CN109971789A - A kind of gene editing system and its application in new gold mycobacteria - Google Patents
A kind of gene editing system and its application in new gold mycobacteria Download PDFInfo
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Abstract
The invention discloses a kind of gene editing system and its applications in new gold mycobacteria, belong to gene engineering technology field and technical field of bioengineering.This system can reach the purpose that gene editing is carried out to new gold mycobacteria, its working mechanism is as follows: first importing pML-Cpf1 plasmid in new gold mycobacteria, so that the Cpf1 gene on pML-Cpf1 plasmid, the gene expression of the gene and coding DNA ligase LigD of coding DNA terminal binding protein mku, then pJM-crRNA plasmid is imported in new gold mycobacteria, so that pJM-crRNA plasmid transcription generates crRNA sequence, crRNA sequence can form compound in conjunction with Cpf1 gene, this compound can be targeted and be cut and need to edit gene and to need the gene of editor to be knocked in new gold mycobacteria.
Description
Technical field
The present invention relates to a kind of gene editing system and its applications in new gold mycobacteria, belong to genetic engineering skill
Art field and technical field of bioengineering.
Background technique
Steroid drugs status in chemical drug industry is prominent, is the second major class drug for being only second to antibiotic, plays to body
Important adjustment effect, including improve protein metabolism, recovery and enhance physical strength, diuretic antihypertensive etc.；Also, steroid drugs may be used also
Treat the endocrine system diseases such as skin diseases and prostate, Ai Disenshi such as rheumatic arthritis, eczema；In addition, steroid drugs is also
It can be used for practising contraception, the fields such as miscarriage prevention and surgery anesthesia, clinically have a wide range of applications.
Currently, China using steroid drugs new resources develop as pharmaceuticals industry the direction developed in the recent period and emphasis it
One, the outlet of steroid drugs and Steroid medicine intermediates also has become the important species that China's drug goes to the world.But due to
Steroid drugs synthesis step is more, reaction is complicated, yield is low, isolates and purifies difficulty, and China is in steroid drugs research, production and clinic
Research aspect with advanced country in the world compared with there are also a certain distance, the existing steroid drugs kind in China be only foreign countries on
The one third of the steroid drugs in city, and be mostly low-and-medium-grade products.Therefore, it is badly in need of promoting the production water of China's steroid drugs
It is flat.
Androstane-4-alkene-3,17- diketone (AD) and androstane -1,4- diene -3,17- diketone (ADD) are important steroid drugs
Intermediate can synthesize almost all of steroidal drug by the chemical modification in structure, the status in steroid drugs industry
It becomes increasingly conspicuous.Currently, China uses microbe transformation method to produce ADD more, however, since substrate feed concentrations are low, microbe conversion
Period is long and the problems such as to the deficiency of steroidal microbial metabolism mechanism cognition, and China is still bright in the production of steroidal microorganism conversion
Aobvious to lag behind the countries such as America and Europe, this seriously constrains the paces of China's steroid drugs industry Green Transformation.Therefore, it is badly in need of promoting me
The production level of state Steroid medicine intermediates ADD is to promote the production level of China's steroid drugs.
New gold mycobacteria (Mycobacterium neoaurum) can transformation phytosterin synthesize ADD, and there is life
Long fast advantage, is a kind of ADD production bacterium of great potential, if ADD can be synthesized to new gold mycobacteria transformation phytosterin
Key enzyme 3- sterone-Δ 1- dehydrogenase (KSDD), cholesterol oxidase (Cho M), sterone C27 monooxygenase in the process
(SMO) and -9 α of 3- sterone-hydroxylase (KSH) is studied, and metabolic engineering strategies is combined to carry out new gold mycobacteria
Transformation, it is most likely that obtain the new gold mycobacteria for capableing of efficient accumulation ADD, this modified new gold mycobacteria is undoubtedly
It is an important breakthrough for promoting China's Steroid medicine intermediates ADD production level.
However, the protection mechanism exclusiveness as present in new gold mycobacteria itself is stronger, foreign gene is not easy
It is stabilized and expresses in new gold mycobacteria, therefore, the gene editing method for being applicable to new gold mycobacteria is less,
For example, homologous recombination, still, since the incidence of mycobacteria homologous recombination is obviously more much lower than other bacteriums, only 10-6~
10-5, also, new gold mycobacteria progress gene editing is also needed to bring resistance label into the genome using homologous recombination,
Carrying out gene editing to new gold mycobacteria using homologous recombination has that low efficiency, period are long, screening amount is big, complicated for operation etc.
Disadvantage；Suicide vector system (pNIL/pGOAL series plasmids) although etc. other gene editing methods can be to new golden branch bar
Bacterium carries out gene editing, but efficiency is not also high, does not have any advantage compared with homologous recombination.
Therefore, gene editing is undoubtedly a difficult point being transformed to the metabolic engineering of new gold mycobacteria, we
There is an urgent need to improve for new gold mycobacteria gene editing system, so as to efficiently to new gold mycobacteria into
Row transformation.
Summary of the invention
[technical problem]
The technical problem to be solved in the present invention is to provide a kind of bases with strong points to new gold mycobacteria and high-efficient
Because of editing system.
[technical solution]
To solve the above problems, the present invention provides a kind of plasmid that can be used for gene editing, the plasmid includes
PMV261 expression vector, Cpf1 gene, Pj23119 promoter and NHEJ revision points, are named as pML-Cpf1 plasmid；It is described
Hsp60 promoter on pMV261 expression vector is replaced for Tac promoter；The NHEJ revision points include coding DNA
The gene of terminal binding protein mku and the gene of coding DNA ligase LigD；The Cpf1 gene is located at pMV261 expression and carries
The downstream of the Tac promoter of body and the rrnB terminator upstream of pMV261 expression vector are driven by Tac promoter and are expressed；Institute
State the downstream that Pj23119 promoter is located at the KanR resistant gene of pMV261 expression vector；The NHEJ revision points are located at
The upstream of the replication orgin (ori) in the downstream and pMV261 expression vector of Pj23119 promoter, is driven by Pj23119 promoter
Dynamic expression.
In one embodiment of the invention, the nucleotide sequence of the Cpf1 gene is as shown in SEQ ID NO:1.
In one embodiment of the invention, the nucleotide sequence such as SEQ of the gene of the Pj23119 promoter is encoded
Shown in ID NO:2.
In one embodiment of the invention, the nucleotide sequence of the gene of the end the DNA collection hop protein mku is encoded
As shown in SEQ ID NO:3.
In one embodiment of the invention, the nucleotide sequence such as SEQ of the gene of the DNA ligase LigD is encoded
Shown in ID NO:4.
In one embodiment of the invention, the nucleotide sequence such as SEQ ID of the gene of the Tac promoter is encoded
Shown in NO:5.
The present invention also provides a kind of plasmid that can be used for gene editing, the plasmid successively include OriM replicon,
PMB1 replicon, Pj23119 promoter, crRNA sequence, rrnB terminator and aadA resistant gene, are named as pJM-crRNA
Plasmid；The crRNA sequence is targeting sequence, can selectively targeted target sequence；The target sequence refers to the core that need to edit gene
Nucleotide sequence.
In one embodiment of the invention, the nucleotide sequence such as SEQ ID of the gene of the OriM replicon is encoded
Shown in NO:6.
In one embodiment of the invention, the nucleotide sequence such as SEQ ID of the gene of the pMB1 replicon is encoded
Shown in NO:7.
In one embodiment of the invention, the nucleotide sequence such as SEQ of the gene of the Pj23119 promoter is encoded
Shown in ID NO:2.
In one embodiment of the invention, the nucleotide sequence such as SEQ ID of the gene of the rrnB terminator is encoded
Shown in NO:8.
In one embodiment of the invention, the nucleotide sequence of the aadA resistant gene such as SEQ ID NO:9 institute
Show.
The present invention also provides a kind of double-mass model CRISPR/Cpf1 gene editing systems, and the system contains above-mentioned simultaneously
PML-Cpf1 plasmid and pJM-crRNA plasmid.
The present invention also provides a kind of method for editing new gold mycobacteria gene, the method is to use above-mentioned one kind
Double-mass model CRISPR/Cpf1 gene editing system.
In one embodiment of the invention, the method is first by a kind of above-mentioned double-mass model CRISPR/Cpf1 gene
PML-Cpf1 plasmid in editing system imports in new gold mycobacteria, so that Cpf1 gene, volume on pML-Cpf1 plasmid
The gene expression of the gene and coding DNA ligase LigD of code DNA terminal binding protein mku, then by a kind of above-mentioned double matter
PJM-crRNA plasmid in grain CRISPR/Cpf1 gene editing system imports in new gold mycobacteria, so that pJM-crRNA
Plasmid transcription generates crRNA sequence, and crRNA sequence can form compound in conjunction with Cpf1 gene, this compound can be targeted and be cut
Gene need to be edited by, which cutting, is knocked the gene that editor is needed in new gold mycobacteria, need to edit the knockout meeting of gene so that new gold
Color Mycobacterium tuberculosis genes generate DSB fracture, at this point, the DNA terminal binding protein mku of the expression on pML-Cpf1 plasmid and
DNA ligase LigD can repair DSB fracture, complete the editing process of new gold mycobacteria gene.
The present invention also provides above-mentioned pML-Cpf1 plasmid or above-mentioned pJM-crRNA plasmids or a kind of above-mentioned double-mass model
CRISPR/Cpf1 gene editing system or a kind of above-mentioned method for editing new gold mycobacteria gene are in the new golden branch of editor
Application in terms of bacillus gene.
[beneficial effect]
(1) the CRISPR/Cpf1 gene editing system based on double-mass model that the present invention provides a kind of, this system can reach pair
New gold mycobacteria carries out the purpose of gene editing, and working mechanism is as follows: pML-Cpf1 plasmid first being imported new gold point
In branch bacillus, so that the gene and coding DNA of Cpf1 gene, coding DNA terminal binding protein mku on pML-Cpf1 plasmid
Then the gene expression of ligase LigD imports pJM-crRNA plasmid in new gold mycobacteria, so that pJM-crRNA matter
Grain transcription generates crRNA sequence, and crRNA sequence can form compound in conjunction with Cpf1 gene, this compound can be targeted and be cut
Gene, which need to be edited, is knocked the gene that editor is needed in new gold mycobacteria, need to edit the knockout meeting of gene so that new golden
Mycobacterium tuberculosis genes generate DSB fracture, at this point, DNA the terminal binding protein mku and DNA of the expression on pML-Cpf1 plasmid
Ligase LigD can repair DSB fracture, complete the editing process of new gold mycobacteria gene；
(2) the pML-Cpf1 plasmid and pJM-crRNA matter that CRISPR/Cpf1 gene editing service system of the invention uses
Grain can be stabilized in new gold mycobacteria, express, will not be by the protection present in new gold mycobacteria itself
Mechanism is repelled；
(3) gene volume is carried out to new gold mycobacteria using CRISPR/Cpf1 gene editing service system of the invention
Volume, knockout efficiency is higher, up to 40%.
Detailed description of the invention
The map of Fig. 1: pMV261 expression vector.
The map of Fig. 2: pML-Cpf1 plasmid.
The map of Fig. 3: pJM-crRNA plasmid.
Fig. 4: -9 α of new gold mycobacteria JC-12 and new gold mycobacteria JC-12/KshA fermentation gained 3- sterone-hydroxyl
Change the enzyme activity comparison of enzyme.
Specific embodiment
The present invention is further limited combined with specific embodiments below.
PCas9 expression vector, pMV261 expression vector involved in following embodiments, ptrc99A expression vector are purchased from excellent
Precious biology；PJYS1Ptac plasmid involved in following embodiments is purchased from Nanjing Genscript Biotechnology Co., Ltd.；Following implementations
ClonExpress II One Step Cloning Kit involved in example is purchased from Nanjing Vazyme Biotechnology Co., Ltd.；
New gold mycobacteria (Mycobacterium neoaurum) involved in following embodiments receives biology purchased from north, resource number
For ATCC 25795.
Culture medium involved in following embodiments is as follows:
LB liquid medium: glucose 10g/L, yeast powder 5g/L, NaCl 10g/L.
Inclined-plane/solid medium: glucose 10g/L, tryptone 6g/L, yeast powder 5g/L, NaCl 10g/L, agar powder
20g/L, pH 7.5.
Seed culture medium: glucose 20g/L, tryptone 10g/L, yeast powder 6g/L, NaCl 10g/L, pH 7.5.
Competence culture medium: glucose 20g/L, tryptone 10g/L, yeast powder 6g/L, NaCl 10g/L, Tween-
800.2% (V/V), pH 7.5.
Detection method involved in following embodiments is as follows:
- 9 α of 3- sterone-hydroxylase enzyme activity detection method:
Most suitable KSH measurement system includes: 105 μM of NADH, 200 μM of steroidal substrate 4-ADs (AD)
(being dissolved in 100% isopropanol), 50mM Tris-HC1 (200 μ L, pH 7.0) and 30 μ g KSH enzymes；Substrate causes anti-after being added
It answers, microplate reader SpectraMax 190 (Molecular Devices) is recorded at 35 DEG C, light absorption value of the NADH at 340nm
Change (ε=6.22mMcm-1), software Soft-max PRO is to analyze enzymatic initial velocity of reaction；
- 9 α of 3- sterone-hydroxylase enzyme activity is defined as: enzyme amount required for 1min internal oxidition 1nmol steroidal substrate indicates
For enzyme amount required for 1min internal oxidition 1nmol NADH, unit U.
- 9 α of 3- sterone-hydroxylase specific enzyme activity detection method:
- 9 α of 3- sterone-hydroxylase specific enzyme activity calculation formula is as follows: -9 α of 3- sterone-hydroxylase specific enzyme activity=(3- sterone -
9 α-hydroxylase enzyme activity/- 9 α of 3- sterone-hydroxylase weight), unit Umg-1。
The detection method of gene knockout efficiency:
The calculation formula of gene knockout efficiency is as follows: gene knockout efficiency=(verifying knocks out successful colony counts/picking
The total quantity of transformant) × 100%.
New gold mycobacteria JC-12 competent cell the preparation method is as follows:
By the new gold mycobacteria JC-12 inoculation in frozen pipe as in 10mL LB liquid medium, 30 DEG C of shaking table cultures
48h obtains seed liquor；Seed liquor is forwarded in 50mL competence culture medium with 1% inoculum concentration, 30 DEG C of shaking table cultures 4~
6h obtains bacterium solution；The 50mL centrifuge tube of bacterium solution, 10% glycerol, drying sterile electric shock cup and sterilizing is placed in and is pre-chilled on ice
30min；The bacterium solution of pre-cooling is fitted into centrifuge tube in superclean bench, 8000rpm, 4 DEG C of refrigerated centrifuge 5min are discarded supernatant,
10% glycerol of 10mL is added and thallus suspends, 8000rpm, 4 DEG C of refrigerated centrifuge 5min repeat previous step, discard
Clearly, 10% glycerol of 2mL is added and thallus suspends, dispenses into EP pipe, it is thin to obtain JC-12 competence by every 80 μ L of pipe
Born of the same parents.
The method for transformation of new gold mycobacteria JC-12 is as follows:
Plasmid (double steaming sterile waters are de-) need to be converted by being added in JC-12 competent cell in sterile superclean bench, be mixed
It is even, 30min is placed on ice；After 30min, competent cell is all transferred in the electric shock cup of pre-cooling, dries a glass outer wall rapidly, set
In electroporation apparatus, 2200V, 5ms are electroporated；After electric shock, 1mL seed culture medium is added into electric shock cup, mixes, entirely
Portion is transferred in EP pipe, 30 DEG C of 4~6h of shaking tables recovery；After recovery, cell is collected by centrifugation, removes part supernatant, is coated on containing anti-
On inclined-plane/solid medium of property, 30 DEG C culture carton upside down culture 5~7 days, the new gold mycobacteria JC- after being converted
12。
The building and use of embodiment 1:CRISPR/Cpf1 gene editing system
Specific step is as follows:
1, the building of CRISPR/Cpf1 gene editing system
(1) Hsp60 on pMV261 expression vector is opened by restriction enzyme Xba I and BamH I and T4 ligase
Mover replaces with Tac promoter, and is carried out by restriction enzyme Xba I and HindIII to pMV261 expression vector digestion
Linearisation obtains basic framework；
(2) using pJYS1Ptac plasmid as template, the P1 primer and P2 primer progress PCR amplification (PCR amplification in table 1 are used
2) reaction system is shown in Table, obtain Cpf1 gene, i.e. Frag1；
(3) gene (the nucleotide sequence such as SEQ ID of coding DNA end collection hop protein mku is obtained by chemical synthesis
Shown in NO:3) and coding DNA ligase LigD gene (nucleotide sequence is as shown in SEQ ID NO:4)；With chemical synthesis
Obtained coding DNA end integrates the gene of hop protein mku and the gene of coding DNA ligase LigD as template, uses respectively
P3 primer, P4 primer and P5 primer, P6 primer in table 1 are cloned, and the coding DNA end collection hop protein of linearisation is obtained
The gene of mku and the gene of coding DNA ligase LigD；By the coding DNA end collection hop protein mku's of the linearisation of acquisition
The gene of gene and coding DNA ligase LigD are merged by P3 and P6 primer PCR, are obtained fusion segment NHEJ and are repaired base
Cause；Obtained NHEJ revision points are connected to by ClonExpress II One Step Cloning Kit C112
On pMD18T carrier, recombination pMD18T-LigD::mku carrier is obtained；It is with obtained recombination pMD18T-LigD::mku carrier
Template, using in table 1 P7 primer and P8 primer cloned, obtain Pj23119-LigD::mku express frame, i.e. Frag2；
(4) Frag1 obtained in the basic framework obtained in (1) and (2) is passed through into ClonExpress II One Step
Cloning Kit C112 assembles (Gibson assembly and connection reaction system is shown in Table 3), obtains annular carrier；Pass through restriction enzyme
Enzyme Spe I linearizes the annular carrier digestion of acquisition, obtains linear carrier；It will be obtained in the linear carrier of acquisition and (3)
The Frag2 obtained passes through ClonExpress II One Step Cloning Kit C112 assembling (Gibson assembly and connection reaction
3) system is shown in Table, obtain pML-Cpf1 plasmid (plasmid map is shown in Fig. 1), wherein Cpf1 gene is located at pMV261 expression vector
The downstream of Hsp60 promoter and the rrnB terminator upstream of pMV261 expression vector are driven by Hsp60 promoter and are expressed；
Pj23119 promoter is located at the downstream of the KanR resistant gene of pMV261 expression vector；NHEJ revision points are opened positioned at Pj23119
The upstream of the replication orgin (ori) in the downstream and pMV261 expression vector of mover is driven by Pj23119 promoter and is expressed；
(5) using pMV261 expression vector as template, using in table 1 P11 primer and P12 primer cloned to obtain OriM
Replicon, i.e. Frag3；
(6) using ptrc99A carrier as template, using in table 1 P9 primer and P10 primer cloned to obtain pMB1 and replicated
Son and aadA resistant gene, i.e. Frag4；
(7) to need the sequence of editor's gene as target sequence in new gold mycobacteria, design can selectively targeted target sequence
Targeting sequence, obtain crRNA sequence；This crRNA sequence is synthesized by Jin Weizhi biotechnology company and is connected to pMD18T load
On body, recombination pMD18T-crRNA carrier is obtained；Using obtained recombination pMD18T-crRNA carrier as template, using in table 1
P13 primer and P14 primer are cloned, and are obtained Pj23119-crRNA-rrnB and are expressed frame, i.e. Frag5；
(8) Frag5 for obtaining Frag4 that (6) obtain, (7) passes through ClonExpress II One Step Cloning
The Frag3 that Kit C112 assembling (Gibson assembly and connection reaction system is shown in Table 3) is obtained with (5) afterwards passes through ClonExpress II
One Step Cloning Kit C112 assembles (Gibson assembly and connection reaction system is shown in Table 3), obtains pJM-crRNA plasmid
(plasmid map is shown in Fig. 2), wherein pJM-crRNA plasmid successively includes OriM replicon, pMB1 replicon, Pj23119 starting
Son, crRNA sequence, rrnB terminator and aadA resistant gene.
2, the use of CRISPR/Cpf1 gene editing system
CRISPR/Cpf1 gene editing system contains pML-Cpf1 plasmid and pJM-crRNA plasmid simultaneously, this
CRISPR/Cpf1 gene editing system when in use, needs first to import pML-Cpf1 plasmid in new gold mycobacteria, to be screened
After being proved to be successful, then competence done with the new gold mycobacteria transformant screened, pJM-crRNA plasmid is imported into this turn
In beggar.
1 primer of table and its sequence
Table 2PCR amplification reaction system
| ddH20 | upto50μL |
| 2×PhantaMaxMasterMix | 25μL |
| Upstream primer | 2μL |
| Downstream primer | 2μL |
| Template DNA | 1μL |
Table 3Gibson assembly and connection reaction system
| Linearized vector | 90ng |
| Insert Fragment | 180ng |
| 5×CEIIBuffer | 4μL |
| ExnaseII | 2μL |
| ddH2O | addto20μL |
Embodiment 2:CRISPR/Cpf1 gene editing systematic difference
Specific step is as follows:
Scheme one:
(1) building of CRISPR/Cpf1 gene editing system
Using the KshA gene of new gold mycobacteria JC-12 as gene need to be knocked out, the nucleotides sequence of gene need to be knocked out
It is classified as target sequence, selectively targeted target sequence crRNA (nucleotide sequence such as SEQ ID NO:24 institute is designed for according to target sequence
Show)；According to embodiment 1, construct to obtain the CRISPR/Cpf1 gene editing system of scheme one according to the crRNA that design obtains；
(2) gene editing of new gold mycobacteria JC-12
Using the new gold mycobacteria JC-12 of unused CRISPR/Cpf1 gene editing system editor as blank control,
PML-Cpf1 plasmid in the CRISPR/Cpf1 gene editing system for the scheme one that (1) obtains is transformed into new golden branch bar
In bacterium JC-12 competent cell, it is coated on inclined-plane/solid medium (containing 50 μ g/mL kanamycins), 30 DEG C of cultures 3~4
It；Picking transformant carries out PCR preliminary identification with the P15 primer of table 4 and P16 primer, and the transformant that picking is proved to be successful, which is done, to be felt
By state, 0.5mM IPTG is added while preparing competence and is induced in 30 DEG C, the CRISPR/ for the scheme one that (1) is obtained
PJM-crRNA plasmid in Cpf1 gene editing system is transformed into transformant competent cell, is coated on inclined-plane/solid culture
On base (containing 50 μ g/mL kanamycins and 50 μ g/mL streptomysins), 30 DEG C are cultivated 3~4 days；Picking transformant is drawn with the P15 of table 4
Object and P16 primer carry out PCR preliminary identification, the transformant sequence verification that picking is proved to be successful.
The CRISPR/Cpf1 gene editing system of detection operational version one carries out new gold mycobacteria JC-12 gene
The gene editing efficiency of editor, testing result are as follows: the knockout efficiency of KshA can reach 40%.
The new gold mycobacteria JC-12 of picking blank control group and the CRISPR/Cpf1 gene of operational version one are compiled
The system of collecting knocks out the single colonie of the new gold mycobacteria JC-12/KshA after KshA gene, is inoculated in seed culture medium respectively
In 30 DEG C of culture 48h, bacterium solution is obtained；Bacterium solution is crushed by cell crushing instrument, crude enzyme liquid is obtained, crude enzyme liquid centrifuging and taking is sunk
It forms sediment, obtains -9 α of 3- sterone-hydroxylase, the new gold mycobacteria JC-12 for detecting blank control group ferments to obtain -9 α of 3- sterone -
The CRISPR/Cpf1 gene editing system of hydroxylase and operational version one knocks out the new gold mycobacteria after KshA gene
JC-12/KshA ferments to obtain the specific enzyme activity of -9 α of 3- sterone-hydroxylase, testing result are as follows: the CRISPR/Cpf1 of operational version one
New gold mycobacteria JC-12/KshA after gene editing system knockout KshA gene ferments to obtain -9 α of 3- sterone-hydroxylase
Specific enzyme activity ferment to obtain -9 α of 3- sterone-hydroxylase specific enzyme activity decline compared with the new gold mycobacteria JC-12 of blank control group
88% (specific visible Fig. 3).
4 primer of table and its sequence
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Sequence table
<110>Southern Yangtze University
<120>a kind of gene editing system and its application in new gold mycobacteria
<160> 26
<170> PatentIn version 3.3
<210> 1
<211> 3903
<212> DNA
<213>artificial sequence
<400> 1
atgtccatct accaagagtt tgtgaataaa tactccctgt ccaagaccct ccgttttgag 60
ctgatccccc aaggcaagac cctcgaaaac atcaaggcac gcggcctcat cctggatgac 120
gaaaagcgcg ctaaggatta caagaaggca aagcagatca tcgacaagta ccaccagttc 180
ttcatcgaag agatcctgtc ctccgtgtgc atctccgagg acctgctcca gaactactcc 240
gatgtctact tcaagctcaa gaagtccgat gacgataacc tgcagaagga cttcaagtcc 300
gctaaggata ccatcaagaa gcagatctcc gaatacatca aggattccga gaagttcaag 360
aacctcttca accagaacct gatcgacgca aagaagggcc aggaatccga tctcatcctg 420
tggctcaagc agtccaagga taacggcatc gagctcttca aggccaactc cgacatcacc 480
gacatcgatg aagctctgga gatcatcaag tccttcaagg gctggaccac ctacttcaag 540
ggcttccacg aaaaccgcaa gaacgtgtac tcctccaacg atatcccaac ctctatcatc 600
taccgcatcg tcgacgataa cctgccaaag ttcctcgaaa acaaggcaaa gtacgagtcc 660
ctgaaggata aggccccaga agctatcaac tacgagcaga tcaagaagga cctggccgaa 720
gagctcacct tcgacatcga ttacaagacc tctgaagtga accagcgcgt cttctccctc 780
gatgaagtgt tcgagatcgc caacttcaac aactacctga accagtccgg catcaccaag 840
ttcaacacca tcatcggcgg caagttcgtc aacggcgaaa acaccaagcg caagggcatc 900
aacgagtaca tcaacctcta ctcccagcag atcaacgata agaccctgaa gaagtacaag 960
atgtccgtgc tcttcaagca gatcctgtcc gacaccgaat ccaagtcctt cgtcatcgac 1020
aagctggagg acgattccga tgtggtcacc accatgcagt ccttctacga acagatcgca 1080
gccttcaaga ccgtggaaga gaagtccatc aaggagaccc tctccctgct cttcgacgat 1140
ctgaaggctc agaagctgga tctctccaag atctacttca agaacgacaa gtccctgacc 1200
gatctctccc agcaggtctt cgacgattac tccgtgatcg gcaccgcagt cctggaatac 1260
atcacccagc agatcgcccc aaagaacctc gataacccat ccaagaagga acaggagctg 1320
atcgccaaga agaccgaaaa ggctaagtac ctgtccctcg agaccatcaa gctggctctc 1380
gaagagttca acaagcaccg cgacatcgat aagcagtgcc gcttcgaaga gatcctcgca 1440
aacttcgctg caatcccaat gatcttcgac gaaatcgcac agaacaagga taacctggcc 1500
cagatctcca tcaagtacca gaaccagggc aagaaggatc tgctccaggc ctccgctgag 1560
gacgatgtga aggcaatcaa ggacctgctc gatcagacca acaacctgct ccacaagctg 1620
aagatcttcc acatctccca gtccgaagac aaggccaaca tcctcgacaa ggatgagcac 1680
ttctacctgg tgttcgaaga gtgctacttc gaactcgcta acatcgtccc actgtacaac 1740
aagatccgca actacatcac ccagaagcca tactccgatg aaaagttcaa gctcaacttc 1800
gagaactcca ccctggcaaa cggctgggac aagaacaagg aaccagataa caccgccatc 1860
ctcttcatca aggacgataa gtactacctg ggcgtgatga acaagaagaa caacaagatc 1920
ttcgacgata aggccatcaa ggaaaacaag ggcgagggct acaagaagat cgtgtacaag 1980
ctgctcccag gcgctaacaa gatgctccca aaggtcttct tctccgcaaa gtccatcaag 2040
ttctacaacc catccgaaga tatcctgcgc atccgcaacc actccaccca caccaagaac 2100
ggctccccac agaagggcta cgaaaagttc gagttcaaca tcgaagactg ccgcaagttc 2160
atcgatttct acaagcagtc catctccaag cacccagagt ggaaggactt cggcttccgc 2220
ttctccgata cccagcgcta caactccatc gatgaattct accgcgaagt ggagaaccag 2280
ggctacaagc tgaccttcga aaacatctcc gagtcctaca tcgattccgt ggtcaaccag 2340
ggcaagctgt acctcttcca gatctacaac aaggacttct ccgcttactc caagggccgc 2400
ccaaacctgc acaccctcta ctggaaggca ctcttcgacg aacgcaacct gcaggatgtg 2460
gtctacaagc tcaacggcga agcagagctg ttctaccgca agcagtccat cccaaagaag 2520
atcacccacc cagccaagga agcaatcgcc aacaagaaca aggataaccc aaagaaggaa 2580
tccgtgttcg agtacgacct gatcaaggat aagcgcttca ccgaggacaa gttcttcttc 2640
cactgcccaa tcaccatcaa cttcaagtcc tccggcgcca acaagttcaa cgatgaaatc 2700
aacctgctcc tgaaggagaa ggctaacgac gtgcacatcc tgtccatcga tcgcggcgaa 2760
cgccacctcg cctactacac cctggtcgac ggcaagggca acatcatcaa gcaggacacc 2820
ttcaacatca tcggcaacga tcgcatgaag accaactacc acgacaagct ggccgctatc 2880
gagaaggacc gcgattccgc tcgcaaggat tggaagaaga tcaacaacat caaggaaatg 2940
aaggaaggct acctctccca ggtggtccac gaaatcgcta agctggtgat cgagtacaac 3000
gcaatcgtgg tcttcgaaga cctgaacttc ggcttcaagc gcggccgctt caaggtggag 3060
aagcaggtct accagaagct ggaaaagatg ctcatcgaga agctgaacta cctcgtgttc 3120
aaggacaacg aattcgataa gaccggcggc gtcctccgtg cataccagct gaccgcccca 3180
ttcgagacct tcaagaagat gggcaagcag accggcatca tctactacgt gccagctggc 3240
ttcacctcta agatctgccc agtgaccggc ttcgtcaacc agctctaccc aaagtacgaa 3300
tccgtctcca agtcccagga gttcttctcc aagttcgaca agatctgcta caacctggat 3360
aagggctact tcgaattctc cttcgactac aagaacttcg gcgataaggc agccaagggc 3420
aagtggacca tcgcatcctt cggctcccgc ctcatcaact tccgcaactc cgacaagaac 3480
cacaactggg atacccgcga agtgtaccca accaaggaac tggagaagct cctgaaggat 3540
tactccatcg aatacggcca cggcgagtgc atcaaggctg caatctgcgg cgaatccgac 3600
aagaagttct tcgcaaagct gacctctgtg ctcaacacca tcctgcagat gcgcaactcc 3660
aagaccggca ccgagctgga ttacctcatc tccccagtgg ccgacgtcaa cggcaacttc 3720
ttcgattccc gccaggctcc aaagaacatg ccacaggacg ctgatgcaaa cggcgcctac 3780
cacatcggtc tgaagggtct catgctcctg ggtcgcatca agaacaacca ggaaggcaag 3840
aagctgaatc tcgtcattaa gaacgaagaa tactttgaat ttgtccagaa ccgcaataac 3900
taa 3903
<210> 2
<211> 29
<212> DNA
<213>artificial sequence
<400> 2
ttgacagcta gctcagtcct aggtataat 29
<210> 3
<211> 822
<212> DNA
<213>artificial sequence
<400> 3
atgcgagcca tttggacggg ttcgatcgcc ttcgggctgg tgaacgtgcc ggtcaaggtg 60
tacagcgcta ccgcagacca cgacatcagg ttccaccagg tgcacgccaa ggacaacgga 120
cgcatccggt acaagcgcgt ctgcgaggcg tgtggcgagg tggtcgacta ccgcgatctt 180
gcccgggcct acgagtccgg cgacggccaa atggtggcga tcaccgacga cgacatcgcc 240
agcttgcctg aagaacgcag ccgggagatc gaggtgttgg agttcgtccc cgccgccgac 300
gtggacccga tgatgttcga ccgcagctac tttttggagc ctgattcgaa gtcgtcgaaa 360
tcgtatgtgc tgctggctaa gacactcgcc gagaccgacc ggatggcgat cgtgcatttc 420
acgctgcgca acaagaccag gctggcggcg ttgcgcgtca aggatttcgg caagcgagag 480
gtgatgatgg tgcacacgtt gctgtggccc gatgagatcc gcgaccccga cttcccggtg 540
ctggaccaga aggtggagat caaacccgcg gaactcaaga tggccggcca ggtggtggac 600
tcgatggccg acgacttcaa tccggaccgc taccacgaca cctaccagga gcagttacag 660
gagctgatcg acaccaaact cgaaggtggg caggcattta ccgccgagga ccaaccgagg 720
ttgctggacg agcccgaaga cgtctccgac ctgctcgcca agctggaggc cagcgtgaag 780
gcgcgctcga aggccaactc aaacgtccca acgcctccgt ga 822
<210> 4
<211> 2280
<212> DNA
<213>artificial sequence
<400> 4
atgggttcgg cgtcggagca acgggtgacg ctgaccaacg ccgacaaggt gctctatccc 60
gccaccggga ccacaaagtc cgatatcttc gactactacg ccggtgttgc cgaagtcatg 120
ctcggccaca tcgcgggacg gccggcgacg cgcaagcgct ggcctaacgg cgtcgaccaa 180
cccgcgttct tcgaaaagca gttggcgttg tcggcgccgc cttggctgtc acgtgcaacg 240
gtggcgcacc ggtccgggac gacgacctat ccgatcatcg atagcgcaac cgggctggcc 300
tggatcgccc aacaggcggc gctggaggtg cacgtgccgc agtggcggtt tgtcgccgag 360
cccggatcag gtgagttaaa tccgggcccg gcaacgcgtt tggtgttcga cctggacccg 420
ggcgaaggcg tgatgatggc ccagctggcc gaggtggcgc gcgcggttcg tgatcttctc 480
gccgatatcg ggttggtcac cttcccggtc accagcggca gcaagggatt gcatctgtac 540
acaccgctgg atgagccggt gagcagcagg ggagccacgg tgttggccaa gcgcgtcgcg 600
cagcgattgg agcaggcgat gcccgcgttg gtcacctcga ccatgaccaa aagcctgcgg 660
gccgggaagg tgtttgtgga ctggagccag aacagcggct cgaagaccac catcgcgccg 720
tactcactac gtggccggac gcatccgacc gtcgcggcgc cacgcacctg ggcggagctc 780
gacgaccccg cactgcgtca gctctcctac gacgaggtgc tgacccggat tgcccgcgac 840
ggcgatctgc tcgagcggct ggatgccgac gctccggtag cggaccggtt gacccgatac 900
cgccgcatgc gcgacgcatc gaaaactccc gagccgattc ccacggcgaa acccgttacc 960
ggagacggca atacgttcgt catccaggag catcacgcgc gtcggccgca ctacgatttc 1020
cggctggaat gcgacggcgt gctggtctcg tgggcggtac cgaaaaacct gcccgacaac 1080
acatcggtta accatctagc gatacacacc gaggaccacc cgctggaata cgccacgttc 1140
gagggcgcga ttcccagcgg ggagtacggc gccggcaagg tgatcatctg ggactccggc 1200
acttacgaca ccgagaagtt ccacgatgac ccgcacacgg gggaggtcat cgtgaatctg 1260
cacggcggcc ggatctctgg gcgttatgcg ctgattcgga ccaacggcga tcggtggctg 1320
gcgcaccgcc taaagaatca gaaagaccag aaggtgttcg agttcgacaa tctggcccca 1380
atgcttgcca cgcacggcac ggtggccggt ctaaaggcca gccagtgggc gttcgaaggc 1440
aagtgggacg gctaccggtt gctggttgag gctgaccacg gcgccgtgcg gctgcggtcc 1500
cgcagcgggc gcgatgtcac cgccgagtat ccgcaattgc gggcattggc ggaggatctc 1560
gccgatcacc acgtggtgct ggacggcgag gccgtcgtac ttgactcctc tggtgtgccc 1620
agcttcagcc agatgcagaa tcggggccgc gacacccgtg tcgagttctg ggcgttcgac 1680
ctgctctacc tcgacggccg cgcgctgcta ggcacccgct accaagaccg gcgtaagctg 1740
ctcgaaaccc tagctaacgc aaccagtctc accgttcccg agctgctgcc cggtgacggc 1800
gcccaagcgt ttgcgtgctc gcgcaagcac ggctgggagg gcgtgatcgc caagaggcgt 1860
gactcgcgct atcagccggg ccggcgctgc gcgtcgtggg tcaaggacaa gcactggaac 1920
acccaggaag tcgtcattgg tggctggcgc gccggggaag gcgggcgcag cagtggcgtc 1980
gggtcgctgc tcatgggcat ccccggtcca ggtgggctgc agttcgccgg gcgggtcggt 2040
accggcctca gcgaacgcga actggccaac ctcaaggaga tgctggcgcc gctgcatacc 2100
gacgagtccc ccttcgacgt accactgccc gcgcgtgacg ccaagggcat cacatatgtc 2160
aagccggcgc tggttgcaga ggtgcgctac agcgagtgga ctccggaggg ccggctgcgt 2220
caatcaagct ggcgtgggct gcggccggac aagaaaccca gtgaggtggt gcgcgaatga 2280
<210> 5
<211> 256
<212> DNA
<213>artificial sequence
<400> 5
ggagcttatc gactgcacgg tgcaccaatg cttctggcgt caggcagcca tcggaagctg 60
tggtatggct gtgcaggtcg taaatcactg cataattcgt gtcgctcaag gcgcactccc 120
gttctggata atgttttttg cgccgacatc ataacggttc tggcaaatat tctgaaatga 180
gctgttgaca attaatcatc gtgtggtacc atgtgtggaa ttgtgagcgg ataacaattt 240
cacacaggaa acagaa 256
<210> 6
<211> 1896
<212> DNA
<213>artificial sequence
<400> 6
gtgagcccac cagctccgta agttcgggtg ctgtgtggct cgtacccgcg cattcaggcg 60
gcagggggtc taacgggtct aaggcggcgt gtacggccgc cacagcggct cttagcggcc 120
cggaaacgtc ctcgaaacga cgcatgtgtt cctcctggtt ggtacaggtg gttgggggtg 180
ctcggctgtc gctggtgttt catcatcagg gctcgacggg agagcggggg agtgtgcagt 240
tgtggggtgg cccctcagcg aaatatctga cttggagctc gtgtcggacc atacaccggt 300
gattaatcgt ggtttattat caagcgtgag ccacgtcgcc gacgaatttg agcagctctg 360
gctgccgtac tggtccctgg caagcgacga tctgctcgag gggatctacc gccaaagccg 420
cgcgtcggcc ctaggccgcc ggtacatcga ggcgaaccca acagcgctgg caaacctgct 480
ggtcgtggac gtagaccatc cagacgcagc gctccgagcg ctcagcgccc gggggtccca 540
tccgctgccc aacgcgatcg tgggcaatcg cgccaacggc cacgcacacg cagtgtgggc 600
actcaacgcc cctgttccac gcaccgaata cgcgcggcgt aagccgctcg catacatggc 660
ggcgtgcgcc gaaggccttc ggcgcgccgt cgatggcgac cgcagttact caggcctcat 720
gaccaaaaac cccggccaca tcgcctggga aacggaatgg ctccactcag atctctacac 780
actcagccac atcgaggccg agctcggcgc gaacatgcca ccgccgcgct ggcgtcagca 840
gaccacgtac aaagcggctc cgacgccgct agggcggaat tgcgcactgt tcgattccgt 900
caggttgtgg gcctatcttc ccgccctcat gcggatctac ctgccgaccc ggaacgtgga 960
cggactcggc cgcgcgatct atgccgagtg ccacgcgcga aacgccgaat ttccgtgcaa 1020
cgacgtgtgt cccggaccgc taccggacag cgaggtccgc gccatcgcca acagcatttg 1080
gcgttggatc acaaccaagt cgcgcatttg ggcggacggg atcgtggtct acgaggccac 1140
actcagtgcg cgccatgcgg ccatctcgcg gaagggcgca gcagcgcgca cggcggcgag 1200
cacagttgcg cggcgcgcaa agtccgcgtc agccatggag gcattgctat gagcgacggc 1260
tacagcgacg gctacagcga cggctacaac tggcagccga ctgtccgcaa aaagcggcgc 1320
gtgaccgccg ccgaaggcgc tcgaatcacc ggactatccg aacgccacgt cgtccggctc 1380
gtggcgcagg aacgcagcga gtggttcgcc gagcaggctg cacgccgcga acgcatccgc 1440
gcctatcacg acgacgaggg ccactcttgg ccgcaaacgg ccaaacattt cgggctgcat 1500
ctggacaccg ttaagcgact cggctatcgg gcgaggaaag agcgtgcggc agaacaggaa 1560
gcggctcaaa aggcccacaa cgaagccgac aatccaccgc tgttctaacg caattgggga 1620
gcgggtgtcg cgggggttcc gtggggggtt ccgttgcaac gggtcggaca ggtaaaagtc 1680
ctggtagacg ctagttttct ggtttgggcc atgcctgtct cgttgcgtgt ttcgttgcgc 1740
ccgttttgaa taccagccag acgagacggg gttctacgaa tcttggtcga taccaagcca 1800
tttccgctga atatcgggga gctcaccgcc agaatcggtg gttgtggtga tgtacgtggc 1860
gaactccgtt gtagtgcctg tggtggcatc cgtggc 1896
<210> 7
<211> 589
<212> DNA
<213>artificial sequence
<400> 7
ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 60
agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 120
cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 180
caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 240
tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 300
ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 360
ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 420
gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 480
gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 540
tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaa 589
<210> 8
<211> 45
<212> DNA
<213>artificial sequence
<400> 8
caaataaaac gaaaggctca gtcgaaagac tgggcctttc gtttt 45
<210> 9
<211> 792
<212> DNA
<213>artificial sequence
<400> 9
atgagggaag cggtgatcgc cgaagtatcg actcaactat cagaggtagt tggcgtcatc 60
gagcgccatc tcgaaccgac gttgctggcc gtacatttgt acggctccgc agtggatggc 120
ggcctgaagc cacacagtga tattgatttg ctggttacgg tgaccgtaag gcttgatgaa 180
acaacgcggc gagctttgat caacgacctt ttggaaactt cggcttcccc tggagagagc 240
gagattctcc gcgctgtaga agtcaccatt gttgtgcacg acgacatcat tccgtggcgt 300
tatccagcta agcgcgaact gcaatttgga gaatggcagc gcaatgacat tcttgcaggt 360
atcttcgagc cagccacgat cgacattgat ctggctatct tgctgacaaa agcaagagaa 420
catagcgttg ccttggtagg tccagcggcg gaggaactct ttgatccggt tcctgaacag 480
gatctatttg aggcgctaaa tgaaacctta acgctatgga actcgccgcc cgactgggct 540
ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc agtaaccggc 600
aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat 660
cagcccgtca tacttgaagc tagacaggct tatcttggac aagaagaaga tcgcttggcc 720
tcgcgcgcag atcagttgga agaatttgtc cactacgtga aaggcgagat caccaaggta 780
gtcggcaaat aa 792
<210> 10
<211> 42
<212> DNA
<213>artificial sequence
<400> 10
tgcagaattc gaagcttatg tccatctacc aagagtttgt ga 42
<210> 11
<211> 42
<212> DNA
<213>artificial sequence
<400> 11
atttgtccag aaccgcaata actaaatcga tgtcgacgta gt 42
<210> 12
<211> 29
<212> DNA
<213>artificial sequence
<400> 12
atgggttcgg cgtcggagca acgggtgac 29
<210> 13
<211> 51
<212> DNA
<213>artificial sequence
<400> 13
ctagtattct cctctttaat ctctagtatc attcgcgcac cacctcactg g 51
<210> 14
<211> 51
<212> DNA
<213>artificial sequence
<400> 14
attaaagagg agaatactag atgcgagcca tttggacggg ttcgatcgcc t 51
<210> 15
<211> 29
<212> DNA
<213>artificial sequence
<400> 15
tcacggaggc gttgggacgt ttgagttgg 29
<210> 16
<211> 87
<212> DNA
<213>artificial sequence
<400> 16
tctagattga cagctagctc agtcctaggt ataatgctag ctactagaga aagaggagaa 60
atactagatg ggttcggcgt cggagca 87
<210> 17
<211> 48
<212> DNA
<213>artificial sequence
<400> 17
ttcgaattct gcagcttcac ggaggcgttg ggacgtttga gttggcct 48
<210> 18
<211> 30
<212> DNA
<213>artificial sequence
<400> 18
atgagggaag cggtgatcgc cgaagtatcg 30
<210> 19
<211> 28
<212> DNA
<213>artificial sequence
<400> 19
tttccatagg ctccgccccc ctgacgag 28
<210> 20
<211> 28
<212> DNA
<213>artificial sequence
<400> 20
gtgagcccac cagctccgta agttcggg 28
<210> 21
<211> 24
<212> DNA
<213>artificial sequence
<400> 21
gccacggatg ccaccacagg cact 24
<210> 22
<211> 45
<212> DNA
<213>artificial sequence
<400> 22
aacaacgtcg acatggcgca caggcaacca tagggcagga aattt 45
<210> 23
<211> 49
<212> DNA
<213>artificial sequence
<400> 23
tgcgccatgt cgacgttgtt attataccta ggactgagct agctgtcaa 49
<210> 24
<211> 137
<212> DNA
<213>artificial sequence
<400> 24
ggatccgaat ttctactgtt gtagatcagg caaccatagg gcaggaaatt taaataaaac 60
gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc 120
tcctgagtag gacaaat 137
<210> 25
<211> 20
<212> DNA
<213>artificial sequence
<400> 25
aatacctgag cggccagccc 20
<210> 26
<211> 19
<212> DNA
<213>artificial sequence
<400> 26
cggggctccg ctgcggtcg 19
Claims (10)
1. a kind of plasmid that can be used for gene editing, which is characterized in that the plasmid includes pMV261 expression vector, Cpf1 base
Cause, Pj23119 promoter and NHEJ revision points, are named as pML-Cpf1 plasmid；On the pMV261 expression vector
Hsp60 promoter is replaced for Tac promoter；The NHEJ revision points include the base of coding DNA terminal binding protein mku
The gene of cause and coding DNA ligase LigD；The Cpf1 gene is located at the downstream of the Tac promoter of pMV261 expression vector
And the rrnB terminator upstream of pMV261 expression vector, it is driven and is expressed by Tac promoter；The Pj23119 promoter is located at
The downstream of the KanR resistant gene of pMV261 expression vector；The NHEJ revision points be located at the downstream of Pj23119 promoter with
And the upstream of the replication orgin (ori) of pMV261 expression vector, it is driven and is expressed by Pj23119 promoter.
2. a kind of plasmid that can be used for gene editing as described in claim 1, which is characterized in that the nucleosides of the Cpf1 gene
Acid sequence is as shown in SEQ ID NO:1.
3. a kind of plasmid that can be used for gene editing as claimed in claim 1 or 2, which is characterized in that encode the end DNA
Collect the nucleotide sequence of the gene of hop protein mku as shown in SEQ ID NO:3.
4. a kind of plasmid that can be used for gene editing a method according to any one of claims 1-3, which is characterized in that encode the DNA
The nucleotide sequence of the gene of ligase LigD is as shown in SEQ ID NO:4.
5. a kind of plasmid that can be used for gene editing as described in claim 1-4 is any, which is characterized in that encode the Tac
The nucleotide sequence of the gene of promoter is as shown in SEQ ID NO:5.
6. a kind of plasmid that can be used for gene editing, which is characterized in that the plasmid successively includes OriM replicon, pMB1 duplication
Son, Pj23119 promoter, crRNA sequence, rrnB terminator and aadA resistant gene, are named as pJM-crRNA plasmid；Institute
Stating crRNA sequence is targeting sequence, can selectively targeted target sequence；The target sequence refers to the nucleotides sequence that need to edit gene
Column.
7. a kind of double-mass model CRISPR/Cpf1 gene editing system, which is characterized in that the system contains claim 1- simultaneously
A kind of 5 any plasmids that can be used for gene editing and a kind of matter that can be used for gene editing as claimed in claim 6
Grain.
8. a kind of method for editing new gold mycobacteria gene, which is characterized in that the method is using described in claim 7
A kind of double-mass model CRISPR/Cpf1 gene editing system.
9. a kind of method for editing new gold mycobacteria gene as claimed in claim 8, which is characterized in that the method is
The pML-Cpf1 plasmid in a kind of double-mass model CRISPR/Cpf1 gene editing system as claimed in claim 7 is first imported into new gold
In color mycobacteria, so that the gene and volume of Cpf1 gene, coding DNA terminal binding protein mku on pML-Cpf1 plasmid
The gene expression of code DNA ligase LigD, then by a kind of double-mass model CRISPR/Cpf1 gene editing as claimed in claim 7
PJM-crRNA plasmid in system imports in new gold mycobacteria, so that pJM-crRNA plasmid transcription generates crRNA sequence,
CrRNA sequence can form compound in conjunction with Cpf1 gene, this compound can be targeted and be cut and need to edit gene and make new gold
It needs the gene of editor to be knocked in mycobacteria, the knockout meeting of gene need to be edited so that new gold mycobacteria gene generates DSB
Fracture, at this point, the DNA terminal binding protein mku and DNA ligase LigD of the expression on pML-Cpf1 plasmid can repair DSB
Fracture, completes the editing process of new gold mycobacteria gene.
10. a kind of any plasmid that can be used for gene editing or as claimed in claim 6 a kind of available of claim 1-5
In the plasmid of gene editing or a kind of double-mass model CRISPR/Cpf1 gene editing system as claimed in claim 7 or claim 8
Or a kind of application of the method for the new gold mycobacteria gene of editor described in 9 in terms of editing new gold mycobacteria gene.
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