CN112779288A - Gene knockout and kit - Google Patents
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- CN112779288A CN112779288A CN202110111949.9A CN202110111949A CN112779288A CN 112779288 A CN112779288 A CN 112779288A CN 202110111949 A CN202110111949 A CN 202110111949A CN 112779288 A CN112779288 A CN 112779288A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knockout animals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
Abstract
Gene knockout and a kit. Selecting at least two target sequences of a coding region of a gene to be knocked out, and enabling the target sequences to respectively comprise complete target codons CAA, CAG or CGA; utilizing at least two sgRNA sequences to position BE3 to a corresponding target sequence so that a target single base C in a target codon becomes T, and correspondingly introducing a stop codon TAA or TAG, TGA to realize gene knockout; the at least two sgRNA sequences are sequences that correspond complementarily to the at least two target sequences, respectively, wherein the at least two sgRNAs are co-injected to achieve effective knock-out of the same gene. The present invention provides a more efficient, highly accurate, and low off-target gene knockout method.
Description
The application is a divisional application based on the invention with the application number of ' 201811113109.0 ', the application date of ' 2018, 9, 25 and the name of ' realizing effective knockout of the same gene by co-injection of multiple sgrnas '.
Technical Field
The invention relates to improvement of a gene knockout method strategy based on base editing and application thereof.
Background
Gene editing techniques refer to the process of "editing" a gene of interest to introduce a desired change to a target site of genomic DNA. Early gene editing was achieved by the discovery of endogenous homologous recombination. However, the traditional eukaryotic targeted gene operation has the defects of low gene targeting efficiency, limited application range and the like. The discovery and application of a type II CRISPR/Cas9(Clustered regulated Short Palindromic repeat-associated Cas9 endonuse) system breaks the original limitation, and the system is proved to be a multipurpose gene editing tool [ Le et al, 2013; patrick et al, 2014 ].
The CRISPR/Cas9 system-mediated specific gene knockout (knock-out) is realized by utilizing sgRNA (single-stranded RNA) to guide the positioning and shearing of double-stranded DNA of Cas9 protein through target sequence complementation, non-homologous end joining (NHEJ) repair is carried out, and frame shift mutation (frameshift mutation) is caused to cause gene knockout, and the method mainly has the following defects in practical application: first, the non-homologous end joining mechanism easily generates random insertions and deletions (indels), so that new bases may be randomly introduced near the breakpoint, resulting in inaccurate gene editing. Second, CRISPR/Cas 9-mediated gene editing has always some off-target effects [ Gorski et al, 2017 ].
Recent research shows that the fusion protein of Cas9 and deaminase constructed based on CRISPR/Cas9 technology can BE used as a Base Editor (BE). The first base editing (BE1) was performed by fusing the rat cytidine deaminase ApOBEC1 to dCas9, which changes the C/G base pair to A/T by deamination. Since various modifications of the BE system were made to improve its editing efficiency, BE3, which is currently widely used, can introduce C-T nucleotide substitutions in a window of 4-8 bases at the position of the non-binding strand of the sgRNA [ Komor et al, 2016 ]. This provides a new idea for gene knockout by introducing a stop codon through CT mutation, for example, CAA, CAG, CGA into a stop codon TAA, TAG, TGA, or TGG into a stop codon TAA, TGA, TAG through GA mutation, and terminating translation of the encoded gene, thereby realizing gene knockout. It provides a safer and more precise knockout strategy than Cas 9-mediated NHEJ [ Komor et al, 2017; kim et al, 2017 ]. However, there is no example of obtaining an effective gene knock-out by this method at present due to the limitation by the editing efficiency of BE3 or the like.
Disclosure of Invention
One of the purposes of the invention is to provide a high-efficiency and accurate gene knockout strategy.
According to a first aspect of the present invention, there is provided a gene knockout method comprising:
selecting at least two 19-21 bp-NGG target sequences of a coding region of a gene to be knocked out, and enabling the target sequences to respectively comprise complete target codons CAA, CAG or CGA, wherein a target single base C is located at the 4 th-8 th position of the target sequence, and the target codons are separated from NGG by 12-14 bp;
utilizing at least two sgRNA sequences to position BE3 to a corresponding target sequence so that a target single base C in a target codon becomes T, and correspondingly introducing a stop codon TAA or TAG, TGA to realize gene knockout;
the at least two sgRNA sequences are 19-21 bp sequences which are respectively complementary and correspond to the at least two target sequences,
wherein the at least two sgrnas are co-injected to achieve effective knock-out of the same gene.
In the alternative scheme, a CCN-19-21 bp target sequence of a coding region of a gene to be knocked out can be selected to contain a complete target codon TGG, a target single base G is preferably positioned at the 4 th-8 th position (right end) of the target sequence, and the target codon is separated from the NGG by 12-14 bp.
According to the invention, BE3 can BE rAPOBEC-SpCas 9-NLS-UGI-NLS.
The method according to the invention can be used to knock out two target genes: mouse Pcdc1 and Tyr.
According to a second aspect of the invention, there is provided the use of the above method for knock-out of the mouse Pcdc1 and Tyr genes in the cell line N2 a.
According to a third aspect of the present invention, there is provided the use of sgrnas obtained according to the above-described use, which are effective in introducing a stop codon to achieve gene knockout, for murine Pcdc1 and Tyr gene knockout in mouse embryonic cells.
According to a fourth aspect of the present invention, there is provided a kit for gene knock-out, including the sgRNA described above, BE3, and an amplification reagent.
The invention utilizes a base editing technology developed on the basis of CRISPR/Cas9, creates a stop codon through accurate CT or GA single base mutation, and improves gene knockout efficiency by combining with co-injection of a plurality of sgRNAs, thereby establishing a gene knockout strategy with high efficiency, accuracy and low off-target effect.
Drawings
FIG. 1 is a schematic diagram showing that the objective gene is knocked out by using CT mutation according to the present invention (bold underline indicates PAM; italics indicates a mutant codon; italics bold underline indicates a mutant base);
FIG. 2 is a schematic structural view of BE 3;
FIG. 3 is a graph showing the efficiency of gene knockout on mouse embryos;
FIG. 4 is a graph showing the efficiency of gene knockout in mice.
Detailed Description
First, sgRNA was designed. Base site-directed editing is the use of sgrnas to target the location of BE3 to the target site, and the selection and design of the sgrnas specific to the target gene is the key point of the present invention. The sgRNA is selected and designed as follows:
selecting a 19-21 bp-NGG target sequence of a coding region of a gene to be knocked out to enable the target sequence to comprise complete target codons CAA, CAG or CGA, wherein a target single base C is positioned at the 4 th-8 th position of the target sequence, and the target codon is separated from NGG by 12-14 bp;
utilizing the sgRNA sequence to position BE3 to a target sequence so that a target single base C in a target codon becomes T to correspondingly introduce a stop codon TAA or TAG, TGA;
in the alternative scheme, a CCN-19-21 bp target sequence of a coding region of a gene to be knocked out is selected to contain a complete target codon TGG, a target single base G is preferably positioned at the 4 th-8 th position (right end) of the target sequence, and the target codon is preferably spaced from the CCN by 12-14 bp.
Aiming at two target genes, namely mouse Pcdc1 and Tyr, the invention selects the following target gene sequences to design corresponding sgRNA (bold underline indicates PAM; italic underline indicates candidate mutant codon):
1.Pcdc1
Sg-1:aaaacaggccgccttctgtaatgg(SEQ ID NO.1)
Sg-2:cggtttcaaggcatggtcattgg(SEQ ID NO.2)
Sg-3:cctggtcattcacttaagctgtg(SEQ ID NO.3)
2.Tyr
Sg-1:tgcggccagctttcaggcagagg(SEQ ID NO.4)
Sg-2:ccttcttctcctcctggcaggta(SEQ ID NO.5)
Sg-3:ccagggtttctgccttggcacag(SEQ ID NO.6)
aiming at the selected target gene sequences, mice Pcdc1(3 pieces) and Tyr (3 pieces), corresponding sgRNA expression vectors are constructed, and different sgRNAs are respectively introduced into pGL 3-U6-EGFP-sgRNA.
Example 1
BE3 mediated base editing is carried out on a cell line, and a stop codon is introduced to realize gene knockout. Knockout of the cell line (by electroporation or lipofection) is carried out by conventional procedures, and lipofection is exemplified.
(1) Taking N2a cells as an example, the invention carries out the culture and transfection of eukaryotic cells: n2a cells were seeded and cultured in DMEM high-glucose medium (HyClone, SH30022.01B) supplemented with 10% FBS, which contained penicillin (100U/ml) and streptomycin (100. mu.g/ml).
(2) The cells were divided into 6-well plates before transfection, and transfection was performed when the density reached 70% -80%.
(3) Transfection is exemplified by lipofection. According to LipofectamineTMThe operating manual of 2000 Transfection Reagent (Invitrogen,11668-019) for example SpCas9 nickase, 2. mu.g of BE3 plasmid was mixed with 1. mu.g of pGL3-U6-EGFP-sgRNA plasmid, and the mixture was co-transfected into cells in each well at 6-8 small sizesThe liquid is changed after the time, and the cells are collected by flow sorting after 72 hours.
(4) Genotyping analysis
A. A portion of the cells was lysed in a lysis buffer (10. mu.M Tris-HCl,0.4M NaCl, 2. mu.M EDTA, 1% SDS) with 100. mu.g/ml proteinase K, extracted with phenol-chloroform and dissolved in 50. mu.l of deionized water.
B. Performing PCR amplification by using a pair of primers N-For and N-Rev, and purifying by using an AxyPrep PCR clean kit (AXYGEN, AP-PCR-250G) to obtain a PCR recovery product, wherein the PCR reaction system is as follows:
300-400ng genomic DNA
25μl 2X Buffer
1μl dNTP
2μl N-For(10μm)
2μl N-Rev(10μm)
1μl DNA Polymerase(Vazyme,P505-d3)
Water was added to the reaction system to 50. mu.l.
C. The PCR product was recovered and subjected to A-addition reaction using rTaq. The reaction system of adding A is as follows:
700-800ng PCR recovery product
5μl 10 X Buffer(Mg2+PLUS)
4μl dNTP
0.5μl rTaq(TAKARA,R001 AM)
Water was added to the reaction system to 50. mu.l.
After 30 min incubation at 72 ℃, 2. mu.l of the product was ligated to pMD19-T vector (TAKARA,3271) in the ligation reaction:
2. mu.l of product A
0.5μl pMD19-T vector
2.5μl Solution Ι(TAKARA,6022)
After 30 min incubation at 16 ℃ 2. mu.l of the product were taken to transform DH5 competent cells (TransGen, CD 201).
D. The single clone is picked, the universal primer M13-F is used for sequencing each target gene mutation, and the sequencing result is as follows (the bold underline represents PAM; the italic represents the mutant code molecule; the italic bold underline represents the mutant base):
1.Pcdc1
Sg-1:aaaacaggccgccttctgtaatgg
Mut:aaaataggccgccttctgtaatgg
Sg-2:cggtttcaaggcatggtcattgg
Mut:cggttttaaggcatggtcattgg
Sg-3:cctggtcattcacttaagctgtg
Mut:cctggtcattcacttgggctgtg
2.Tyr
Sg-1:tgcggccagctttcaggcagagg
Mut:tgcggctagctttcaggcagagg
Sg-2:ccttcttctcctcctggcaggta
Mut:ccttcttctcctcctaacaggta
Sg-3:ccagggtttctgccttggcacag
Mut:ccagggtttctgccttaacacag
the results show that: the target gene has sgRNA targeted base mutation, a stop codon is introduced, and Pcdc1 and Tyr genes are successfully knocked out.
Example 2
BE3 mediated base editing is carried out on a mouse embryo, and a stop codon is introduced to realize gene knockout.
(1) In vitro transcription: the BE3 plasmid was linearized with AgeI enzyme (NEB, R3552L) and transcribed in vitro with T7 ULTRA (Ambion, AM 1345). BE3 mRNA was purified using RNeasy Mini kit (QIAGEN, 74104). sgRNA nucleotides were annealed to pUC57sgRNA expression vector with T7 promoter (Addgene, 51132). The sgRNA was then amplified and transcribed with the megashortscript T7 kit (Ambion, AM 1345). sgRNA was purified using megaclean kit (Ambion, AM1908) and recovered by ethanol precipitation.
(2) Microinjection: superovulation treated female C57BL/6J mice were mated with C57BL/6J male mice and fertilized eggs were collected from the oviduct on day 0.5. Fertilized eggs were injected with BE3 mRNA (50 ng/. mu.l) and specific sgRNA (25 ng/. mu.l), respectively. The injection conditions were as follows:
1.Pcdc1
combination 1:BE3 mRNA+PD1-sg1
combination 2:BE3 mRNA+PD1-sg1+ 2
combination 3:BE3 mRNA+PD1-sg1+ 2+3
2.Tyr
combination 1:BE3 mRNA+Tyr-sg1
combination 2:BE3 mRNA+Tyr-sg1+ 2
(3) genotyping analysis
A. The injected fertilized eggs were cultured into blastocysts for genomic analysis: embryo genome DNA was extracted by rapid extraction (Luxige, QE09050), and 10. mu.L of the rapid extract was added to the embryo for digestion.
B. Performing PCR amplification by using a pair of primers N-For and N-Rev, and purifying by using an AxyPrep PCR clean kit (AXYGEN, AP-PCR-250G) to obtain a PCR recovery product, wherein the PCR reaction system is as follows:
300-400ng genomic DNA
25μl 2X Buffer
1μl dNTP
2μl N-For(10μm)
2μl N-Rev(10μm)
1μl DNA Polymerase(Vazyme,P505-d3)
Water was added to the reaction system to 50. mu.l.
C. The PCR product was recovered and subjected to A-addition reaction using rTaq. The reaction system of adding A is as follows:
700-800ng PCR recovery product
5μl 10 X Buffer(Mg2+PLUS)
4μl dNTP
0.5μl rTaq(TAKARA,R001 AM)
Water was added to the reaction system to 50. mu.l.
After 30 min incubation at 72 ℃, 2. mu.l of the product was ligated to pMD19-T vector (TAKARA,3271) in the ligation reaction:
2. mu.l of product A
0.5μl pMD19-T vector
2.5μl SolutionΙ(TAKARA,6022)
After 30 min incubation at 16 ℃ 2. mu.l of the product were taken to transform DH5 competent cells (TransGen, CD 201).
D. Monoclonals are picked, the mutation of each target gene is sequenced by using a universal primer M13-F, the number of embryos which are successfully knocked out, the number of embryos which are edited but not edited into i-stop and the number of embryos which are not edited are counted according to the sequencing result, and the result is shown in figure 3 (the black part represents the number of embryos which are successfully knocked out, the dark gray part represents the number of embryos which are edited but not edited into i-stop, and the light gray part represents the number of embryos which are not edited).
The results show that: the target gene has sgRNA targeted base mutation, a stop codon is introduced, and Pcdc1 and Tyr genes are successfully knocked out; in addition, co-injection of multiple sgrnas can improve gene knockout efficiency on embryos.
Example 3
BE 3-mediated knockout mice were constructed.
The embryo collection, microinjection, embryo culture, embryo transplantation and the like of the mice are carried out according to the conventional operation.
(1) Microinjection: fertilized eggs were injected with BE3 mRNA and specific sgRNA (Experimental 1: PD1-sg1+ 2+3, Tyr-sg1+2), or BE3 mRNA and specific sgRNA (Experimental 2: PD1-sg1+ 2+3, Tyr-sg1+ 2+3), respectively. Performing embryo transplantation conventionally;
(2) genotype analysis:
A. extracting genome DNA by conventional mouse tail shearing: after digestion with 100. mu.g/ml proteinase K in lysis buffer (10. mu.M Tris-HCl,0.4M NaCl, 2. mu.M EDTA, 1% SDS), the mixture was extracted with phenol-chloroform and dissolved in 50. mu.l of deionized water.
B. Performing PCR amplification by using a pair of primers N-For and N-Rev, and purifying by using an AxyPrep PCR clean kit (AXYGEN, AP-PCR-250G) to obtain a PCR recovery product, wherein the PCR reaction system is as follows:
300-400ng genomic DNA
25μl 2X Buffer
1μl dNTP
2μl N-For(10μm)
2μl N-Rev(10μm)
1μl DNA Polymerase(Vazyme,P505-d3)
Water was added to the reaction system to 50. mu.l.
C. The PCR product was recovered and subjected to A-addition reaction using rTaq. The reaction system of adding A is as follows:
700-800ng PCR recovery product
5μl 10 X Buffer(Mg2+PLUS)
4μl dNTP
0.5μl rTaq(TAKARA,R001 AM)
Water was added to the reaction system to 50. mu.l.
After 30 min incubation at 72 ℃, 2. mu.l of the product was ligated to pMD19-T vector (TAKARA,3271) in the ligation reaction:
2. mu.l of product A
0.5μl pMD19-T vector
2.5μl SolutionΙ(TAKARA,6022)
After 30 min incubation at 16 ℃ 2. mu.l of the product were taken to transform DH5 competent cells (TransGen, CD 201).
D. Single clones were picked, each target gene mutation was sequenced using the universal primer M13-F, and the results of sequencing were used to count mice with successful gene knockout, mice with editing but not editing as i-stop, and mice without editing, as shown in FIG. 4 (black parts indicate mice with successful gene knockout, dark gray parts indicate mice with editing but not editing as i-stop, and light gray parts indicate mice without editing).
The results confirm that sgRNA targeted base mutation occurs in the target gene, a stop codon is introduced, and the Pcdc1 and Tyr genes are successfully knocked out; in addition, co-injection of multiple sgrnas can improve gene knockout efficiency on embryos and achieve complete gene knockout on mice.
Sequence listing
<110> Zhaoqing Huaxia Kaiqi Biotech Co., Ltd
<120> gene knockout and kit
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> Artificial sequence ()
<400> 1
aaaacaggcc gccttctgta atgg 24
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence ()
<400> 2
cggtttcaag gcatggtcat tgg 23
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence ()
<400> 3
cctggtcatt cacttaagct gtg 23
<210> 4
<211> 23
<212> DNA
<213> Artificial sequence ()
<400> 4
tgcggccagc tttcaggcag agg 23
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence ()
<400> 5
ccttcttctc ctcctggcag gta 23
<210> 6
<211> 23
<212> DNA
<213> Artificial sequence ()
<400> 6
ccagggtttc tgccttggca cag 23
Claims (5)
1. A mouse Tyr gene knockout method, comprising:
selecting at least two 19-21 bp-NGG target sequences of a coding region of a gene to be knocked out, and enabling the target sequences to respectively comprise complete target codons CAA, CAG or CGA, wherein a target single base C is located at the 4 th-8 th position of the target sequence, and the target codons are separated from NGG by 12-14 bp;
utilizing at least two sgRNA sequences to position BE3 to a corresponding target sequence so that a target single base C in a target codon becomes T, and correspondingly introducing a stop codon TAA or TAG, TGA to realize gene knockout;
the at least two sgRNA sequences are 19-21 bp sequences which are respectively complementary and correspond to the at least two target sequences,
wherein the at least two sgrnas are co-injected to achieve effective knock-out of the same gene.
2. The method of claim 1, wherein BE3 is rAPOBEC-SpCas 9-NLS-UGI-NLS.
3. Use of the method of claim 1 for knockout of the murine Tyr gene in cell line N2 a.
4. Use of the method of claim 1 for knockout of the murine Tyr gene in mouse embryonic cells.
5. A kit for gene knock-out, comprising the sgRNA of claim 1, BE3, and an amplification reagent.
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| CN109234316A (en) | 2019-01-18 |
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