CN110066829A - A kind of CRISPR/Cas9 gene editing system and its application - Google Patents
A kind of CRISPR/Cas9 gene editing system and its application Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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Abstract
The invention discloses a kind of CRISPR/Cas9 gene editing system and its applications, 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；The pML-Cas9 plasmid and pJM-sgRNA plasmid that this system uses can be stabilized in new gold mycobacteria, express, and will not lose with the passage of new gold mycobacteria, will not be repelled by the protection mechanism present in new gold mycobacteria itself；Gene editing is carried out to new gold mycobacteria using the CRISPR/Cas9 gene editing service system of this system, knockout efficiency is higher, up to 50%.
Description
Technical field
The present invention relates to a kind of CRISPR/Cas9 gene editing system and its application, belong to gene engineering technology field with
And technical field of bioengineering.
Background technique
Steroid drugs belongs to hormone medicine, is the endogenous material found when studying mammal endocrine system,
With epochmaking pharmaceutical value, sustain life, adjusting sexual function, body development, immunological regulation, treating skin disease and life
Educating control aspect has important role.These are acted on so that steroid drugs, which becomes in chemical drug industry, is only second to the of antibiotic
Two major class drugs, but also steroid drugs becomes one of the emphasis of China's drug new resources exploitation, more make steroid drugs and
Steroid medicine intermediates become the important species of China's medicine outlet.
But since China compares in the production level of steroid drugs with advanced country in the world there are also a certain distance,
The existing steroid drugs kind in China is only the one third of the external steroid drugs listed, and is mostly low and middle-grade productions
Product, profit are lower.Therefore, it is badly in need of promoting the production level of China's steroid drugs.
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, occupy in the production of steroid drugs
Critical role, however, since substrate feed concentrations are low, the microbe conversion period is long and to the cognition of steroidal microbial metabolism mechanism
The problems such as insufficient, China also lag significantly behind advanced country in the world in the production of steroidal microorganism conversion.Therefore, it is badly in need of promoting me
The production level of state Steroid medicine intermediates ADD, and then promote the production level of China's steroid drugs.
New gold mycobacteria (Mycobacterium neoaurum) can transformation phytosterin synthesize androstane-4-alkene-3,
17- diketone (AD) and Androsta-1,4-diene-3,17-dione (ADD), and have and grow fast advantage, it is a kind of great potential
ADD produces bacterium, if 3- sterone-Δ 1- dehydrogenase, gallbladder during ADD can be synthesized to new gold mycobacteria transformation phytosterin
The key enzymes such as sterol oxidizing ferment, sterone C27 monooxygenase and 3- sterone -9 α-hydroxylase are studied, and combine metabolic engineering
Strategy is transformed the ADD synthesis relational approach of new gold mycobacteria, it is most likely that the new of efficient accumulation ADD is capable of in acquisition
Gold mycobacteria, this modified new gold mycobacteria, which is undoubtedly, promotes China's Steroid medicine intermediates ADD production level
An important breakthrough, also provide more possibility to promote the production level of China's steroid drugs.
However, the protection mechanism exclusiveness as present in new gold mycobacteria itself is stronger, foreign gene is difficult
Be stabilized and express in new gold mycobacteria, this allow for being applicable to the gene editing method of new gold mycobacteria compared with
It is few, for example, the incidence due to mycobacteria homologous recombination is obviously more much lower than other bacteriums, only 10-6~10-5, also,
Carrying out gene editing to new gold mycobacteria using homologous recombination also needs to bring resistance label into the genome, therefore, utilizes
Homologous recombination, which carries out gene editing to new gold mycobacteria, has that low efficiency, period are long, screening amount is big, complicated for operation etc. bad
Gesture；Suicide vector system (pNIL/pGOAL series plasmids) although etc. other gene editing methods can be to new gold mycobacteria
Gene editing is carried out, but efficiency is not also high, does not have any advantage compared with homologous recombination.
Therefore, gene editing is undoubtedly the difficult point that metabolic engineering is carried out to new gold mycobacteria, has been badly in need of
The kind gene editing system for new gold mycobacteria, to be efficiently transformed to new gold mycobacteria.
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 CRISPR/Cas9 gene editing system, the CRISPR/
Cas9 gene editing system includes pML-Cas9 plasmid and pJM-sgRNA plasmid；
The pML-Cas9 plasmid includes that pMV261 expression vector, Cas9 gene, Pj23119 promoter and NHEJ are repaired
Gene；Hsp60 promoter on the pMV261 expression vector is replaced for Tac promoter；The NHEJ revision points packet
The gene of the mku of terminal binding protein containing coding DNA and the gene of coding DNA ligase LigD；The Cas9 gene is located at
The downstream of the Tac promoter of pMV261 expression vector and the rrnB terminator upstream of pMV261 expression vector, by Tac promoter
Driving expression；The Pj23119 promoter is located at the downstream of the KanR resistant gene of pMV261 expression vector；The NHEJ is repaired
Gene is located at the upstream in the downstream of Pj23119 promoter and the replication orgin (ori) of pMV261 expression vector, by Pj23119
Promoter driving expression；
The pJM-sgRNA plasmid successively includes OriM replicon, pMB1 replicon, Pj23119 promoter, sgRNA sequence
Column, rrnB terminator and aadA resistant gene, are named as pJM-sgRNA plasmid；The sgRNA sequence is targeting sequence, can
Selectively targeted target sequence；The target sequence refers to the nucleotide sequence that need to edit gene.
In one embodiment of the invention, the nucleotide sequence of the Cas9 gene is as shown in SEQ ID NO:1.
In one embodiment of the invention, it encodes in the pML-Cas9 plasmid and pJM-sgRNA plasmid
The nucleotide sequence of the gene of Pj23119 promoter is as shown in SEQ 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.
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 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 gene editing method that can be used for new gold mycobacteria, the method is using upper
State a kind of CRISPR/Cas9 gene editing system.
In one embodiment of the invention, the method is first by a kind of above-mentioned CRISPR/Cas9 gene editing system
PML-Cas9 plasmid in system imports in new gold mycobacteria, so that Cas9 gene, coding DNA end on pML-Cas9 plasmid
The gene expression for holding the gene and coding DNA ligase LigD of binding protein mku, then by a kind of above-mentioned CRISPR/Cas9
PJM-sgRNA plasmid in gene editing system imports in new gold mycobacteria, so that pJM-sgRNA plasmid transcription generates
SgRNA sequence, Cas9 handle and Terminator in sgRNA sequence will form loop-stem structure, and Cas9 gene can identify this
For loop-stem structure to form compound in conjunction with sgRNA sequence, this compound, which can target edit gene and be anchored on, need to edit base
Because upper, after anchoring, Cas9 gene, which can play activity cutting, need to edit the gene that gene to need editor in new gold mycobacteria
It is knocked, the knockout meeting of gene need to be edited so that new gold mycobacteria gene generates DSB fracture, at this point, pML-Cas9 plasmid
On expression DNA terminal binding protein mku and DNA ligase LigD can repair DSB fracture, complete new gold mycobacteria
The editing process of gene.
The present invention also provides a kind of plasmid, the plasmid is opened comprising pMV261 expression vector, Cas9 gene, Pj23119
Mover and NHEJ revision points, are named as pML-Cas9 plasmid；Hsp60 promoter on the pMV261 expression vector is replaced
It has been changed to Tac promoter；The NHEJ revision points include that the gene of coding DNA terminal binding protein mku and coding DNA connect
Connect the gene of enzyme LigD；The Cas9 gene is located at downstream and the pMV261 expression of the Tac promoter of pMV261 expression vector
The rrnB terminator upstream of carrier is driven by Tac promoter and is expressed；The Pj23119 promoter is located at pMV261 expression vector
KanR resistant gene downstream；The NHEJ revision points are located at the downstream of Pj23119 promoter and pMV261 expression carries
The upstream of the replication orgin (ori) of body is driven by Pj23119 promoter and is expressed.
In one embodiment of the invention, the nucleotide sequence of the Cas9 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, the plasmid successively includes OriM replicon, pMB1 replicon, Pj23119
Promoter, sgRNA sequence, rrnB terminator and aadA resistant gene, are named as pJM-sgRNA plasmid；The sgRNA sequence
It is targeting sequence, it can selectively targeted target sequence；The target sequence refers to the nucleotide sequence that need to edit gene.
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.
Described in a kind of any described CRISPR/Cas9 gene editing system of claim 1-5 or claim 6 or 7
A kind of gene editing method can be used for new gold mycobacteria or a kind of plasmid according to any one of claims 8 or claim 9 institute
A kind of application of the plasmid stated in terms of editing new gold mycobacteria gene
The present invention also provides a kind of above-mentioned CRISPR/Cas9 gene editing systems or above-mentioned one kind can be used for new gold point
The gene editing method of branch bacillus or a kind of above-mentioned pML-Cas9 plasmid or a kind of above-mentioned pJM-sgRNA plasmid are new golden in editor
Application in terms of Mycobacterium tuberculosis genes.
[beneficial effect]
(1) the CRISPR/Cas9 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-Cas9 plasmid first being imported new gold point
In branch bacillus, so that the gene and coding DNA of Cas9 gene, coding DNA terminal binding protein mku on pML-Cas9 plasmid
Then the gene expression of ligase LigD imports pJM-sgRNA plasmid in new gold mycobacteria, so that pJM-sgRNA matter
Grain transcription generates sgRNA sequence, and Cas9 handle and Terminator in sgRNA sequence will form loop-stem structure, Cas9 base
Because that can identify this loop-stem structure to form compound in conjunction with sgRNA sequence, this compound, which can target, need to edit gene and be anchored
It need to edit on gene, after anchoring, Cas9 gene can play activity cutting and need to edit gene and to need in new gold mycobacteria
The gene of editor is knocked, and need to edit the knockout meeting of gene so that new gold mycobacteria gene generates DSB fracture, at this point,
The DNA terminal binding protein mku and DNA ligase LigD of expression on pML-Cas9 plasmid can repair DSB fracture, complete new
The editing process of gold mycobacteria gene；
(2) the pML-Cas9 plasmid and pJM-sgRNA plasmid that CRISPR/Cas9 gene editing system of the invention uses are just
It can be stabilized, express in new gold mycobacteria, will not be lost with the passage of new gold mycobacteria, it will not be by
Protection mechanism present in new gold mycobacteria itself is repelled；
(3) gene volume is carried out to new gold mycobacteria using CRISPR/Cas9 gene editing service system of the invention
Volume, knockout efficiency is higher, up to 50%.
Detailed description of the invention
Fig. 1: pML-Cas9 plasmid map.
Fig. 2: pJM-sgRNA plasmid map.
Specific embodiment
The present invention is further limited combined with specific embodiments below.
PMD18T carrier, pMV261 expression vector involved in following embodiments, ptrc99A expression vector are purchased from excellent precious raw
Object；ClonExpress II One Step Cloning Kit involved in following embodiments only praises biological section purchased from Nanjing promise
Skill Co., Ltd；New gold mycobacteria (Mycobacterium neoaurum) involved in following embodiments is raw purchased from Bei Na
Object, resource number are 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.
Fermentation medium: phytosterol 1g/L, glucose 20g/L, tryptone 10g/L, yeast powder 6g/L, NaCl
10g/L、K2HPO43g/L、MgSO40.3g/L、MnCl25×10-4G/L, beta-cyclodextrin 3g/L, pH 7.5.
Competence culture medium: glucose 20g/L, tryptone 10g/L, yeast powder 6g/L, NaCl 10g/L, Tween-80
0.2% (V/V), pH 7.5.
Detection method involved in following embodiments is as follows:
3- sterone-Δ 1- dehydrogenase enzyme activity detection method:
Most suitable KSDD measurement system (3mL) includes: crude enzyme liquid (100 μ L), the Tris-HC1 (pH 7.0) of final concentration 50mM,
The DCPIP that 40 μM of final concentration, the PMS of final concentration 1.5mM, 500 μM of steroidal substrate 4-ADs (AD) of final concentration
(being dissolved in 100% isopropanol)；2h, light absorption value variation of the detected reaction solution at 600nm are reacted at 30 DEG C；
3- sterone-Δ 1- dehydrogenase enzyme activity is defined as: enzyme amount needed for restoring 1 μm of ol DCPIP in 1min defines
For an enzyme-activity unit, unit U.
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,
10mL10% glycerol 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/Cas9 gene editing system
Specific step is as follows:
1, the building of CRISPR/Cas9 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 Hind III to pMV261 expression vector digestion
Linearisation obtains basic framework；
(2) Cas9 gene (nucleotide sequence is as shown in SEQ ID NO:1), i.e. Frag1 are obtained by chemical synthesis；
(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
P1 primer, P2 primer and P3 primer, P4 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 P1 and P4 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 P5 primer and P6 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-Cas9 plasmid (plasmid map is shown in Fig. 1), wherein Cas9 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 P9 primer and P10 primer cloned to obtain OriM
Replicon, i.e. Frag3；
(6) using ptrc99A carrier as template, using in table 1 P7 primer and P8 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 sgRNA sequence；This sgRNA sequence is synthesized by Jin Weizhi biotechnology company and is connected to pMD18T load
On body, recombination pMD18T-sgRNA carrier is obtained；Using obtained recombination pMD18T-sgRNA carrier as template, using in table 1
P11 primer and P12 primer are cloned, and are obtained Pj23119-sgRNA-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-sgRNA plasmid
(plasmid map is shown in Fig. 2), wherein pJM-sgRNA plasmid successively includes OriM replicon, pMB1 replicon, Pj23119 starting
Son, sgRNA sequence, rrnB terminator and aadA resistant gene.
2, the use of CRISPR/Cas9 gene editing system
CRISPR/Cas9 gene editing system contains pML-Cas9 plasmid and pJM-sgRNA plasmid simultaneously, this
CRISPR/Cas9 gene editing system when in use, needs first to import pML-Cas9 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-sgRNA plasmid is imported into this turn
In beggar.
1 primer of table and its sequence
2 pcr amplification reaction system of table
| ddH20 | up to 50μL |
| 2×Phanta Max Master Mix | 25μL |
| Upstream primer | 2μL |
| Downstream primer | 2μL |
| Template DNA | 1μL |
3 Gibson assembly and connection reaction system of table
| Linearized vector | 90ng |
| Insert Fragment | 180ng |
| 5×CE II Buffer | 4μL |
| Exnase II | 2μL |
| ddH2O | add to 20μL |
Embodiment 2:CRISPR/Cas9 gene editing systematic difference
Specific step is as follows:
(1) building of CRISPR/Cas9 gene editing system
Using the ksdd 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 sgRNA (nucleotide sequence such as SEQ ID NO:24 institute is designed for according to target sequence
Show)；According to embodiment 1, construct to obtain CRISPR/Cas9 gene editing system according to the sgRNA that design obtains；
(2) gene editing of new gold mycobacteria JC-12
Using the new gold mycobacteria JC-12 of unused CRISPR/Cas9 gene editing system editor as blank control,
PML-Cas9 plasmid in CRISPR/Cas9 gene editing system that (1) obtains is transformed into new gold mycobacteria JC-12 sense
By in state cell, being coated on inclined-plane/solid medium (containing 50 μ g/mL kanamycins), 30 DEG C are cultivated 3~4 days；Picking conversion
Son carries out PCR preliminary identification with the P13 primer of table 4 and P14 primer, and the transformant that picking is proved to be successful does competence, is preparing
0.5mM IPTG is added while competence to induce in 30 DEG C, the CRISPR/Cas9 gene editing system for the scheme one that (1) is obtained
PJM-sgRNA plasmid in system is transformed into transformant competent cell, is coated on inclined-plane/solid medium (containing 50 μ g/mL cards
That mycin and 50 μ g/mL streptomysins) on, 30 DEG C are cultivated 3~4 days；Picking transformant, with the P13 primer of table 4 and P14 primer into
Row PCR preliminary identification, the transformant sequence verification that picking is proved to be successful.
Detect the base edited using CRISPR/Cas9 gene editing system to new gold mycobacteria JC-12 gene
Because of editorial efficiency, testing result are as follows: the knockout efficiency of ksdd can reach 50%.
It the new gold mycobacteria JC-12 of picking blank control group and is struck using CRISPR/Cas9 gene editing system
Except the single colonie of the new gold mycobacteria JC-12/ksdd after ksdd gene, it is inoculated in seed culture medium and is trained in 30 DEG C respectively
48h is supported, seed liquor is obtained；Seed liquor is seeded in fermentation medium according to 10% inoculum concentration in 30 DEG C, 160rpm culture
168h obtains fermentation liquid；By fermentation liquid, thalline were collected by centrifugation；Thallus is crushed by cell crushing instrument, obtains crude enzyme liquid；It will be thick
Enzyme solution centrifuging and taking precipitating, obtains 3- sterone-Δ 1- dehydrogenase, detects the new gold mycobacteria JC-12 fermentation of blank control group
Obtained crude enzyme liquid and use CRISPR/Cas9 gene editing system knocks out the new gold mycobacteria JC- after ksdd gene
The enzyme activity for the crude enzyme liquid that 12/ksdd ferments, testing result are as follows: the CRISPR/Cas9 gene editing system used knocks out
The 3- sterone for the crude enzyme liquid that new gold mycobacteria JC-12/ksdd after ksdd gene ferments-Δ 1- dehydrogenase enzyme activity compared with
The 3- sterone for the crude enzyme liquid that the new gold mycobacteria JC-12 of blank control group ferments-Δ 1- dehydrogenase enzyme activity has dropped
80%.
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 CRISPR/Cas9 gene editing system and its application
<160> 24
<170> PatentIn version 3.3
<210> 1
<211> 4107
<212> DNA
<213>artificial sequence
<400> 1
atggataaaa agtattccat tggcctggac atcggcacca attctgtggg ttgggcagtc 60
atcaccgacg aatacaaggt cccatccaag aagttcaagg tgctcggtaa taccgatcgc 120
cactctatca agaaaaacct gatcggcgcc ctgctcttcg actccggcga aaccgcagaa 180
gcaacccgtc tcaagcgtac cgcacgtcgc cgctacaccc gccgtaagaa tcgcatctgc 240
tacctccagg aaatcttctc taatgagatg gcaaaggtgg atgactcctt tttccaccgc 300
ctcgaagagt ccttcctggt ggaagaggac aagaaacacg agcgccatcc tatcttcggc 360
aatattgtcg atgaagtcgc atatcatgaa aaatacccaa ccatttacca tctccgtaaa 420
aaactcgtcg attccaccga taaggcagat ctccgcctga tctatctggc actggcccac 480
atgatcaagt ttcgtggcca cttcctgatc gaaggtgacc tcaatccaga caattccgac 540
gtggacaagc tgttcatcca gctggtgcaa acctacaacc agctctttga ggaaaaccca 600
atcaacgcat ctggcgtcga cgcaaaagcc atcctgtccg cccgtctctc caagtctcgt 660
cgcctcgaaa acctcattgc ccagctccct ggcgagaaga aaaacggtct gttcggcaat 720
ctgatcgccc tgtctctggg tctgacccca aatttcaaat ccaactttga tctcgcagaa 780
gatgccaagc tgcagctctc taaggacacc tacgatgatg acctggataa cctcctcgcc 840
cagatcggcg accagtacgc cgatctcttc ctcgcagcca agaacctctc tgacgcaatt 900
ctgctgtccg acatcctgcg cgtgaacacc gaaatcacta aggcaccact ctctgcctcc 960
atgattaagc gctacgacga gcatcatcag gatctcactc tcctcaaagc cctggtccgc 1020
cagcagctcc cagagaagta caaggaaatc tttttcgacc aatccaaaaa cggctacgca 1080
ggttacatcg atggcggcgc ctctcaggaa gagttttaca aattcattaa gccaatcctc 1140
gaaaagatgg acggcaccga ggaactgctg gtgaaactca accgtgaaga tctcctgcgc 1200
aaacagcgca ccttcgacaa cggttccatt cctcaccaga tccacctggg cgaactgcac 1260
gcaatcctcc gccgtcaaga ggacttctac ccattcctga aggacaaccg tgaaaagatc 1320
gaaaagattc tcaccttccg catcccttac tacgtgggtc ctctcgcccg tggcaattcc 1380
cgcttcgcct ggatgactcg caaatctgaa gagaccatca ccccttggaa ctttgaggaa 1440
gtcgtcgata aaggcgcctc tgcccaatcc ttcatcgagc gcatgaccaa cttcgacaag 1500
aacctcccaa acgagaaagt gctgcctaag cattccctcc tgtacgagta ctttactgtc 1560
tacaatgagc tgactaaggt gaagtatgtc actgaaggca tgcgtaagcc agcctttctc 1620
tccggcgagc agaaaaaggc aatcgtcgac ctcctgttta aaaccaaccg caaagtgact 1680
gtgaaacagc tcaaggaaga ttactttaaa aaaatcgaat gcttcgattc tgtggaaatc 1740
tccggcgtgg aggatcgttt caacgcctcc ctgggtacct atcacgacct cctgaagatt 1800
atcaaggaca aagattttct ggataacgag gaaaacgagg acattctcga ggacattgtg 1860
ctgaccctga ccctcttcga ggaccgcgag atgatcgagg agcgcctgaa gacctatgcc 1920
cacctctttg acgacaaggt catgaagcaa ctcaagcgcc gccgctatac cggttggggc 1980
cgtctctccc gtaagctcat caatggtatc cgcgacaagc aatccggcaa gactatcctg 2040
gactttctga agtctgacgg cttcgccaac cgcaatttta tgcaactgat ccacgacgat 2100
tccctgacct tcaaagagga catccagaaa gcccaagtgt ccggtcaagg cgactccctg 2160
cacgaacaca tcgccaatct ggcaggttcc ccagcaatca agaagggcat cctgcagacc 2220
gtcaaggtgg tggacgaact cgtcaaagtg atgggtcgcc acaaaccaga aaacatcgtc 2280
atcgagatgg cccgtgagaa ccagaccacc cagaaaggcc agaaaaactc ccgtgagcgc 2340
atgaagcgca ttgaagaagg cattaaagag ctcggctctc agatcctgaa agagcatcct 2400
gtcgagaaca cccaactgca gaatgagaag ctgtatctgt attatctcca gaacggccgc 2460
gacatgtacg tcgaccagga actggacatc aaccgtctct ctgattacga tgtggaccat 2520
atcgtccctc agtctttcct gaaagacgac tctattgaca acaaagtcct cacccgctcc 2580
gacaagaacc gcggcaagtc cgataacgtg ccatccgagg aggtcgtgaa gaagatgaag 2640
aactactggc gccagctgct caacgccaag ctgatcactc agcgcaagtt cgataacctg 2700
accaaggccg aacgtggtgg tctctccgag ctcgacaagg caggctttat caagcgccaa 2760
ctcgtggaga ctcgccaaat cactaaacac gtcgcccaga tcctcgactc ccgcatgaat 2820
accaagtacg atgaaaatga caagctcatc cgcgaagtga aagtcattac cctgaagtcc 2880
aaactggtct ctgactttcg caaggatttc cagttctaca aggtccgcga gattaataac 2940
tatcatcatg cacacgatgc atacctcaac gcagtcgtgg gtaccgcact gatcaagaag 3000
taccctaaac tggagtccga gttcgtctat ggcgactaca aggtgtacga cgtccgcaaa 3060
atgattgcca agtccgagca ggagatcggc aaagcaactg ccaaatattt cttttactcc 3120
aacatcatga acttcttcaa gaccgaaatc accctcgcca acggtgaaat ccgcaaacgt 3180
ccactcatcg agactaatgg tgaaaccggc gagatcgtct gggacaaggg ccgtgacttc 3240
gcaaccgtcc gcaaggtcct ctccatgcca caggtcaaca tcgtgaaaaa gaccgaggtg 3300
caaaccggcg gcttctccaa ggagtctatc ctgcctaaac gcaattccga taagctcatt 3360
gcacgcaaaa aggactggga ccctaaaaaa tacggcggtt tcgactcccc aactgtcgca 3420
tattctgtgc tcgtggtcgc caaagtggaa aagggcaaat ccaaaaagct caagtccgtc 3480
aaggaactcc tgggtatcac catcatggaa cgctcctcct ttgagaagaa ccctatcgat 3540
ttcctcgaag caaaaggtta caaggaggtg aagaaagatc tgatcatcaa gctccctaaa 3600
tactccctct tcgagctcga gaacggccgc aagcgtatgc tggcctccgc aggtgaactg 3660
caaaaaggta acgagctggc actcccatcc aagtatgtca actttctcta cctggcctcc 3720
cactacgaaa agctgaaagg ttccccagaa gacaacgagc agaaacagct gttcgtggag 3780
cagcacaagc actacctgga cgaaatcatc gagcagatct ccgagttctc taaacgcgtc 3840
attctggccg atgccaacct cgataaagtg ctctccgcct acaataagca tcgtgataag 3900
ccaatccgtg agcaggcaga gaacatcatt cacctgttca ctctcaccaa cctgggtgca 3960
ccagccgcct ttaagtactt cgacaccacc atcgaccgca agcgctatac ctccactaag 4020
gaggtgctcg atgcaaccct gatccaccag tctatcaccg gcctctacga gactcgcatc 4080
gatctctccc agctgggtgg cgactaa 4107
<210> 2
<211> 35
<212> DNA
<213>artificial sequence
<400> 2
ttgacagcta gctcagtcct aggtataatg ctagc 35
<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> 165
<212> DNA
<213>artificial sequence
<400> 5
aattcgtgtc gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata 60
acggttctgg caaatattct gaaatgagct gttgacaatt aatcatcggc tcgtataatg 120
tgtggaattg tgagcggata acaatttcac acaggaaaca gaatt 165
<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> 792
<212> DNA
<213>artificial sequence
<400> 8
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> 9
<211> 45
<212> DNA
<213>artificial sequence
<400> 9
caaataaaac gaaaggctca gtcgaaagac tgggcctttc gtttt 45
<210> 10
<211> 29
<212> DNA
<213>artificial sequence
<400> 10
atgggttcgg cgtcggagca acgggtgac 29
<210> 11
<211> 51
<212> DNA
<213>artificial sequence
<400> 11
ctagtattct cctctttaat ctctagtatc attcgcgcac cacctcactg g 51
<210> 12
<211> 51
<212> DNA
<213>artificial sequence
<400> 12
attaaagagg agaatactag atgcgagcca tttggacggg ttcgatcgcc t 51
<210> 13
<211> 29
<212> DNA
<213>artificial sequence
<400> 13
tcacggaggc gttgggacgt ttgagttgg 29
<210> 14
<211> 87
<212> DNA
<213>artificial sequence
<400> 14
tctagattga cagctagctc agtcctaggt ataatgctag ctactagaga aagaggagaa 60
atactagatg ggttcggcgt cggagca 87
<210> 15
<211> 48
<212> DNA
<213>artificial sequence
<400> 15
ttcgaattct gcagcttcac ggaggcgttg ggacgtttga gttggcct 48
<210> 16
<211> 30
<212> DNA
<213>artificial sequence
<400> 16
atgagggaag cggtgatcgc cgaagtatcg 30
<210> 17
<211> 28
<212> DNA
<213>artificial sequence
<400> 17
tttccatagg ctccgccccc ctgacgag 28
<210> 18
<211> 28
<212> DNA
<213>artificial sequence
<400> 18
gtgagcccac cagctccgta agttcggg 28
<210> 19
<211> 24
<212> DNA
<213>artificial sequence
<400> 19
gccacggatg ccaccacagg cact 24
<210> 20
<211> 55
<212> DNA
<213>artificial sequence
<400> 20
gcctgcaggt cgactctaga ggatccttga cagctagctc agtcctaggt ataat 55
<210> 21
<211> 42
<212> DNA
<213>artificial sequence
<400> 21
catgattacg aattcaaaaa aagcaccgac tcggtgccac tt 42
<210> 22
<211> 22
<212> DNA
<213>artificial sequence
<400> 22
cggctcattg agcaaccgat ac 22
<210> 23
<211> 19
<212> DNA
<213>artificial sequence
<400> 23
cgcccgacac agcctttca 19
<210> 24
<211> 102
<212> DNA
<213>artificial sequence
<400> 24
ctcgacagta gtcgttgaga gttttagagc tagaaatagc aagttaaaat aaggctagtc 60
cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tt 102
Claims (10)
1. a kind of CRISPR/Cas9 gene editing system, which is characterized in that the CRISPR/Cas9 gene editing system includes
PML-Cas9 plasmid and pJM-sgRNA plasmid；
The pML-Cas9 plasmid includes that pMV261 expression vector, Cas9 gene, Pj23119 promoter and NHEJ repair base
Cause；Hsp60 promoter on the pMV261 expression vector is replaced for Tac promoter；The NHEJ revision points include
The gene of coding DNA terminal binding protein mku and the gene of coding DNA ligase LigD；The Cas9 gene is located at
The downstream of the Tac promoter of pMV261 expression vector and the rrnB terminator upstream of pMV261 expression vector, by Tac promoter
Driving expression；The Pj23119 promoter is located at the downstream of the KanR resistant gene of pMV261 expression vector；The NHEJ is repaired
Gene is located at the upstream in the downstream of Pj23119 promoter and the replication orgin (ori) of pMV261 expression vector, by Pj23119
Promoter driving expression；
The pJM-sgRNA plasmid successively include OriM replicon, pMB1 replicon, Pj23119 promoter, sgRNA sequence,
RrnB terminator and aadA resistant gene, are named as pJM-sgRNA plasmid；The sgRNA sequence is targeting sequence, can be special
Property targeting target sequence；The target sequence refers to the nucleotide sequence that need to edit gene.
2. a kind of CRISPR/Cas9 gene editing system as described in claim 1, which is characterized in that the Cas9 gene
Nucleotide sequence is as shown in SEQ ID NO:1.
3. a kind of CRISPR/Cas9 gene editing system as claimed in claim 1 or 2, which is characterized in that encode the pML-
The nucleotide sequence of the gene of Pj23119 promoter in Cas9 plasmid and pJM-sgRNA plasmid such as SEQ ID NO:2 institute
Show.
4. a kind of CRISPR/Cas9 gene editing system a method according to any one of claims 1-3, which is characterized in that described in coding
The nucleotide sequence of the gene of the end DNA collection hop protein mku is as shown in SEQ ID NO:3.
5. a kind of CRISPR/Cas9 gene editing system as described in claim 1-4 is any, which is characterized in that described in coding
The nucleotide sequence of the gene of DNA ligase LigD is as shown in SEQ ID NO:4.
6. a kind of gene editing method that can be used for new gold mycobacteria, which is characterized in that the method is to be wanted using right
Seek a kind of any CRISPR/Cas9 gene editing system of 1-5.
7. a kind of gene editing method that can be used for new gold mycobacteria as claimed in claim 6, which is characterized in that described
Method is first to lead the pML-Cas9 plasmid in a kind of any CRISPR/Cas9 gene editing system of claim 1-5
Enter in new gold mycobacteria, so that the gene of Cas9 gene, coding DNA terminal binding protein mku on pML-Cas9 plasmid
And the gene expression of coding DNA ligase LigD, then by a kind of any CRISPR/Cas9 base of claim 1-5
Because the pJM-sgRNA plasmid in editing system imports in new gold mycobacteria, so that pJM-sgRNA plasmid transcription generates
SgRNA sequence, Cas9handle and Terminator in sgRNA sequence will form loop-stem structure, and Cas9 gene can identify this
For loop-stem structure to form compound in conjunction with sgRNA sequence, this compound, which can target edit gene and be anchored on, need to edit base
Because upper, after anchoring, Cas9 gene, which can play activity cutting, need to edit the gene that gene to need editor in new gold mycobacteria
It is knocked, the knockout meeting of gene need to be edited so that new gold mycobacteria gene generates DSB fracture, at this point, pML-Cas9 plasmid
On expression DNA terminal binding protein mku and DNA ligase LigD can repair DSB fracture, complete new gold mycobacteria
The editing process of gene.
8. a kind of plasmid, which is characterized in that the plasmid include pMV261 expression vector, Cas9 gene, Pj23119 promoter with
And NHEJ revision points, it is named as pML-Cas9 plasmid；Hsp60 promoter on the pMV261 expression vector be replaced in order to
Tac promoter；The NHEJ revision points include the gene and coding DNA ligase of coding DNA terminal binding protein mku
The gene of LigD；The Cas9 gene is located at downstream and the pMV261 expression vector of the Tac promoter of pMV261 expression vector
RrnB terminator upstream, by Tac promoter drive express；The Pj23119 promoter is located at pMV261 expression vector
The downstream of KanR resistant gene；The NHEJ revision points are located at downstream and the pMV261 expression vector of Pj23119 promoter
Replication orgin (ori) upstream, by Pj23119 promoter drive express.
9. a kind of plasmid, which is characterized in that the plasmid successively includes OriM replicon, pMB1 replicon, Pj23119 starting
Son, sgRNA sequence, rrnB terminator and aadA resistant gene, are named as pJM-sgRNA plasmid；The sgRNA sequence is target
It, can selectively targeted target sequence to sequence；The target sequence refers to the nucleotide sequence that need to edit gene.
10. one described in a kind of any described CRISPR/Cas9 gene editing system of claim 1-5 or claim 6 or 7
Kind can be used for described in the gene editing method or a kind of plasmid according to any one of claims 8 or claim 9 of new gold mycobacteria
A kind of application of plasmid in terms of editing new gold mycobacteria gene.
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| CN114277047A (en) * | 2021-12-28 | 2022-04-05 | 苏州金唯智生物科技有限公司 | Application of high-throughput screening tool for enabling escherichia coli to obtain effective NHEJ system in escherichia coli gene editing |
| CN114277047B (en) * | 2021-12-28 | 2023-10-03 | 苏州金唯智生物科技有限公司 | Application of high-throughput screening tool for obtaining effective NHEJ system from escherichia coli in escherichia coli gene editing |
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