CN105647962A - Gene editing method for knocking out rice MIRNA393b stem-loop sequences with application of CRISPR(clustered regulatory interspersed short palindromic repeat)-Cas9 system - Google Patents
Gene editing method for knocking out rice MIRNA393b stem-loop sequences with application of CRISPR(clustered regulatory interspersed short palindromic repeat)-Cas9 system Download PDFInfo
- Publication number
- CN105647962A CN105647962A CN201610085619.6A CN201610085619A CN105647962A CN 105647962 A CN105647962 A CN 105647962A CN 201610085619 A CN201610085619 A CN 201610085619A CN 105647962 A CN105647962 A CN 105647962A
- Authority
- CN
- China
- Prior art keywords
- pcr
- product
- rice
- mir393b
- mirna393b
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
-
- 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
- C12N2310/14—Type of nucleic acid interfering N.A.
- C12N2310/141—MicroRNAs, miRNAs
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
-
- 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
- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/10—Vectors comprising a non-peptidic targeting moiety
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to construction of rice transgenic materials and aims to provide a gene editing method for knocking out rice MIRNA393b stem-loop sequences with application of a CRISPR(clustered regulatory interspersed short palindromic repeat)-Cas9 system. The gene editing method comprises steps as follows: gRNA target sites are selected for cloning and GG linking, enzyme digestion is performed after amplification, and a product is linked with a pGREB 32 vector; escherichia coli competent cells are transformed; plasmids with a correct sequencing result are used for transforming agrobacteria, transgenic plants are obtained through mediated transformation of rice calli, and transgenic positive lines are obtained; the T0-generation mutant plant seeds are collected for seeding, and the T1-generation plants are subjected to homozygote screening; homozygous lines which are discovered to be negative through MIRNA393b expression are rice mutants completely losing the MIRNA393b stem-loop sequences and MIRNA393b stem-loop sequence expression. According to the gene editing method, MIRNA stem-loop sequences can be effectively knocked out, and loss-of-function mutants of different members in the same MIRNA family can be prepared; the mutant plant propagates to obtain a large number of seeds and is an ideal material for acquiring rice MIRNA393b gene functions successfully.
Description
Technical field
The present invention relates to the structure of Transgenic Rice material, particularly to the gene editing method using CRISPR-Cas9 system to knock out MIR393b stem ring sequence.
Background technology
Oryza sativa L. is cereal crops important in the world, is also the model organism of monocotyledon research. MiRNA393 is a conservative microRNA family in plant, regulates and controls auxin signal path by post-transcriptional level negative regulation growth hormone receptor TIR1/AFBs family protein. Its functional study at present mainly includes plant immunization reaction, growth promoter and three aspects of Stress responses. And the function of miR393 is also known little about it in Oryza sativa L., mainly lack the mutant material of this gene.
In the several years in past, the main transgenic approach adopting simulation competition target gene carries out the functional study of Oryza sativa L. miRNA. But the MIM393 strain of simulation competition target gene can not be completely eliminated the effect of MIR393 gene, and cannot distinguish between the function of MIR393a and MIR393b gene, it is therefore desirable to build gene knockout strain each special for its MIR393a and MIR393b, the biological function that this family member's MIR393a/b gene is real just can be illustrated. But not yet report lacks the rice mutant of MIR393a or MIR393b gene so far.
CRISPR (clusteredregulatoryinterspersedshortpalindromicrepeat) sequence is one section has multiple repetitive sequences of palindrome with intervening sequence, and it comes from procaryotic a kind of acquired immune system. In recent years, CRISPR-Cas system based on RNA mediation is flourish, this system adjustable point modifies specific gene order in (deletion, interpolation, activation, suppression) target cell, for targeting editor's genome sequence effective technological means of offer. But, there is no the report using this technology to knock out Oryza sativa L. MIRNA gene stem ring sequence at present.
Summary of the invention
The problem to be solved in the present invention is, overcome deficiency of the prior art, a kind of CRISPR-Cas9 of utilization system is provided to knock out the gene editing method of Oryza sativa L. MIRNA393b stem ring sequence, to obtain the ideal abrupt body completely losing miR393b stem ring sequence and expression thereof.
For solving technical problem, the solution of the present invention is:
There is provided a kind of CRISPR-Cas9 of utilization system to knock out the gene editing method of Oryza sativa L. MIRNA393b stem ring sequence, comprise the following steps:
(1) selection of gRNA target site
Owing to MIR393b gene is positioned on No. four chromosome of rice genome, the principle of the design target site according to CRISPR-Cas9 technology, first target site is designed in the precursor stem ring sequence of MIR393b gene, and second target site is held at stem ring sequence downstream 3 ';
(2) the fragment clone of gRNA
With plasmid pGTR for template, cloning three fragment L1, the partly overlapping fragments of L2, L3 by PCR method, primer sequence is as follows, and wherein F and R represents forward and reverse primer respectively:
L1F:cgggtctcaggcaggatgggcagtctgggcaacaaagcaccagtgg
L1R:cgggtctcagcgttgggcttctgcaccagccggg
L2F:taggtctccacgctagcacacgttttagagctaggaa
L2R:cgggtctcattcctcaggccgtgcaccagccggg
L3F:taggtctccggaaactagtgggttttagagctagaa
L3R:taggtctccaaacggatgagcgacagcaaacaaaaaaaaaagcaccgactcg
PCR system is: Phusion enzyme 0.5 �� l; 5 �� Buffer10 �� l; The each 2.5 �� l of primer; Water 30 �� l; DNTP4 �� l; PGTRplasmid0.5 �� l;
PCR reaction condition is: denaturation 95 DEG C, 5min; Degeneration 95 DEG C, 30s; Anneal 60 DEG C, 30s; Extend 72 DEG C, 30s; Totally 33 circulations; Finally extend 10min;
After PCR reaction, taking 5-10 �� l product, after the solidifying electrophoresis detection of the agarose with 2%, purification reclaims purpose fragment, measures the concentration of three PCR primer L1, L2, L3;
(3) carry out GG (gRNA-gRNA) to connect
According to the PCR concentration measured, by three fragment mixed in equal amounts, T7 enzyme coupled reaction and BsaI enzyme action carry out simultaneously;
Take each 2 �� l of L1, L2, L3, mix with 10 �� lT7ligasebuffer, 1 �� lBsaI HF, 0.5 �� lT7ligase, 2.5 �� l water; PCR instrument carries out reaction as follows: 37 DEG C, 5min; 20 DEG C, 10min; 30-50 circulation;
(4) connect product and carry out pcr amplification;
After coupled reaction terminates, take connection product 1 �� l, add 19 �� l water dilutions, using the product after dilution as template, do pcr amplification with L1F, L3R for primer; After PCR terminates, take 5 �� l products and carry out electrophoresis detection, and by product purification; Product is sized to 350bp;
(5) by the product of previous step purification, FokI enzyme action exposes cohesive end, simultaneously FokI enzyme action empty carrier pGREB32;
Enzyme action system is 20 �� l, including substrate 5 �� l, FokI5 �� l, Buffer10 �� l; Substrate includes GG purified product and empty carrier pGREB32;
Enzyme action time 3-4h, enzyme action temperature 37 DEG C; Detect digestion products with 1% agarose gel, and reclaim target product, measure concentration;
(6) taking GG product and pGREB32 carrier mixed in equal amounts that enzyme action processes, be attached, ligase is T4 ligase, and 4 DEG C connect overnight;
(7) by the vector competent escherichia coli cell after connection, plated overnight; Picking list bacterium colony, shakes bacterium 4-6h, extracts plasmid, takes sample and check order; By plasmid correct for sequencing result, convert Agrobacterium EHA105;
(8) agrobacterium mediation converted Rice Callus obtains transfer-gen plant
With wild rice, Japan is fine for material callus induction, carries out agriculture bacillus mediated rice conversion experiment; Carrying out infecting with EHA105 Agrobacterium and convert, screen through hygromycin resistance, resistant calli differentiation and regeneration obtains transgenic positive strain;
(9) after obtaining positive strain, extract genomic DNA, design primer in the both sides of two gRNA sequences, purpose fragment is carried out pcr amplification, utilizes vertical polyacrylate hydrogel electrophoresis detection mutant;
(10) above mutating strain series PCR primer is purified recovery, connects carrier T and check order;
(11) detection that miR393b expresses;
Collect T0For mutant plants seed, send out Seedling, to T1Homozygote screening is carried out for plant; Extracting homozygote plant leaf blade tissue RNA, miR393b precursor carries out qRT-PCR detection and analyzes, primer is as follows:
osa-miR393b-qRT-up:5��CGGCCTGAGGAAACTAGTGGA3��
osa-miR393b-qRT-dn:5��GGAAGATGAGGAGGCGGAAG3��
Product size: 152bp
(12) through the homozygous lines that miR393b detection of expression is negative, it is the rice mutant completely losing miR393b stem ring sequence and expression thereof.
Compared with prior art, the beneficial effects of the present invention is:
The main transgenic approach adopting simulation competition target gene carried out the function inhibitio research of Oryza sativa L. miRNA in the past. But the strain of simulation competition target gene can not be completely eliminated the effect of MicroRNA gene, and cannot distinguish between the function of tiny RNA family gene member. And this technology adopt CRISPR-Cas9 method can targeting knock out specific dna sequence, especially can knock out the whole stem ring sequence of certain specific MicroRNA gene so that its function completely loses, thus obtaining effective afunction mutant. Therefore, this technology has the advantage that the stem ring sequence effectively knocking out tiny RNA, and can prepare the afunction mutant of same tiny RNA family different members. Carry out a large amount of seed of expanding propagation with mutant plants, be the ideal material being successfully obtained Study On Rice MIRNA393b gene function. So far, not yet there is the report of gene editing method and this rice mutant material knocking out tiny RNA stem ring sequence.
Accompanying drawing explanation
Fig. 1 is MIR393b gene structure and 2 gRNA sites;
Fig. 2 is that first round PAGE gel electrophoresis filters out Heterozygous mutants;
Fig. 3 second takes turns PAGE gel electrophoresis and filters out Mutants homozygous;
Fig. 4 is the comparison (black rectangle represents disappearance base, and single underscore represents insertion base, and double underline represents replacement base) of mutating strain series purpose sequencing fragment result;
Fig. 5 is the qRT-PCR detection of Mutants homozygous miR393b precursor expression level.
Detailed description of the invention
The acquisition of embodiment 1 Oryza sativa L. MIRNA393b stem ring sequence knockouts strain and qualification
The turned rice varieties of the present invention is Japan fine (Oryza.SativaL.spp.japonica, varNipponbare).
The selection of 1.gRNA target site
Owing to MIR393b gene is positioned on No. four chromosome of rice genome, the principle of the design target site according to CRISPR-Cas9 technology, the present invention designs first target site in the precursor stem ring sequence of MIR393b gene, and second target site is held at stem ring sequence downstream 3 '. See Fig. 1.
The clone of 2.gRNA fragment and vector construction
2.1 with plasmid pGTR for template, clones three fragment L1, the partly overlapping fragments of L2, L3 by PCR method, and primer sequence is as follows:
L1F:cgggtctcaggcaggatgggcagtctgggcaacaaagcaccagtgg (as shown in SEQIDNO:1)
L1F:cgggtctcagcgttgggcttctgcaccagccggg (as shown in SEQIDNO:2)
L2F:taggtctccacgctagcacacgttttagagctaggaa (as shown in SEQIDNO:3)
L2R:cgggtctcattcctcaggccgtgcaccagccggg (as shown in SEQIDNO:4)
L3F:taggtctccggaaactagtgggttttagagctagaa (as shown in SEQIDNO:5)
L3R:taggtctccaaacggatgagcgacagcaaacaaaaaaaaaagcaccgactcg (as shown in SEQIDNO:6)
Pcr amplification L1 fragment system is as follows:
| Phusion enzyme | 0.5��l |
| 5��Buffer | 10��l |
| L1F | 2.5��l |
| L1R | 2.5��l |
| Water | 30��l |
| dNTP | 4��l |
| Template (pGTR plasmid) | 0.5��l |
| Amount to | 50��l |
Pcr amplification L2 fragment system is as follows:
Pcr amplification L3 fragmentSystem is as follows:
| Phusion enzyme | 0.5��l |
| 5��Buffer | 10��l |
| L3F | 2.5��l |
| L3R | 2.5��l |
| Water | 30��l |
| dNTP | 4��l |
| Template (pGTR plasmid) | 0.5��l |
| Amount to | 50��l |
PCR response procedures is: 95 DEG C of 5min of denaturation;95 DEG C of 30s of degeneration, 60 DEG C of 30s of annealing, extension 72 DEG C of 30s, totally 33 circulations. Last 72 DEG C extend 10min.
After PCR reaction, take 5-10 �� l product, be purified recovery purpose fragment after the solidifying electrophoresis detection of the agarose with 2%, measure production concentration. L1, L2, L3 product size is about 130bp, 200bp, 150bp.
2.2 carry out GG (gRNA-gRNA) connects.
According to the production concentration that upper pacing is fixed, by 3 fragment mixed in equal amounts, T7 enzyme connects. Reaction system is as follows:
| Reagent | Volume (�� l) |
| L1 | 2 |
| L2 | 2 |
| L3 | 2 |
| 2��T7 ligase buffer | 10 |
| BsaI-HF | 1 |
| T7 ligase | 0.5 |
| Water | 2.5 |
| Cumulative volume | 20 |
Above coupled reaction carries out in PCR instrument: 37 DEG C, 5min; 20 DEG C, 10min; 30-50 circulation.
2.3 connect product carries out pcr amplification
After coupled reaction terminates, take connection product 1 �� l, add 19 �� l water dilutions, using the product after dilution as template, do pcr amplification with L1F, L3R for primer.
PCR system is as follows:
| Reagent | Volume (�� l) |
| GG product after dilution | 2.5 |
| L1L | 2.5 |
| L3R | 2.5 |
| 2��TaqMaster | 25 |
| Water | 17.5 |
| Cumulative volume | 50 |
PCR response procedures is: 95 DEG C of 5min of denaturation; 95 DEG C of 30s of degeneration, 60 DEG C of 30s of annealing, extension 72 DEG C of 30s, totally 33 circulations. Finally extend 10min.
Taking 5-10 �� lPCR product electrophoresis detection, product size is about 350bp, and by product purification.
2.4 by the product of previous step purification, and FokI enzyme action exposes cohesive end, simultaneously FokI enzyme action empty carrier pGREB32.
Enzyme action system is 20 �� l, wherein substrate (respectively GG purified product, empty carrier pGREB32) 5 �� l, FokI5 �� l, Buffer10 �� l.
Enzyme action time 3-4h, enzyme action temperature 37 DEG C. Detect digestion products with 1% agarose gel, and reclaim target product, measure concentration.
GG product after enzyme action and carrier pGREB32 T4 enzyme are attached by 2.5,4 DEG C, connect overnight.
Wherein GG product and each 4 �� l, T4DNAligase1 �� l, the T4DNAligasebuffer1 �� l of carrier.
3. by the vector competent escherichia coli cell Trans10 after connection, plated overnight, picking list bacterium colony, shake bacterium 4-6h, extract plasmid, take sample segment and check order.
Sequencing primer is U3-F, UGW-gRNA-R. Sequence is as follows:
U3-F:agtaccacctcggctatccaca is (such as SEQIDNO: 7Shown in)
UGW-gRNA-R:ggacctgcaggcatgcacgcgctaaaaacggactagc (such as SEQIDNO:8 institutesShow)
4. will connect correct plasmid, convert Agrobacterium EHA105.
5. agrobacterium mediation converted Rice Callus obtains transfer-gen plant
With wild rice Japan fine (Oryza.SativaL.spp.japonica, varNipponbare) for material callus induction, carry out agriculture bacillus mediated rice conversion experiment. Carrying out infecting with EHA105 Agrobacterium and convert, screen through hygromycin resistance, resistant calli differentiation and regeneration obtains transgenic positive strain.
6. the detection of large fragment deletion mutant in transgenic paddy rice
The detection primer of 6.1 purpose of design genes: according to genes of interest, the upstream and downstream two gRNA sequences separately designs primer, and primer sequence is respectively as follows:
F1: gctggctgcaacaaacattct is (such as SEQIDNO: 9 institutesShow)
R1: tgcttacacaaattagatgccatt (such as SEQIDNO:10 institutesShow)
The 6.2 transgenic positive plant that will obtain, extract genomic DNA respectively, carry out PCR reaction. After Standard PCR reaction terminates, then PCR primer is carried out the reaction of high-temperature denatured then renaturation, PCR program and degeneration renaturation step such as following table:
6.3 above-mentioned PCR primer carry out vertical PAGE gel detection gene mutation strain.Altogether through two-wheeled PAGE electrophoresis.
The first round filters out Heterozygous mutants: takes 10 �� lPCR products, carries out PAGE gel electrophoresis. Electrophoresis sets: constant voltage 200V, 150min, electrophoresis result is shown in Fig. 2. The strain of 3 bands of appearance is Heterozygous mutants, is 4,5,8,10 and 14# strain respectively.
Second takes turns PAGE gel electrophoresis filters out Mutants homozygous. By PCR primer single for band in above-mentioned transgenic line respectively with wild type PCR primer mixed in equal amounts, react through degeneration renaturation, carry out PAGE gel electrophoresis. Result is shown in Fig. 3. Screening obtains 1 strain Mutants homozygous strain 6#.
Two-wheeled electrophoresis filters out heterozygote altogether and suddenlys change 5 strains, and homozygote suddenlys change 1 strain.
7. the gene sequencing of mutating strain series
Above mutating strain series PCR primer is purified recovery, connects carrier T and check order. Order-checking company is that the raw work in Shanghai is biological. Sequencing result is shown in Fig. 4.
The mutated sequence analysis of sequencing result, it was found that 8# strain exists the large fragment deletion of 259bp, it is thus achieved that knock out the ideal abrupt material of the whole stem ring sequence of MIR393b.
The detection of 8.miR393b precursor expression level
Collect T0For 8# plant seed, send out Seedling, to 8#T1Homozygote screening is carried out for plant. Extract T1For homozygote plant leaf tissue RNA, miR393b precursor is carried out quantitative fluorescent PCR (qRT-PCR) detection. Design of primers is within stem ring sequence, and PCR primer size is 152bp, and primer sequence is respectively as follows:
Osa-miR393b-qRT-up:5 ' cggcctgaggaaactagtgga3 ' (such as SEQIDNO:11 institutesShow),
Osa-miR393b-qRT-dn:5 ' ggaagatgaggaggcggaag3 ' is (such as SEQIDNO: 12 institutesShow)
QRT-PCR program setting is as follows:
The qRT-PCR testing result of 8# Mutants homozygous miR393b precursor expression level is shown in Fig. 5. These data show compared with wild type, and in 8# mutant, the expression of miR393b stem ring precursor RNA is almost nil. Prove that 8# is the mutant strain completely losing miR393b stem ring sequence and expression thereof.
Through the homozygous lines that miR393b detection of expression is negative, it is the rice mutant completely losing miR393b stem ring sequence and expression thereof. This mutant plants carries out a large amount of seed of expanding propagation, is the ideal material being successfully obtained Study On Rice MIRNA393b gene function.
Claims (1)
1. use CRISPR-Cas9 system to knock out the gene editing method of Oryza sativa L. MIRNA393b stem ring sequence, it is characterised in that to comprise the following steps:
(1) selection of gRNA target site
Owing to MIR393b gene is positioned on No. four chromosome of rice genome, the principle of the design target site according to CRISPR-Cas9 technology, first target site is designed in the precursor stem ring sequence of MIR393b gene, and second target site is held at stem ring sequence downstream 3 ';
(2) the fragment clone of gRNA
With plasmid pGTR for template, cloning three fragment L1, the partly overlapping fragments of L2, L3 by PCR method, primer sequence is as follows, and wherein F and R represents forward and reverse primer respectively:
L1F:cgggtctcaggcaggatgggcagtctgggcaacaaagcaccagtgg
L1R:cgggtctcagcgttgggcttctgcaccagccggg
L2F:taggtctccacgctagcacacgttttagagctaggaa
L2R:cgggtctcattcctcaggccgtgcaccagccggg
L3F:taggtctccggaaactagtgggttttagagctagaa
L3R:taggtctccaaacggatgagcgacagcaaacaaaaaaaaaagcaccgactcg
PCR system is: Phusion enzyme 0.5 �� l;5 �� Buffer10 �� l; The each 2.5 �� l of primer; Water 30 �� l; DNTP4 �� l; PGTRplasmid0.5 �� l;
PCR reaction condition is: denaturation 95 DEG C, 5min; Degeneration 95 DEG C, 30s; Anneal 60 DEG C, 30s; Extend 72 DEG C, 30s; Totally 33 circulations; Finally extend 10min;
After PCR reaction, taking 5-10 �� l product, after the solidifying electrophoresis detection of the agarose with 2%, purification reclaims purpose fragment, measures the concentration of three PCR primer L1, L2, L3;
(3) carry out GG (gRNA-gRNA) to connect
According to the PCR concentration measured, by three fragment mixed in equal amounts, T7 enzyme coupled reaction and BsaI enzyme action carry out simultaneously;
Take each 2 �� l of L1, L2, L3, mix with 10 �� lT7ligasebuffer, 1 �� lBsaI HF, 0.5 �� lT7ligase, 2.5 �� l water; PCR instrument carries out reaction as follows: 37 DEG C, 5min; 20 DEG C, 10min; 30-50 circulation;
(4) connect product and carry out pcr amplification;
After coupled reaction terminates, take connection product 1 �� l, add 19 �� l water dilutions, using the product after dilution as template, do pcr amplification with L1F, L3R for primer; After PCR terminates, take 5 �� l products and carry out electrophoresis detection, and by product purification; Product is sized to 350bp;
(5) by the product of previous step purification, FokI enzyme action exposes cohesive end, simultaneously FokI enzyme action empty carrier pGREB32;
Enzyme action system is 20 �� l, including substrate 5 �� l, FokI5 �� l, Buffer10 �� l; Substrate includes GG purified product and empty carrier pGREB32;
Enzyme action time 3-4h, enzyme action temperature 37 DEG C; Detect digestion products with 1% agarose gel, and reclaim target product, measure concentration;
(6) taking GG product and pGREB32 carrier mixed in equal amounts that enzyme action processes, be attached, ligase is T4 ligase, and 4 DEG C connect overnight;
(7) by the vector competent escherichia coli cell after connection, plated overnight; Picking list bacterium colony, shakes bacterium 4-6h, extracts plasmid, takes sample and check order; By plasmid correct for sequencing result, convert Agrobacterium EHA105;
(8) agrobacterium mediation converted Rice Callus obtains transfer-gen plant
With wild rice, Japan is fine for material callus induction, carries out agriculture bacillus mediated rice conversion experiment; Carrying out infecting with EHA105 Agrobacterium and convert, screen through hygromycin resistance, resistant calli differentiation and regeneration obtains transgenic positive strain;
(9) after obtaining positive strain, extract genomic DNA, design primer in the both sides of two gRNA sequences, purpose fragment is carried out pcr amplification, utilizes vertical polyacrylate hydrogel electrophoresis detection mutant;
(10) above mutating strain series PCR primer is purified recovery, connects carrier T and check order;
(11) detection that miR393b expresses;
Collect T0For mutant plants seed, send out Seedling, to T1Homozygote screening is carried out for plant; Extracting homozygote plant leaf blade tissue RNA, miR393b precursor carries out qRT-PCR detection and analyzes, primer is as follows:
osa-miR393b-qRT-up:5��CGGCCTGAGGAAACTAGTGGA3��
osa-miR393b-qRT-dn:5��GGAAGATGAGGAGGCGGAAG3��
Product size: 152bp
(12) through the homozygous lines that miR393b detection of expression is negative, it is the rice mutant completely losing miR393b stem ring sequence and expression thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610085619.6A CN105647962A (en) | 2016-02-15 | 2016-02-15 | Gene editing method for knocking out rice MIRNA393b stem-loop sequences with application of CRISPR(clustered regulatory interspersed short palindromic repeat)-Cas9 system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610085619.6A CN105647962A (en) | 2016-02-15 | 2016-02-15 | Gene editing method for knocking out rice MIRNA393b stem-loop sequences with application of CRISPR(clustered regulatory interspersed short palindromic repeat)-Cas9 system |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105647962A true CN105647962A (en) | 2016-06-08 |
Family
ID=56488409
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610085619.6A Pending CN105647962A (en) | 2016-02-15 | 2016-02-15 | Gene editing method for knocking out rice MIRNA393b stem-loop sequences with application of CRISPR(clustered regulatory interspersed short palindromic repeat)-Cas9 system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105647962A (en) |
Cited By (38)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| CN106367435A (en) * | 2016-09-07 | 2017-02-01 | 电子科技大学 | Method for directionally knocking out miRNA (micro Ribonucleic Acid) of paddy rice |
| CN106636184A (en) * | 2016-11-17 | 2017-05-10 | 中国科学院东北地理与农业生态研究所 | Application of rice heading-date gene vector |
| CN106906240A (en) * | 2017-03-29 | 2017-06-30 | 浙江大学 | The method that the key gene HPT in barley VE synthesis paths is knocked out with CRISPR Cas9 systems |
| CN107446924A (en) * | 2017-08-16 | 2017-12-08 | 中国科学院华南植物园 | A kind of Kiwi berry Gene A cPDS based on CRISPR Cas9 edits carrier and its construction method and application |
| US9840699B2 (en) | 2013-12-12 | 2017-12-12 | President And Fellows Of Harvard College | Methods for nucleic acid editing |
| WO2018018979A1 (en) * | 2016-07-26 | 2018-02-01 | 浙江大学 | Recombinant plant vector and method for screening non-transgenic gene-edited strain |
| CN108103092A (en) * | 2018-01-05 | 2018-06-01 | 中国农业科学院作物科学研究所 | System and its application for downgrading rice are obtained using CRISPR-Cas systems modification OsHPH genes |
| US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| CN109161558A (en) * | 2018-09-19 | 2019-01-08 | 深圳大学 | A kind of construction method of the efficient over-express vector of monocotyledon miRNA |
| WO2019058255A1 (en) * | 2017-09-19 | 2019-03-28 | Tropic Biosciences UK Limited | Modifying the specificity of plant non-coding rna molecules for silencing gene expression |
| CN109652442A (en) * | 2019-01-18 | 2019-04-19 | 深圳大学 | Efficient CRISPR-CAS9 gene editing carrier and construction method in arabidopsis |
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| WO2019214736A1 (en) * | 2018-05-10 | 2019-11-14 | 中国科学院上海生命科学研究院 | Rice mutant mir393am having high brown planthopper resistance and salt tolerance, and application thereof |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| CN110904109A (en) * | 2019-12-16 | 2020-03-24 | 河南农业大学 | miR1866 gene for controlling rice seed germination, overexpression vector, gRNA expression vector, preparation method and application thereof |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| CN111979258A (en) * | 2020-08-04 | 2020-11-24 | 华中农业大学 | Editing method of high-throughput gene |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| CN112813081A (en) * | 2021-02-23 | 2021-05-18 | 宁夏农林科学院农业生物技术研究中心（宁夏农业生物技术重点实验室） | Method for obtaining high-linoleic acid content rice line |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| CN113817857A (en) * | 2021-04-09 | 2021-12-21 | 中国热带农业科学院三亚研究院 | Transgenic component detection method based on CRISPR Cas enzyme gene editing technology, reaction system and application thereof |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| CN116536357A (en) * | 2023-04-17 | 2023-08-04 | 中国医学科学院输血研究所 | Method for constructing sgRNA shearing activity screening system in CRISPR/Cas12a |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651398A (en) * | 2014-12-24 | 2015-05-27 | 杭州师范大学 | Method for knocking out microRNA gene family by utilizing CRISPR-Cas9 specificity |
-
2016
- 2016-02-15 CN CN201610085619.6A patent/CN105647962A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651398A (en) * | 2014-12-24 | 2015-05-27 | 杭州师范大学 | Method for knocking out microRNA gene family by utilizing CRISPR-Cas9 specificity |
Non-Patent Citations (5)
| Title |
|---|
| HONGWU BIAN ET AL.: "Distinctive expression patterns and roles of the miRNA393⁄TIR1 homolog module in regulating flag leaf inclination and primary and crown root growth in rice (Oryza sativa)", 《NEW PHYTOLOGIST》 * |
| KABIN XIE ET AL.: "Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system", 《PNAS》 * |
| 袁君婷等: "microRNA-483在人体细胞中生物学功能的研究", 《遗传多样性：前沿与挑战——中国的遗传学研究（2013-2015）——2015中国遗传学会大会论文摘要汇编》 * |
| 解亚坤: "水稻miR393基因家族的表达模式及其对植株生长发育的影响", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 邹键等: "含四个串联核酶和GFP基因的转基因质粒的构建", 《中国病毒学》 * |
Cited By (65)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| US11920181B2 (en) | 2013-08-09 | 2024-03-05 | President And Fellows Of Harvard College | Nuclease profiling system |
| US10954548B2 (en) | 2013-08-09 | 2021-03-23 | President And Fellows Of Harvard College | Nuclease profiling system |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US10912833B2 (en) | 2013-09-06 | 2021-02-09 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US9737604B2 (en) | 2013-09-06 | 2017-08-22 | President And Fellows Of Harvard College | Use of cationic lipids to deliver CAS9 |
| US11299755B2 (en) | 2013-09-06 | 2022-04-12 | President And Fellows Of Harvard College | Switchable CAS9 nucleases and uses thereof |
| US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US9840699B2 (en) | 2013-12-12 | 2017-12-12 | President And Fellows Of Harvard College | Methods for nucleic acid editing |
| US11124782B2 (en) | 2013-12-12 | 2021-09-21 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US11053481B2 (en) | 2013-12-12 | 2021-07-06 | President And Fellows Of Harvard College | Fusions of Cas9 domains and nucleic acid-editing domains |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US11578343B2 (en) | 2014-07-30 | 2023-02-14 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| WO2018018979A1 (en) * | 2016-07-26 | 2018-02-01 | 浙江大学 | Recombinant plant vector and method for screening non-transgenic gene-edited strain |
| US10947530B2 (en) | 2016-08-03 | 2021-03-16 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11702651B2 (en) | 2016-08-03 | 2023-07-18 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| CN106367435B (en) * | 2016-09-07 | 2019-11-08 | 电子科技大学 | A kind of method that rice miRNA orientation knocks out |
| CN106367435A (en) * | 2016-09-07 | 2017-02-01 | 电子科技大学 | Method for directionally knocking out miRNA (micro Ribonucleic Acid) of paddy rice |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| CN106636184A (en) * | 2016-11-17 | 2017-05-10 | 中国科学院东北地理与农业生态研究所 | Application of rice heading-date gene vector |
| US11820969B2 (en) | 2016-12-23 | 2023-11-21 | President And Fellows Of Harvard College | Editing of CCR2 receptor gene to protect against HIV infection |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| CN106906240A (en) * | 2017-03-29 | 2017-06-30 | 浙江大学 | The method that the key gene HPT in barley VE synthesis paths is knocked out with CRISPR Cas9 systems |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| CN107446924A (en) * | 2017-08-16 | 2017-12-08 | 中国科学院华南植物园 | A kind of Kiwi berry Gene A cPDS based on CRISPR Cas9 edits carrier and its construction method and application |
| CN107446924B (en) * | 2017-08-16 | 2020-01-14 | 中国科学院华南植物园 | Kiwi fruit gene AcPDS editing vector based on CRISPR-Cas9 and construction method and application thereof |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11932884B2 (en) | 2017-08-30 | 2024-03-19 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11555199B2 (en) | 2017-09-19 | 2023-01-17 | Tropic Biosciences UK Limited | Modifying the specificity of plant non-coding RNA molecules for silencing gene expression |
| WO2019058255A1 (en) * | 2017-09-19 | 2019-03-28 | Tropic Biosciences UK Limited | Modifying the specificity of plant non-coding rna molecules for silencing gene expression |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| CN108103092B (en) * | 2018-01-05 | 2021-02-12 | 中国农业科学院作物科学研究所 | System for modifying OsHPH gene by using CRISPR-Cas system to obtain dwarf rice and application thereof |
| CN108103092A (en) * | 2018-01-05 | 2018-06-01 | 中国农业科学院作物科学研究所 | System and its application for downgrading rice are obtained using CRISPR-Cas systems modification OsHPH genes |
| WO2019214736A1 (en) * | 2018-05-10 | 2019-11-14 | 中国科学院上海生命科学研究院 | Rice mutant mir393am having high brown planthopper resistance and salt tolerance, and application thereof |
| CN109161558A (en) * | 2018-09-19 | 2019-01-08 | 深圳大学 | A kind of construction method of the efficient over-express vector of monocotyledon miRNA |
| CN109161558B (en) * | 2018-09-19 | 2022-05-06 | 深圳大学 | Construction method of monocotyledon miRNA high-efficiency overexpression vector |
| CN109652442A (en) * | 2019-01-18 | 2019-04-19 | 深圳大学 | Efficient CRISPR-CAS9 gene editing carrier and construction method in arabidopsis |
| US11795452B2 (en) | 2019-03-19 | 2023-10-24 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11643652B2 (en) | 2019-03-19 | 2023-05-09 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| CN110904109A (en) * | 2019-12-16 | 2020-03-24 | 河南农业大学 | miR1866 gene for controlling rice seed germination, overexpression vector, gRNA expression vector, preparation method and application thereof |
| CN110904109B (en) * | 2019-12-16 | 2023-04-11 | 河南农业大学 | miR1866 gene for controlling rice seed germination, overexpression vector, gRNA expression vector, preparation method and application thereof |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| CN111979258B (en) * | 2020-08-04 | 2022-04-19 | 华中农业大学 | High-throughput gene editing method |
| CN111979258A (en) * | 2020-08-04 | 2020-11-24 | 华中农业大学 | Editing method of high-throughput gene |
| CN112813081B (en) * | 2021-02-23 | 2022-10-28 | 宁夏农林科学院农业生物技术研究中心（宁夏农业生物技术重点实验室） | Method for obtaining high-linoleic acid content rice line |
| CN112813081A (en) * | 2021-02-23 | 2021-05-18 | 宁夏农林科学院农业生物技术研究中心（宁夏农业生物技术重点实验室） | Method for obtaining high-linoleic acid content rice line |
| CN113817857A (en) * | 2021-04-09 | 2021-12-21 | 中国热带农业科学院三亚研究院 | Transgenic component detection method based on CRISPR Cas enzyme gene editing technology, reaction system and application thereof |
| CN116536357A (en) * | 2023-04-17 | 2023-08-04 | 中国医学科学院输血研究所 | Method for constructing sgRNA shearing activity screening system in CRISPR/Cas12a |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105647962A (en) | Gene editing method for knocking out rice MIRNA393b stem-loop sequences with application of CRISPR(clustered regulatory interspersed short palindromic repeat)-Cas9 system | |
| CN105063061B (en) | A kind of rice mass of 1000 kernel gene tgw6 mutant and the preparation method and application thereof | |
| CN104846010B (en) | A kind of method for deleting transgenic paddy rice riddled basins | |
| CN107164401A (en) | A kind of method and application that rice Os PIL15 mutant is prepared based on CRISPR/Cas9 technologies | |
| CN110317828B (en) | Method for cultivating broad-spectrum bacterial leaf blight resistant rice by modifying rice OsSWEET gene promoter | |
| CN108034671B (en) | Plasmid vector and method for establishing plant population by using same | |
| CN111254142B (en) | Molecular marker of corn seed cadmium low accumulation control gene ZmCD1 and application | |
| CN108642065A (en) | A kind of paddy endosperm silty related gene OsSecY2 and its coding protein and application | |
| CN104593380B (en) | For the gene ZmHKT1 for the coding corn HKT transport proteins for improving plant salt endurance;1a and its application | |
| CN104628839B (en) | A kind of paddy endosperm amyloplast development associated protein and its encoding gene and application | |
| CN104593381B (en) | A kind of corn resistant gene of salt and its application | |
| CN106480084B (en) | Application of OsLAC13 and miR397a/b in cultivation of high-setting-rate or high-yield rice | |
| Marlin et al. | Molecular diversity of the flowering related gene (LEAFY) on shallot (Allium cepa var. aggregatum) and Allium relatives. | |
| Li et al. | Rapid generation of selectable marker-free transgenic rice with three target genes by co-transformation and anther culture | |
| CN112662681B (en) | Application of OsNRAMP1 in improving disease resistance of rice | |
| CN109609510A (en) | The application of soybean PHR transcription factor encoding gene GmPHRb | |
| CN105925587B (en) | Early rice chloroplast development gene subjected to low-temperature response and detection method and application thereof | |
| CN114349835A (en) | Application of GhREM protein and coding gene thereof in regulating and controlling aphid resistance of cotton | |
| CN110904109B (en) | miR1866 gene for controlling rice seed germination, overexpression vector, gRNA expression vector, preparation method and application thereof | |
| CN113801891A (en) | Construction method and application of beet BvCENH3 gene haploid induction line | |
| CN109082425B (en) | Rape boron high-efficiency gene BnA3NIP5; transposon insertion fragment TEQ of 1Q and primer and application thereof | |
| CN102533762B (en) | Method for obtaining novel desensitization transgenic soybean material | |
| CN106434742B (en) | Utilize the method for soybean expression canine distemper albumen | |
| CN117230107B (en) | mRNA variable shear-luciferase reporting system and application thereof | |
| CN103243114B (en) | Fusion gene, protein expressed by same and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160608 |
|
| RJ01 | Rejection of invention patent application after publication |