out digestion, 10 μ with Stu I (Fermentas article No. FD0424)
L systems：
Gibson Assembly react 1 using 2 × premix reaction system (article No. E2611S) of NEB, 20 μ L in 50 DEG C
Hour：
Reaction product converts Escherichia coli, and screening positive clone.
5, carrier pNTU33102 is integrated into streptomyces coelicolor genome (experiment step using the method for engagement transfer
Suddenly referring to Tobias Kieser, Mervyn J.Bibb, Mark J.Buttner, Keith F.Chater, David
A.Hopwood,Practical Streptomyces Genetics,John Innes Foundation(2000),249-250
Page), i.e., pNTU33102 plasmids are imported into the Streptomyces coelicolor M145 bacterial strain of competence.
Engaging shift experiment step is：
(1) streptomyces coelicolor Fresh spores are suspended in 1mL sterile waters.
(2) 50 DEG C of water-bath heat shocks are placed in room temperature after ten minutes.
(3) overnight by the Escherichia coli ET12567 bacterial strains (streptomycete laboratory is widely used) containing pNTU33102 plasmids
Culture is in being added isometric spore suspension.
(4) MS culture medium flat plates are coated with, in 28 DEG C of cultures.
(5) after cultivating 14 hours, culture is covered with the 1mL sterile waters containing 0.5mg nalidixic acids and 1mg apramycins
Base, standing is cultivated 4~5 days after drying in 28 DEG C, after zygote waiting is grown, screens the joint element with apramycin resistance.Cause
It because can not autonomous replication for plasmid, it is necessary to Chromosome recombination, it is possible to screen connecing for engagement transfer with apramycin
Zygote.
MS culture mediums (1L)：20 grams of mannitol (Mannitol), 20 grams of soybean cake powder, 20 grams of agar powder.
6, with YEME fluid nutrient medium cultures there is the joint element of apramycin resistance to add after being cultivated 48 hours at 28 DEG C
Entering 1 μM of thiostrepton induction CRISPR-Cas9 system, the recognition site 4 of the Cas9 of target DNA fragments both sides is cut,
The chromosome sequence for carrying the segment of target dna and being interrupted is generated, the two passes through the first recombination site (2) and second respectively
Homologous recombination occurs for recombination site (3) site, generates the chromosome that the carrier for carrying target dna and target dna are knocked.
YEME culture mediums (1L)：3 grams of yeast extract (Yeast Extract), bacto peptone (Bacto-peptone) 5
Gram, 3 grams of malt extract (Malt Extract), 10 grams of glucose, 100 grams of sucrose.
7, plasmid is extracted, the plasmid is transferred in Escherichia coli again, is coated on the tablet containing apramycin, it is right
Obtained clone screens, and obtains the clone for carrying target DNA fragments.The digestion of positive colony and sequence verification
The plasmid of positive colony is extracted, electrophoresis is run, according to the identification of plasmid band size to obtained clone, screening, which contains, catches
Obtain the clone of large fragment.Plasmid, further digestion (Fig. 7) and sequence verification (Fig. 8) are extracted to positive colony.In Fig. 7, left figure is
BamHI digestion qualification results, right figure are Bgl II and StuI double digestion qualification results.Fig. 8 is shown to carrying target DNA fragments
Plasmid target DNA fragments both ends sequencing result.As can be seen that target DNA fragments have been cloned into from Fig. 7 and Fig. 8
On carrier.
The simple and practicable feature of present invention combination CRISPR/Cas9 technologies accurate, efficient feature and homologous recombination technique,
Started it is a kind of using CRISPR/Cas9 technologies out of organism Direct Cloning large fragment target dna method.The method walks
It is rapid simple, it is at low cost, it is efficient, it is particularly suitable that big target DNA fragments are cloned from organism.
Above description is only the preferred embodiment of the application and the explanation to institute's application technology principle.People in the art
Member should be appreciated that invention scope involved in the application, however it is not limited to technology made of the specific combination of above-mentioned technical characteristic
Scheme, while should also cover in the case where not departing from the inventive concept, it is carried out by above-mentioned technical characteristic or its equivalent feature
Other technical solutions of arbitrary combination and formation.Such as features described above has similar work(with (but not limited to) disclosed herein
Can technical characteristic replaced mutually and the technical solution that is formed.
Claims (10)
1. a kind of method from biological vivo clone target DNA fragments to carrier includes the following steps：
(1) identification is positioned at the CRISPR-Cas9 recognition sites of the target DNA fragments side, CRISPR-Cas9 identifications position
Point is the sequence of 23nt, including the spacer sequences of 20nt and the PAM sequences of the 3nt positioned at the spacer sequences 3 ' end；
(2) identification is located at the integration site and the first recombination site of the target DNA fragments both sides, wherein described first
Recombination site is between the target DNA fragments and the CRISPR-Cas9 recognition sites；
(3) integration site, the CRISPR-Cas9 recognition sites and first recombination site are cloned into the load
In body, wherein the position of the CRISPR-Cas9 recognition sites on the carrier is located at the integration site and described first
Between recombination site, wherein coding of the carrier with the coded sequence of Cas9, sgRNA sequences and the first resistant gene
Sequence, wherein the Cas9 coded sequences are inducible expressions, the coded sequence of first resistant gene and the sgRNA sequences
Row are constitutive expressions；
(4) the spacer sequences are cloned into 5 ' ends of sgRNA sequences on the carrier；
(5) integration site on the carrier occurs in the organism with the integration site of the target DNA fragments side
Homologous recombination, to by the inhereditary material where the vector integration to the target DNA fragments, then pass through described first
The positive colony of vector integration occurs for resistant gene screening；
(6) expression of the Cas9 coded sequences of the positive colony, cutting is induced to be located at the target DNA fragments both sides
Then two CRISPR-Cas9 recognition sites are located at the same DNA fragmentation two that the cutting of CRISPR-Cas9 systems generates
Homologous recombination occurs between two first recombination sites at end, generates the plasmid for including the target DNA fragments；
(7) plasmid is extracted, is screened by first resistant gene and obtains the plasmid for carrying the target DNA fragments.
2. according to the method described in claim 1, the method further includes：
Second recombination site of the identification positioned at the target DNA fragments side, wherein CRISPR-Cas9 recognition sites position
It is located between second recombination site in first recombination site；And
Second recombination site is also integral in the carrier in step (3), wherein second recombination site is in institute
It states on carrier between the integration site and the CRISPR-Cas9 recognition sites；
In step (6), it is located at two second recombination positions of the fragment ends that CRISPR-Cas9 systems cutting generates
Homologous recombination occurs between point.
3. method according to claim 1 or 2, wherein the carrier includes appointing for pMB1, p15A, pSC101 replicon
The single restriction enzyme site of one or more and one or more restriction enzymes.
4. method according to claim 1 or 2, wherein the carrier is the pCRISPR-Cas9 that SG5 replicons are deleted
Carrier, and being screened with apramycin in step (5) and step (7), induced with thiostrepton in step (6) described in
The expression of Cas9 coded sequences.
5. method according to claim 1 or 2, wherein the target DNA fragments include the regulating and controlling sequence of large fragment, contain
The gene cluster for having the gene of large fragment introne and regulating and controlling sequence or being made of function related gene.
6. method according to claim 1 or 2, wherein the length of the integration site 1 is 2~3kb, first weight
The length of group site and second recombination site is 300~700bp.
7. a kind of carrier for from biological vivo clone target DNA fragments, wherein the carrier includes the code sequence of Cas9
The coded sequence of row, sgRNA sequences and the first resistant gene, wherein the Cas9 coded sequences are inducible expressions, it is described
Resistant gene and sgRNA are constitutive expressions；
There is integration site, CRISPR-Cas9 recognition sites and the first recombination site, the CRISPR- on the carrier
The position of Cas9 recognition sites on the carrier between the integration site and first recombination site, wherein
The CRISPR-Cas9 recognition sites be 23nt sequence, comprising the spacer sequences of 20nt and positioned at described in
The PAM sequences of the 3nt at 3 ' ends of spacer sequences；And
In the organism, the CRISPR-Cas9 recognition sites are located at the side of the target DNA fragments, the integration
Site and the first recombination site are located at the both sides of the target DNA fragments, and first recombination site is positioned at described
Between DNA fragmentation and the CRISPR-Cas9 recognition sites.
8. carrier according to claim 7, wherein be also integrated with the second recombination site on the carrier, wherein described
Two recombination sites are on the carrier between the integration site and the CRISPR-Cas9 recognition sites；
And in the organism, the CRISPR-Cas9 recognition sites be located at first recombination site be located at and it is described
Between second recombination site.
9. carrier according to claim 7 or 8, wherein the carrier includes appointing for pMB1, p15A, pSC101 replicon
The single restriction enzyme site of one or more and one or more restriction enzymes.
10. carrier according to claim 7 or 8, wherein the carrier is the pCRISPR- that SG5 replicons are deleted
Cas9 carriers.
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| CN104450745A (en) * | 2013-09-12 | 2015-03-25 | 北京大学 | Method for acquiring specific rice gene mutant and application thereof |
| CN105624146A (en) * | 2015-05-28 | 2016-06-01 | 中国科学院微生物研究所 | Molecular cloning method based on CRISPR/Cas9 and homologous recombination of saccharomyces cerevisiae cell endogenous genes |
| CN105802980A (en) * | 2016-04-08 | 2016-07-27 | 北京大学 | CRISPR/Cas9 system with Gateway compatibility and application of CRISPR/Cas9 system |
| CN105821072A (en) * | 2015-01-23 | 2016-08-03 | 深圳华大基因研究院 | CRISPR-Cas9 system used for assembling DNA and DNA assembly method |
| CN107365786A (en) * | 2016-05-12 | 2017-11-21 | 中国科学院微生物研究所 | A kind of method and its application being cloned into spacer sequences in CRISPR-Cas9 systems |
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| CN104450745A (en) * | 2013-09-12 | 2015-03-25 | 北京大学 | Method for acquiring specific rice gene mutant and application thereof |
| CN105821072A (en) * | 2015-01-23 | 2016-08-03 | 深圳华大基因研究院 | CRISPR-Cas9 system used for assembling DNA and DNA assembly method |
| CN105624146A (en) * | 2015-05-28 | 2016-06-01 | 中国科学院微生物研究所 | Molecular cloning method based on CRISPR/Cas9 and homologous recombination of saccharomyces cerevisiae cell endogenous genes |
| CN105802980A (en) * | 2016-04-08 | 2016-07-27 | 北京大学 | CRISPR/Cas9 system with Gateway compatibility and application of CRISPR/Cas9 system |
| CN107365786A (en) * | 2016-05-12 | 2017-11-21 | 中国科学院微生物研究所 | A kind of method and its application being cloned into spacer sequences in CRISPR-Cas9 systems |
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