CN117384948A - Gene sequence for realizing A/T accurate replacement at target site-4 position and replacement method - Google Patents
Gene sequence for realizing A/T accurate replacement at target site-4 position and replacement method Download PDFInfo
- Publication number
- CN117384948A CN117384948A CN202311283988.2A CN202311283988A CN117384948A CN 117384948 A CN117384948 A CN 117384948A CN 202311283988 A CN202311283988 A CN 202311283988A CN 117384948 A CN117384948 A CN 117384948A
- Authority
- CN
- China
- Prior art keywords
- vector
- spcas9
- gene sequence
- expression cassette
- terminator
- 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
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 14
- 238000000034 method Methods 0.000 title claims abstract description 10
- 241000196324 Embryophyta Species 0.000 claims abstract description 19
- 239000013598 vector Substances 0.000 claims abstract description 19
- 101000910035 Streptococcus pyogenes serotype M1 CRISPR-associated endonuclease Cas9/Csn1 Proteins 0.000 claims abstract description 18
- 241000209094 Oryza Species 0.000 claims abstract description 16
- 235000007164 Oryza sativa Nutrition 0.000 claims abstract description 16
- 235000009566 rice Nutrition 0.000 claims abstract description 16
- 230000008685 targeting Effects 0.000 claims abstract description 11
- 108091027544 Subgenomic mRNA Proteins 0.000 claims abstract description 6
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 22
- 241000589158 Agrobacterium Species 0.000 claims description 14
- 238000012216 screening Methods 0.000 claims description 12
- 239000013604 expression vector Substances 0.000 claims description 7
- 238000006467 substitution reaction Methods 0.000 claims description 7
- 102100035102 E3 ubiquitin-protein ligase MYCBP2 Human genes 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 230000035772 mutation Effects 0.000 claims description 6
- 238000010362 genome editing Methods 0.000 claims description 5
- 241000589155 Agrobacterium tumefaciens Species 0.000 claims description 4
- 239000012882 rooting medium Substances 0.000 claims description 4
- 238000003776 cleavage reaction Methods 0.000 claims description 3
- 208000015181 infectious disease Diseases 0.000 claims description 3
- 230000007017 scission Effects 0.000 claims description 3
- 238000001976 enzyme digestion Methods 0.000 claims description 2
- 238000002744 homologous recombination Methods 0.000 claims description 2
- 230000006801 homologous recombination Effects 0.000 claims description 2
- 230000009977 dual effect Effects 0.000 claims 1
- 230000036438 mutation frequency Effects 0.000 claims 1
- 239000002773 nucleotide Substances 0.000 claims 1
- 125000003729 nucleotide group Chemical group 0.000 claims 1
- 230000009261 transgenic effect Effects 0.000 claims 1
- 238000012217 deletion Methods 0.000 abstract description 9
- 230000037430 deletion Effects 0.000 abstract description 9
- 230000037431 insertion Effects 0.000 abstract description 4
- 238000003780 insertion Methods 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 19
- 239000000203 mixture Substances 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 8
- 239000000725 suspension Substances 0.000 description 8
- 230000006698 induction Effects 0.000 description 7
- 230000004069 differentiation Effects 0.000 description 6
- 230000008929 regeneration Effects 0.000 description 6
- 238000011069 regeneration method Methods 0.000 description 6
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 230000006872 improvement Effects 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 239000005708 Sodium hypochlorite Substances 0.000 description 3
- 230000037429 base substitution Effects 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000007614 genetic variation Effects 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 240000008467 Oryza sativa Japonica Group Species 0.000 description 1
- 101150108119 PDS gene Proteins 0.000 description 1
- 244000184734 Pyrus japonica Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- 108010050181 aleurone Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 101150095418 crop gene Proteins 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000012883 rooting culture medium Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a gene sequence for realizing A/T accurate replacement at a target site-4 position and a replacement method. According to the invention, nmCas9 and SpCas9 double expression frames are introduced into a single vector, experiments prove that SpCas9 is utilized to relatively favor base deletion and NmCas9 relatively favor single base insertion in plants, target sgRNA which is matched with SpCas9 and sgRNA which is matched with NmCas9 and has-4 single base deletion in a target sequence are introduced into genome targets of a co-tangent point, so that a rice targeting editing vector is constructed, rice can be caused to replace A or T at any single base at-4 positions after being introduced into rice cells, and therefore, accurate single base replacement of a DSB template-free plant genome is realized.
Description
Technical Field
The invention relates to the technical fields of biotechnology and plant genetic engineering. Specifically, the invention relates to a gene sequence for realizing A/T accurate replacement at a target site-4, and a method for realizing A/T accurate replacement at the target site-4 by utilizing SpCas9 and NmCas9 double expression frames.
Background
Key to crop breeding is the creation and use of genetic variation. The genetic variation utilized in traditional crop breeding mostly comes from accumulation of natural mutation in the domestication process or artificial physicochemical mutagenesis; however, these mutations are randomly generated in the genome, and the variable resources available for improvement of agronomic traits are extremely limited, severely affecting the speed and effectiveness of variety improvement. The gene editing technology, in particular the CRISPR-Cas9 gene editing technology developed since 2013, can introduce variation at specific loci of genome, and brings 'subversion' breakthrough for crop breeding. The most widely applied crop gene editing system at present is to realize the knockout of target genes by introducing small fragment insertion or deletion at fixed points. However, many important traits of crops are often associated with a particular small number of base variations rather than gene deletions.
In order to achieve accurate editing of eukaryotic genomes, particularly high frequency base substitution, a range of HDR-independent editing tools have been developed and widely used. The single base editing technology is a precise editing technology developed based on a CRISPR-Cas system, single base controllable substitution can be introduced without DSB and donor DNA repair templates, but the single base editing system can only finish 6 types of 12 base conversion types and can only efficiently act in an editing window of about 5bp, so that the targeting capability is doubly limited by PAM and the editing window. The guide editing system is a recently developed precise gene editing technology, and can complete any type of base substitution and precise small fragment insertion and deletion; and a plurality of sites in the PAM distal end sequence can be edited efficiently, the targeting range is basically unlimited, but the efficiency of guiding editing in plants is low. Therefore, expanding the precise editing thought and developing a new precise editing strategy has important significance for realizing precise manipulation of plant genome and accelerating crop breeding improvement.
Disclosure of Invention
Aiming at the problem that the prior art can not accurately realize that any base of a specific site is replaced by A/T, the invention integrates a double expression frame of Nm1Cas9 and SpCas9 on a single plant expression vector, then introduces target sgRNA which is matched with SpCas9 and sgRNA which is matched with Nm1Cas9 and has-4 single base deletion in a target sequence aiming at a rice genome locus with a PAM sequence of NGGNGATT, realizes the base deletion at the A site through the SpCas9 expression frame, and then carries out T site insertion at the deletion position through the Nm1Cas9 expression frame, thereby realizing that the arbitrary single base at the-4 site is replaced by A or T at high frequency, and realizing the accurate single base substitution of the plant genome without a DSB template.
According to the invention, firstly, nm1Cas9 and SpCas9 expression frames are assembled respectively by using a Gibson method and are sequentially subjected to enzyme digestion and connected to the same carrier, on the basis of the carrier, a Goldengate method is adopted for a single target point to obtain a targeting carrier containing Nm1Cas9 and SpCas9, and the mutation efficiency of site-specific substitution A/T is detected through rice genetic transformation and targeted sequencing. Comprising the following steps:
(1) Removing shell of rice seeds, sterilizing, separating embryo, and placing on callus induction medium to generate secondary callus;
(2) Transferring the secondary callus to a new callus induction medium for preculture;
(3) Contacting the callus obtained in step (2) with agrobacterium carrying a targeting vector for SpCas9-Nm1Cas9 for 15 minutes;
(4) Transferring the callus of the step (3) to a culture dish on which three sterile filter papers (2.5-3.5 mL of agrobacterium suspension medium is added) are placed, and culturing at 21-23 ℃ for 48 hours;
(5) Placing the callus in the step (4) on a pre-screening culture medium for culturing for 5-7 days;
(6) Transferring the callus of step (5) onto a screening medium to obtain a resistant callus;
(7) Transferring the resistant callus to a differentiation regeneration medium to differentiate into seedlings; and is also provided with
(8) Transferring the seedlings in the step (7) into a rooting culture medium for rooting.
Wherein the seed in step (1) is a mature seed; the induction medium in the steps (1) and (2) is an induction medium listed in the explanatory table 1; contacting with agrobacterium in step (3) is immersing the callus in the agrobacterium suspension; the agrobacterium suspension medium in step (4) is the suspension medium listed in table 1 of the specification; the pre-screening medium in step (5) is illustrative of the pre-screening medium listed in table 1; the screening media of step (6) are those listed in Table 1; the differentiation and regeneration medium in the step (7) is a differentiation and regeneration medium listed in table 1 of the specification; the rooting medium in step (8) is the rooting medium listed in Table 1.
In a preferred embodiment, wherein the rice is japonica rice, more preferably, the rice is japonica Nipponbare.
Table 1 exemplary formulation of the medium
TABLE 1
In a preferred embodiment, the nucleotide sequences of the NmCas9 and SpCas9 genes are the nucleotide sequences shown in SEQ ID No.1, specifically as follows:
NM1Cas9 expression frame (promoter and terminator)
tgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataaaaaattaccacatattttttttgtcacacttg
tttgaagtgcagtttatctatctttatacatatatttaaactttactctacgaataatataatctatagtactacaataatatcagtgttttagagaat
catataaatgaacagttagacatggtctaaaggacaattgagtattttgacaacaggactctacagttttatctttttagtgtgcatgtgttctc
ctttttttttgcaaatagcttcacctatataatacttcatccattttattagtacatccatttagggtttagggttaatggtttttatagactaattttttt
agtacatctattttattctattttagcctctaaattaagaaaactaaaactctattttagtttttttatttaataatttagatataaaatagaataaaat
aaagtgactaaaaattaaacaaataccctttaagaaattaaaaaaactaaggaaacatttttcttgtttcgagtagataatgccagcctgtta
aacgccgtcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaagcgaagcagacggcacggcatct
ctgtcgctgcctctggacccctctcgagagttccgctccaccgttggacttgctccgctgtcggcatccagaaattgcgtggcggagcg
gcagacgtgagccggcacggcaggcggcctcctcctcctctcacggcaccggcagctacgggggattcctttcccaccgctccttcg
ctttcccttcctcgcccgccgtaataaatagacaccccctccacaccctctttccccaacctcgtgttgttcggagcgcacacacacacaa
ccagatctcccccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctccccccccccccctctctaccttctctagat
cggcgttccggtccatggttagggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgtgttagatccgtgctgctagcgttc
gtacacggatgcgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatcctgggatggctctagccgttc
cgcagacgggatcgatttcatgattttttttgtttcgttgcatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcg
ggtcatcttttcatgcttttttttgtcttggttgtgatgatgtggtctggttgggcggtcgttctagatcggagtagaattctgtttcaaactacct
ggtggatttattaattttggatctgtatgtgtgtgccatacatattcatagttacgaattgaagatgatggatggaaatatcgatctaggatag
gtatacatgttgatgcgggttttactgatgcatatacagagatgctttttgttcgcttggttgtgatgatgtggtgtggttgggcggtcgttcat
tcgttctagatcggagtagaatactgtttcaaactacctggtgtatttattaattttggaactgtatgtgtgtgtcatacatcttcatagttacga
gtttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactgatgcatatacatgatggcatatgcagcatctat
tcatatgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttgatcttgatatacttggatgatggcatatgcagc
agctatatgtggatttttttagccctgccttcatacgctatttatttgcttggtactgtttcttttgtcgatgctcaccctgttgtttggtgttacttct
gcagGCCACCATGGCCCCAAAGAAGAAGCGCAAGGTCGCCGCGTTCAAGCCAAATT
CCATCAATTATATCCTGGGCCTGGATATCGGGATTGCGAGCGTGGGGTGGGCCATG
GTCGAGATTGATGAGGAGGAGAATCCGATCAGGCTCATTGATCTCGGCGTGAGGG
TGTTCGAGAGGGCCGAGGTCCCAAAAACCGGCGACTCCCTGGCCATGGCGAGGA
GGCTGGCGAGGTCCGTGAGGAGGCTGACCAGGAGGCGCGCGCATCGCCTGCTCA
GGACAAGGAGGCTCCTCAAGAGGGAGGGCGTGCTCCAGGCCGCCAACTTCGATG
AGAATGGGCTCATCAAGAGCCTCCCGAACACCCCGTGGCAACTCCGCGCTGCTGC
TCTCGACAGGAAGCTGACACCGCTCGAGTGGTCCGCCGTGCTCCTCCATCTGATCA
AACACCGCGGCTACCTCTCCCAACGCAAAAACGAGGGGGAAACTGCGGACAAGG
AACTGGGGGCCCTCCTGAAAGGCGTCGCCGGCAATGCGCACGCGCTCCAGACCG
GGGACTTCAGGACACCAGCCGAGCTGGCGCTCAACAAGTTCGAAAAGGAATCCG
GCCATATTAGGAACCAGCGCAGCGACTACAGCCACACCTTTAGCAGGAAGGACCT
GCAGGCGGAGCTGATCCTCCTCTTCGAGAAACAGAAGGAGTTCGGCAACCCACAT
GTCAGCGGCGGCCTCAAGGAAGGCATCGAAACACTCCTCATGACCCAACGCCCAG
CCCTGAGCGGCGATGCGGTCCAGAAGATGCTCGGCCATTGTACCTTTGAACCAGC
GGAGCCGAAGGCCGCGAAAAATACCTATACCGCCGAGCGCTTCATTTGGCTGACC
AAGCTCAACAACCTCAGGATCCTCGAACAGGGCAGCGAGAGGCCGCTGACAGAC
ACCGAACGCGCCACCCTGATGGATGAGCCGTACAGGAAGTCCAAGCTCACCTACG
CCCAGGCCCGCAAACTCCTCGGCCTGGAGGATACCGCCTTCTTCAAGGGGCTCCG
CTATGGCAAGGATAATGCCGAAGCGTCCACCCTCATGGAAATGAAGGCGTACCATG
CCATCAGCCGCGCCCTGGAGAAGGAAGGGCTCAAGGACAAGAAGAGCCCGCTGA
ATCTCAGCCCGGAACTGCAGGATGAGATCGGCACAGCCTTCTCCCTGTTCAAAAC
CGATGAGGACATTACCGGCCGCCTGAAAGACCGCATCCAACCAGAAATCCTCGAG
GCCCTCCTGAAGCACATCTCCTTTGATAAATTCGTGCAGATTAGCCTCAAGGCCCT
CAGGAGGATCGTGCCGCTCATGGAGCAAGGGAAAAGGTACGACGAGGCGTGCGC
CGAGATTTACGGCGATCACTACGGGAAGAAGAATACCGAAGAGAAGATTTACCTG
CCGCCGATTCCAGCCGACGAAATCAGGAATCCGGTCGTCCTCAGGGCCCTGTCCC
AGGCGCGCAAGGTGATTAACGGCGTGGTGAGGCGCTACGGCAGCCCGGCCAGGAT
CCACATCGAAACAGCGCGCGAAGTCGGCAAGTCCTTCAAGGACAGGAAGGAGAT
CGAGAAGAGGCAGGAAGAGAATCGCAAGGACAGGGAGAAGGCGGCGGCGAAGT
TTAGGGAATACTTCCCGAACTTTGTGGGGGAGCCAAAGAGCAAGGACATCCTGAA
GCTCAGGCTGTATGAACAACAGCACGGCAAGTGCCTGTATTCCGGCAAGGAGATC
AACCTGGGGAGGCTGAACGAGAAGGGCTATGTGGAGATCGACCATGCGCTCCCGT
TCTCCCGCACCTGGGACGACTCCTTCAACAACAAGGTGCTCGTGCTCGGGAGCGA
AAATCAGAATAAAGGGAACCAAACACCGTACGAGTACTTCAACGGCAAGGACAA
CAGCCGCGAGTGGCAGGAGTTTAAGGCGAGGGTCGAGACATCCCGCTTTCCGCGC
AGCAAGAAGCAGCGCATTCTCCTCCAGAAATTCGACGAGGATGGGTTCAAAGAGC
GCAACCTCAACGATACCAGGTACGTGAACAGGTTCCTCTGCCAATTTGTCGCGGAT
AGGATGCGCCTCACCGGCAAGGGCAAGAAGCGCGTCTTTGCGTCCAATGGGCAGA
TCACAAACCTGCTGAGGGGCTTCTGGGGCCTGAGGAAAGTGCGCGCGGAGAATG
ATAGGCATCACGCGCTGGACGCCGTGGTCGTGGCGTGCTCCACAGTCGCGATGCA
GCAAAAAATCACACGCTTCGTCCGCTACAAAGAGATGAACGCGTTCGACGGCAAA
ACCATCGACAAGGAAACTGGGGAGGTCCTGCACCAGAAGACCCACTTCCCACAA
CCGTGGGAGTTTTTCGCGCAGGAGGTCATGATCAGGGTGTTCGGCAAACCGGACG
GCAAGCCGGAGTTCGAAGAGGCGGATACCCTGGAGAAGCTCAGGACCCTGCTGG
CGGAGAAACTGTCCAGCAGGCCGGAAGCGGTGCATGAGTACGTCACCCCGCTCTT
CGTCTCCAGGGCGCCGAACAGGAAGATGAGCGGCCAAGGCCACATGGAAACAGT
GAAGTCCGCGAAACGCCTCGACGAGGGGGTGAGCGTCCTCAGGGTGCCACTCAC
CCAACTCAAACTCAAGGACCTGGAGAAGATGGTGAATCGCGAGAGGGAGCCAAA
GCTCTATGAGGCCCTGAAGGCCCGCCTGGAAGCCCACAAAGACGACCCAGCGAA
GGCCTTCGCGGAGCCGTTTTATAAGTACGACAAAGCCGGGAACAGGACCCAGCAG
GTCAAAGCCGTGAGGGTCGAACAGGTCCAGAAAACCGGGGTGTGGGTGCGCAAT
CATAACGGGATCGCCGATAATGCCACAATGGTCAGGGTGGATGTGTTCGAGAAAG
GCGATAAGTACTACCTGGTGCCGATCTACAGCTGGCAAGTCGCCAAAGGCATTCTC
CCGGATCGCGCGGTGGTGCAGGGCAAGGATGAAGAGGACTGGCAGCTGATCGAC
GATTCCTTTAATTTCAAGTTCAGCCTGCACCCGAATGACCTCGTGGAGGTCATCAC
CAAGAAAGCCCGCATGTTTGGCTACTTCGCCTCCTGTCACAGGGGCACCGGCAATA
TCAATATCCGCATCCACGACCTCGACCATAAGATCGGCAAAAACGGCATCCTGGAG
GGGATTGGCGTCAAAACAGCGCTGAGCTTCCAGAAGTACCAGATTGACGAATTAG
GCAAAGAGATCAGGCCGTGCAGGCTCAAGAAACGCCCACCGGTCAGGTACCCATA
CGATGTTCCAGATTACGCTAAGCGGCCAGCGGCGACGAAGAAGGCGGGGCAGGC
GAAGAAGAAGAAGTAAGAGCTCaagggtgggcgcgccgacccagctttcttgtacaaagtggtgatatcccgcg
gccatggcggccgggagcatgcgacgtcgatctaactgactagccgcggccatgctagagtccgcaaaaatcaccagtctctctctac
aaatctatctctctctatttttctccagaataatgtgtgagtagttcccagataagggaattagggttcttatagggtttcgctcatgtgttgag
catataagaaacccttagtatgtatttgtatttgtaaaatacttctatcaataaaatttctaattcctaaaaccaaaatccagtgacctgca
SpCas9 expression cassette (promoter and terminator)
aagcttgatatcgaattcctgcagtgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataaaaaatt
accacatattttttttgtcacacttgtttgaagtgcagtttatctatctttatacatatatttaaactttactctacgaataatataatctatagtacta
caataatatcagtgttttagagaatcatataaatgaacagttagacatggtctaaaggacaattgagtattttgacaacaggactctacagtt
ttatctttttagtgtgcatgtgttctcctttttttttgcaaatagcttcacctatataatacttcatccattttattagtacatccatttagggtttaggg
ttaatggtttttatagactaatttttttagtacatctattttattctattttagcctctaaattaagaaaactaaaactctattttagtttttttatttaataa
tttagatataaaatagaataaaataaagtgactaaaaattaaacaaataccctttaagaaattaaaaaaactaaggaaacatttttcttgtttc
gagtagataatgccagcctgttaaacgccgtcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaagc
gaagcagacggcacggcatctctgtcgctgcctctggacccctctcgagagttccgctccaccgttggacttgctccgctgtcggcatc
cagaaatgcgtggcggagcggcagacgtgagccggcacggcaggcggcctcctcctcctctcacggcacggcagctacggggga
ttcctttcccaccgctccttcgctttcccttcctcgcccgccgtaataaatagacaccccctccacaccctctttccccaacctcgtgttgttc
ggagcgcacacacacacaaccagatctcccccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctccccccccc
cccctctctaccttctctagatcggcgttccggtccatggttagggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgtgtt
agatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatc
ctgggatggctctagccgttccgcagacgggatcgatttcatgattttttttgtttcgttgcatagggtttggtttgcccttttcctttatttcaat
atatgccgtgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttggttgtgatgatgtggtctggttgggcggtcgttctagatcggag
tagaattctgtttcaaactacctggtggatttattaattttggatctgtatgtgtgtgccatacatattcatagttacgaattgaagatgatggat
ggaaatatcgatctaggataggtatacatgttgatgcgggttttactgatgcatatacagagatgctttttgttcgcttggttgtgatgatgtg
gtgtggttgggcggtcgttcattcgttctagatcggagtagaatactgtttcaaactacctggtgtatttattaattttggaactgtatgtgtgt
gtcatacatcttcatagttacgagtttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactgatgcatatac
atgatggcatatgcagcatctattcatatgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttgatcttgatata
cttggatgatggcatatgcagcagctatatgtggatttttttagccctgccttcatacgctatttatttgcttggtactgtttcttttgtcgatgctc
accctgttgtttggtgttacttctgcagcccgggggatccccaatacttgtatggccgcggccgcgccaccatggccccaaagaagaag
cgcaaggtcgacaagaagtactccatcggcctcgacatcggcaccaattctgttggctgggccgtgatcaccgacgagtacaaggtg
ccgtccaagaagttcaaggtcctcggcaacaccgaccgccactccatcaagaagaatctcatcggcgccctgctgttcgactctggcg
agacagccgaggctacaaggctcaagaggaccgctagacgcaggtacaccaggcgcaagaaccgcatctgctacctccaagagat
cttctccaacgagatggccaaggtggacgacagcttcttccacaggctcgaggagagcttcctcgtcgaggaggacaagaagcacga
gcgccatccgatcttcggcaacatcgtggatgaggtggcctaccacgagaagtacccgaccatctaccacctccgcaagaagctcgtc
gactccaccgataaggccgacctcaggctcatctacctcgccctcgcccacatgatcaagttcaggggccacttcctcatcgagggcg
acctcaacccggacaactccgatgtggacaagctgttcatccagctcgtgcagacctacaaccagctgttcgaggagaacccgatcaa
cgcctctggcgttgacgccaaggctattctctctgccaggctctctaagtcccgcaggctcgagaatctgatcgcccaacttccgggcg
agaagaagaatggcctcttcggcaacctgatcgccctctctcttggcctcaccccgaacttcaagtccaacttcgacctcgccgaggac
gccaagctccagctttccaaggacacctacgacgacgacctcgacaatctcctcgcccagattggcgatcagtacgccgatctgttcct
cgccgccaagaatctctccgacgccatcctcctcagcgacatcctcagggtgaacaccgagatcaccaaggccccactctccgcctc
catgatcaagaggtacgacgagcaccaccaggacctcacactcctcaaggccctcgtgagacagcagctcccagagaagtacaagg
agatcttcttcgaccagtccaagaacggctacgccggctacatcgatggcggcgcttctcaagaggagttctacaagttcatcaagccg
atcctcgagaagatggacggcaccgaggagctgctcgtgaagctcaatagagaggacctcctccgcaagcagcgcaccttcgataat
ggctccatcccgcaccagatccacctcggcgagcttcatgctatcctccgcaggcaagaggacttctacccgttcctcaaggacaacc
gcgagaagattgagaagatcctcaccttccgcatcccgtactacgtgggcccgctcgccaggggcaactccaggttcgcctggatga
ccagaaagtccgaggagacaatcaccccctggaacttcgaggaggtggtggataagggcgcctctgcccagtctttcatcgagcgca
tgaccaacttcgacaagaacctcccgaacgagaaggtgctcccgaagcactcactcctctacgagtacttcaccgtgtacaacgagct
gaccaaggtgaagtacgtgaccgaggggatgaggaagccagctttccttagcggcgagcaaaagaaggccatcgtcgacctgctgtt
caagaccaaccgcaaggtgaccgtgaagcagctcaaggaggactacttcaagaaaatcgagtgcttcgactccgtcgagatctccgg
cgtcgaggataggttcaatgcctccctcgggacctaccacgacctcctcaagattatcaaggacaaggacttcctcgacaacgaggag
aacgaggacatcctcgaggacatcgtgctcaccctcaccctcttcgaggaccgcgagatgatcgaggagcgcctcaagacatacgcc
cacctcttcgacgacaaggtgatgaagcagctgaagcgcaggcgctataccggctggggcaggctctctaggaagctcatcaacgg
catccgcgacaagcagtccggcaagacgatcctcgacttcctcaagtccgacggcttcgccaaccgcaacttcatgcagctcatccac
gacgactccctcaccttcaaggaggacatccaaaaggcccaggtgtccggccaaggcgattccctccatgaacatatcgccaatctcg
ccggctccccggctatcaagaagggcattctccagaccgtgaaggtggtggacgagctggtgaaggtgatgggcaggcacaagcca
gagaacatcgtgatcgagatggcccgcgagaaccagaccacacagaagggccaaaagaactcccgcgagcgcatgaagaggatc
gaggagggcattaaggagctgggctcccagatcctcaaggagcacccagtcgagaacacccagctccagaacgagaagctctacct
ctactacctccagaacggccgcgacatgtacgtggaccaagagctggacatcaaccgcctctccgactacgacgtggaccatattgtg
ccgcagtccttcctgaaggacgactccatcgacaacaaggtgctcacccgctccgacaagaacaggggcaagtccgataacgtgcc
gtccgaagaggtcgtcaagaagatgaagaactactggcgccagctcctcaacgccaagctcatcacccagaggaagttcgacaacct
caccaaggccgagagaggcggcctttccgagcttgataaggccggcttcatcaagcgccagctcgtcgagacacgccagatcacaa
agcacgtggcccagatcctcgactcccgcatgaacaccaagtacgacgagaacgacaagctcatccgcgaggtgaaggtcatcacc
ctcaagtccaagctcgtgtccgacttccgcaaggacttccagttctacaaggtgcgcgagatcaacaactaccaccacgcccacgacg
cctacctcaatgccgtggtgggcacagccctcatcaagaagtacccaaagctcgagtccgagttcgtgtacggcgactacaaggtgta
cgacgtgcgcaagatgatcgccaagtccgagcaagagatcggcaaggcgaccgccaagtacttcttctactccaacatcatgaatttct
tcaagaccgagatcacgctcgccaacggcgagattaggaagaggccgctcatcgagacaaacggcgagacaggcgagatcgtgtg
ggacaagggcagggatttcgccacagtgcgcaaggtgctctccatgccgcaagtgaacatcgtgaagaagaccgaggttcagaccg
gcggcttctccaaggagtccatcctcccaaagcgcaactccgacaagctgatcgcccgcaagaaggactgggacccgaagaagtat
ggcggcttcgattctccgaccgtggcctactctgtgctcgtggttgccaaggtcgagaagggcaagagcaagaagctcaagtccgtca
aggagctgctgggcatcacgatcatggagcgcagcagcttcgagaagaacccaatcgacttcctcgaggccaagggctacaaggag
gtgaagaaggacctcatcatcaagctcccgaagtacagcctcttcgagcttgagaacggccgcaagagaatgctcgcctctgctggcg
agcttcagaagggcaacgagcttgctctcccgtccaagtacgtgaacttcctctacctcgcctcccactacgagaagctcaagggctcc
ccagaggacaacgagcaaaagcagctgttcgtcgagcagcacaagcactacctcgacgagatcatcgagcagatctccgagttctcc
aagcgcgtgatcctcgccgatgccaacctcgataaggtgctcagcgcctacaacaagcaccgcgataagccaattcgcgagcaggc
cgagaacatcatccacctcttcaccctcaccaacctcggcgctccagccgccttcaagtacttcgacaccaccatcgaccgcaagcgct
acacctctaccaaggaggttctcgacgccaccctcatccaccagtctatcacaggcctctacgagacacgcatcgacctctcacaactc
ggcggcgatTCCGGCGGCAGCccaaagaagaagcggaaggtgTCTGGAGGTTCTcctaagaaaaagagaaa
agtgTCCGGCGGCTCCccgaagaagaagcgcaaggtgTGAGAGCTCcggccgggagcatgcgacgtcgatcta
actgactagccgcggccatgctagagtccgcaaaaatcaccagtctctctctacaaatctatctctctctatttttctccagaataatgtgtg
agtagttcccagataagggaattagggttcttatagggtttcgctcatgtgttgagcatataagaaacccttagtatgtatttgtatttgtaaaa
tacttctatcaataaaatttctaattcctaaaaccaaaatccagtgacct
Drawings
Fig. 1 is a schematic structural diagram of a vector constructed using Nm1Cas9 and SpCas9 according to the present invention;
FIG. 2 is a comparison of the mutant produced by the present invention with a control sequence.
Detailed Description
Embodiments of the present invention are described below with reference to the accompanying drawings. It should be noted that the following examples are only illustrative of exemplary implementations of the present invention and are not intended to limit the present invention in any way. Certain equivalent modifications and obvious improvements to the present invention may be made by those skilled in the art.
The operations in the following detailed description are performed using conventional operations commonly used in the art without additional specificity. The person skilled in the art can easily obtain teachings about such conventional operations from the prior art, for example, see textbooks Sambrook and David Russell, molecular Cloning: A Laboratory Manual,3rd ed., vols1,2; charles Neal Stewart, alicher Touraev, vitaly Citovsky and Tzvi Tzfira, plant Transformation Technologies, etc. The raw materials, reagents, materials and the like used in the following examples are all commercially available products unless otherwise specified.
EXAMPLE 1 construction of expression vectors
The UBI, osSpCas9, osNm1Cas9 and 35S terminator genes were amplified and the T-UBI-OsSpCas9-35S terminator and T-UBI-OsNm1Cas9-35S terminator were assembled by homologous recombination on the intermediate vector T. Connecting UBI-OsSpCas9-35S terminator to a PHUC400 vector existing in a laboratory by using PstI/SacI restriction enzyme site to obtain PHUC-SpCas9, and connecting UBI-OsNm1Cas9-35S terminator to the PHUC-SpCas9 vector by using PmeI restriction enzyme site to obtain PHUC-Sp-Nm1; on this basis, the sgRNA expression cassette was ligated by HindIII cleavage to give the final expression vector PHUC411-Nm1, which is shown in FIG. 1.
Example 2 construction of targeting vectors
A20 bp sequence at the front end of the PAM sequence of NGGNGATT on the rice PDS gene is selected as a target position of SpCas9, such as a fourth exon GCTGCTTGGAAGGATGAAGATGGAGATT sequence, then utilizing SpCas9 tends to produce-44 bit deletion characteristic, nm1Cas9 targeting recognition sequence is GATAGCTGCTTGGAAGGATG-AGATGGAGATTThe underlined part is the PAM sequence. After the identification sequence is determined, a double-targeting primer is synthesized by using an existing intermediate vector MsgScRNA-OsU3 in a laboratory as a template, and amplified and recovered, wherein the primer sequence is as follows:
MScRNA-PDS FP:
AATAATGGTCTCAGGCAGATAGCTGCTTGGAAGGATGAGAGTTGTAGCTCCCTTTCTCATTT
MScRNA-PDS RP:
AATAATGGTCTCAAAACTCTTCATCCTTCCAAGCAGCTGCCACGGATCATCTGCACAACTCTT the above recovered product was fused with the expression vector of PHUC411-Nm1 by GoldGate, plasmids were extracted and sequenced, and the correctly sequenced vector was transferred into Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 strain (kept by the university of Anhui agriculture, agricultural college) for genetic transformation by freeze thawing.
Example 3-acquisition of rice genetic transformation and mutant with double expression cassettes of SpCas9 and NmCas9 as targeting vectors.
1. Induction and preculture of mature embryo callus
Removing shell from mature seeds of Japanese sunny, selecting seeds with normal appearance and clean and no mildew, shaking with 70% alcohol for 90sec, and pouring out alcohol; the seeds were washed with 50% sodium hypochlorite (stock solution available chlorine concentration greater than 4%) containing Tween20, 1 drop of Tween20 was added per 100 ml, and shaken on a shaker for 45min (180 r/min). Pouring out sodium hypochlorite, washing with sterile water for 5-10 times until no sodium hypochlorite smell exists, and finally adding sterile water and soaking overnight at 30 ℃. Embryos were separated along the aleurone layer with a scalpel blade and scutellum placed up on induction medium (composition see table 1), 12 grains/dish, and dark cultured at 30 ℃ to induce callus.
After two weeks, spherical, rough and pale yellow secondary calli appear, and the pre-culture operation can be carried out, namely, the secondary calli are transferred to a new callus induction culture medium, and the secondary calli are subjected to dark culture at 30 ℃ for 5 days. After the preculture is finished, small particles with good states and vigorous division are collected into a 50mL sterile centrifuge tube by a spoon and used for agrobacterium infection.
2. Cultivation of Agrobacterium Strain and suspension preparation
Agrobacterium strain EHA105 containing the targeting vector was streaked on LB plates containing 50mg/L kanamycin (see Table 1 for composition), cultured in the dark at 28℃and after 24h the activated Agrobacterium was inoculated with a sterile inoculating loop onto fresh LB plates of 50mg/L kanamycin for a second activation, and cultured overnight in the dark at 28 ℃. 20-30mL of Agrobacterium suspension medium (composition shown in Table 1) was added to a 50mL sterile centrifuge tube, activated 2 times with an inoculating loop, OD660 was adjusted to about 0.10-0.25, and the mixture was allowed to stand at room temperature for 30min or more.
3. Infection and co-cultivation
To the prepared callus (see step 1), the Agrobacterium suspension was added and soaked for 15min, with gentle shaking in between. Pouring out the liquid after the soaking is finished (the liquid is dripped as much as possible), sucking out the superfluous agrobacterium liquid on the surface of the callus by using sterile filter paper, and drying by using sterile air in an ultra clean bench. Three pieces of sterile filter paper are placed on a disposable sterile petri dish with the thickness of 100 multiplied by 25mm, 2.5mL of agrobacterium suspension medium is added, the callus after the suction is uniformly dispersed on the filter paper, and the callus is cultivated for 48 hours in the dark at the temperature of 23 ℃.
4. Pre-screening and screening culture
After the end of co-cultivation, the co-cultivated calli were evenly spread in pre-screening medium (composition see Table 1), and cultivated in the dark at 30℃for 5 days. After the pre-screening culture is finished, transferring the calli to a screening culture medium (the composition is shown in table 1), inoculating 25 calli to each culture dish, culturing in the dark at 30 ℃, and after 2-3 weeks, obviously growing the resistant calli, and carrying out differentiation and regeneration operation.
5. Differentiation and regeneration
2-3 small fresh particles with good growth state are selected for each independent transformant and transferred to a differentiation regeneration medium (the composition is shown in Table 1). Each dish was inoculated with 5 independent transformants. Culturing at 28deg.C under light with light intensity of 3000-6000lx for 16 hr and 8 hr in dark.
6. Rooting and transplanting
When the buds of the resistant callus grow to about 2cm, only one well-grown seedling is taken from each independent transformant, and the seedlings are transferred to a rooting medium (the composition is shown in table 1), and are cultivated by illumination at 28 ℃ for 16 hours, dark for 8 hours and light intensity of 3000-6000lx. After two weeks, young seedlings with developed root systems are selected, the culture medium is washed off by water, and the young seedlings are transplanted into soil.
Example 4 molecular characterization of Rice genetically transformed plants
Before transplanting, rice leaf samples are taken, and genome DNA of 48 transformed plants is extracted by a CTAB method. The resulting genomic DNA samples were used in HITOM sequencing analysis to detect the frequency of substitution of the site-4 of the target site with A/T in the transformed population. From the HITOM results, it was found that various types of gene mutations occurred in 48 plants for this locus on the fourth exon of rice OsPDS. The plant ratio of the T-4-bit A is 10, the existing single-base editing technology can only realize the C-T or A-G substitution, but can not realize the A-T substitution, and the action site of the guiding editing technology is usually at the-3-bit and the C-terminal, and can not realize the-4-bit substitution. Plants with G replaced by A at the-4 position have a 5-plant ratio, wild-type have a 8-plant ratio, and the rest are single or multiple base deletions. Figure 2 shows a partial mutation situation. At present, no scheme for reporting A-T replacement with high efficiency is known, and the scheme has great significance.
Claims (8)
1. A gene sequence for achieving accurate a/T replacement at the-4 position of a target site, characterized in that the gene sequence comprises a double expression cassette of SpCas9 and NmCas 9.
2. The gene sequence according to claim 1, wherein the double expression cassette is composed of a nucleotide sequence shown as SEQ ID NO.1 of the sequence Listing.
3. An expression cassette comprising the dual SpCas9 and NmCas9 expression cassette of claim 1.
4. An expression vector comprising the gene sequence of claim 1 or the expression cassette of claim 3.
5. Use of the gene of claim 1, the expression cassette of claim 3 or the vector of claim 4, comprising cleaving the rice genome for a PAM sequence NGGNGATT with the double expression cassette to obtain a transgenic plant or plant part comprising an a/T mutation site.
6. A method for inserting a/T mutation sites at a target site of interest using an expression vector comprising the gene sequence of claim 1, comprising:
amplifying UBI, osSpCas9, osNm1Cas9 and 35S terminator genes, and assembling the T-UBI-OsSpCas9-35S terminator and the T-UBI-OsNm1Cas9-35S terminator on an intermediate vector T by a homologous recombination mode;
ligating UBI-OsSpCas9-35S terminator to the gluc 400 vector using the PstI/SacI cleavage site to give gluc-SpCas 9;
ligating the UBI-OsNm1Cas9-35S terminator to the gluc-SpCas 9 vector using a PmeI cleavage site to obtain gluc-Sp-Nm 1; on the basis, a final expression vector PHUC411-Nm1 is obtained by enzyme digestion of HindIII and connection of an sgRNA expression frame;
constructing a targeting vector;
using an agrobacterium strain containing a gluc 411-Nm1 targeting vector for infecting a seed of interest;
co-culturing and screening the obtained callus to obtain edited plant callus, and differentiating the callus into plants.
7. A method for introducing a gene editing tool of the gene sequence of claim 1 into rice cells, comprising transferring a vector containing double expression cassettes of SpCas9 and NmCas9 into agrobacterium tumefaciens, infecting and culturing rice tissues by using agrobacterium tumefaciens, and transferring seedlings obtained by tissue culture into a rooting medium for rooting.
8. The use according to claim 7, characterized in that it comprises the steps of infection, co-cultivation, screening to obtain edited plant calli, then differentiating the calli into plants, detecting the mutation frequency of the substitution of any base at position-4 of the target site with a or T.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311283988.2A CN117384948A (en) | 2023-09-28 | 2023-09-28 | Gene sequence for realizing A/T accurate replacement at target site-4 position and replacement method |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311283988.2A CN117384948A (en) | 2023-09-28 | 2023-09-28 | Gene sequence for realizing A/T accurate replacement at target site-4 position and replacement method |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117384948A true CN117384948A (en) | 2024-01-12 |
Family
ID=89440125
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311283988.2A Pending CN117384948A (en) | 2023-09-28 | 2023-09-28 | Gene sequence for realizing A/T accurate replacement at target site-4 position and replacement method |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117384948A (en) |
-
2023
- 2023-09-28 CN CN202311283988.2A patent/CN117384948A/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109652422B (en) | Efficient single-base editing system OsSpCas9-eCDA and application thereof | |
AU2019297209B2 (en) | Method of obtaining multi-leaf alfalfa material by means of MsPALM1 artificial site-directed mutant | |
CN110283838B (en) | ScCas9 gene with high shearing efficiency and application thereof | |
CN110878305B (en) | Wide-window single-base editing gene and application and breeding method thereof | |
CN112538492B (en) | SpCas9n variant capable of recognizing NRTH (Polyacrylamide) as PAM (Polyacrylamide) sequence and corresponding base editing system | |
CN111718954B (en) | Genome editing tool and application thereof | |
US11365423B2 (en) | Method of obtaining multileaflet Medicago sativa materials by means of MsPALM1 artificial site-directed mutants | |
CN113265403A (en) | Soybean Dt1 gene editing site and application thereof | |
CN113106120A (en) | Needle-leaved tree plant gene editing vector, construction method and application thereof | |
US20230265447A1 (en) | Delivery of developmental regulators to plants for the induction of meristematic tissue with genetic alterations | |
CN116732070A (en) | CGBE single base editor capable of realizing base transversion and application thereof | |
CN109486840B (en) | Codon-plant-modified NmeCas9 gene and application thereof | |
CN114854766B (en) | NtAIDP1 gene mutant for reducing nicotine content in tobacco leaves and application thereof | |
CN116286901A (en) | Male sterile MsGDSL esterase/lipase gene and application thereof | |
CN111534538B (en) | Method for rapidly screening non-transgenic site-directed mutant plants | |
CN115820901A (en) | Visual gene editing detection method in cotton and application thereof | |
CN110257406B (en) | codon-Plant-modified Plant Nme2Cas9 gene and application thereof | |
CN117384948A (en) | Gene sequence for realizing A/T accurate replacement at target site-4 position and replacement method | |
CN112430612A (en) | SpRY gene capable of being efficiently cut and application thereof | |
CN112522299A (en) | Method for obtaining rice with increased tillering by using OsTNC1 gene mutation | |
CN112430613A (en) | SpG gene with wide editing range and application thereof | |
CN113930441A (en) | Method for obtaining transgenic or gene-edited plant body | |
CN115820691B (en) | LbCPf1 variant-based rice base editing system and application | |
CN112575014B (en) | Base editor SpCas9-LjCDAL1 and construction and application thereof | |
CN113862282B (en) | Soybean PCL homologous gene editing site and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |