CN105543270A - Double resistance CRISPR/Cas9 carrier and application - Google Patents
Double resistance CRISPR/Cas9 carrier and application Download PDFInfo
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- CN105543270A CN105543270A CN201510992210.8A CN201510992210A CN105543270A CN 105543270 A CN105543270 A CN 105543270A CN 201510992210 A CN201510992210 A CN 201510992210A CN 105543270 A CN105543270 A CN 105543270A
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- 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
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- 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
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- 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
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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Abstract
The invention relates to a double resistance CRISPR/Cas9 carrier and application. The invention provides the double resistance carrier having both hygromycin B resistance and Basta resistance at the same time; the double resistance carrier is obtained by inserting sequences and fragments derived from multiple different carriers into a sequence of a carrier pFGC5941 by enzyme-cutting and linking up, and constructing. Hygromycin B is used for screening in a conversion process, and Basta is used for screening offspring transgenic plants not containing an external source T-DNA sequence in offspring plants. Two resistances, i.e., the hygromycin B and the Basta are used to respectively serve as screening marks of the transgenic plants for the first time, a feasible manner is provided for screening transgenic offspring not containing the external source T-DNA sequence, determining the existence of external source T-DNA by a complex molecular biology detection experiment is avoided, and the offspring transgenic plants not containing the external source T-DNA sequence can be obtained by directly screening the Basta resistance and performing phenotype combination.
Description
Technical field
The present invention relates to genetically engineered field, specifically, relate to a kind of novel Double CRISPR/Cas9 carrier and application.
Background technology
Paddy rice is as the model plant of farm crop gene functional research, and relative genetics and molecular biology research are subject to the attention of investigators always.The Main Means of Study On Rice gene function is become by the paddy rice of transgenosis acquisition target gene improvement.Although the transgenic technology of paddy rice is ripe at present; but the rice material obtained by transgenosis is owing to cannot remove the external source T-DNA proceeded to; often detrimentally affect be can cause to experimental result, the uncertainty of experimental result and the complicacy of experimentation too increased.
At present for the editing technique develop rapidly of gene.Over the past two years, became by the gene editing technology of CRISPR/Cas9 System-mediated the mainstream technology that organic sphere obtains knock out mutants body.This systemic origin is in the immunity system of bacterium, its principle of work is that crRNA (CRISPR-derivedRNA) is combined by base pairing and tracrRNA (trans-activatingRNA) and forms tracrRNA/crRNA mixture, and this mixture guides the sequence target site that nuclease Cas9 albumen is matching with crRNA to shear double-stranded RNA.And by these two kinds of RNA of engineer, the sgRNA (shortguideRNA) being formed and there is guiding function can be transformed, guide Cas9 to the cutting of DNA fixed point.By the sgRNA of synthetic and target-gene sequence homology, directly can realize the editor to target gene, and this gene by editing mode can genetic stability to of future generation, without the need to the sustainable existence of CRISPR/Cas9 external source T-DNA.
Summary of the invention
The object of this invention is to provide a kind of novel Double CRISPR/Cas9 carrier and application.
Another object of the present invention is to provide the construction process of the paddy rice CRY1a homozygous mutation body plant not containing external source T-DNA sequence.
In order to realize the object of the invention, the Double CRISPR/Cas9 carrier of one of the present invention, it is by pcr amplification 35S promoter and NOS terminator sequence, over-lap PCR is utilized 3 × flag sequence of 35S promoter and NOS terminator sequence and external synthesis to be together in series, between EcoRI and HindIII site tandem sequence being inserted into carrier pFGC5941, obtain carrier I; By pcr amplification gypsy insulator sequence, by increasing, the sequence obtained is inserted between EcoRI and the HindIII site of carrier I, obtains carrier II; By pcr amplification NLS-cas9-NLS sequence, the sequence obtained increasing is inserted into the SpeI site of carrier II, obtains carrier III; Finally by pcr amplification 35S:HPT sequence, the sequence obtained increasing is inserted into the XbaI site of carrier III, namely builds and obtains Double CRISPR/Cas9 carrier.
Wherein, 3 × flag sequence is as shown in SEQIDNO:1, and gypsy insulator sequence is as shown in SEQIDNO:2, and NLS-cas9-NLS sequence is as shown in SEQIDNO:3, and 35S:HPT sequence is as shown in SEQIDNO:4.
The primer of amplification 35S promoter and NOS terminator sequence is respectively as shown in SEQIDNO:5-6 and SEQIDNO:7-8; The primer of amplification gypsy insulator sequence is as shown in SEQIDNO:9-10; The primer of amplification NLS-cas9-NLS sequence is as shown in SEQIDNO:11-12; The primer of amplification 35S:HPT sequence is as shown in SEQIDNO:13-14.
The complete sequence of the Double CRISPR/Cas9 carrier of the present invention is as shown in SEQIDNO:15.The bootable exogenous array of this carrier is in the sequence of host's inediting object target gene.
The present invention also provides engineering bacteria containing described Double CRISPR/Cas9 carrier and transgenic cell line.
The present invention also provides described Double CRISPR/Cas9 carrier obtaining not containing the application in the transfer-gen plant of external source T-DNA sequence.Such as, predicted by the target area of third party software to target gene, then forecasting sequence and U3 promotor are together inserted in dual anti-carrier and target gene is edited.
Particularly, described application is for the sgRNA sequence of the target gene design in plant based on CRISPR/Cas9, DNA fragmentation containing the described sgRNA sequence of coding is cloned in Double CRISPR/Cas9 carrier, conversion of plant, with hygromycin B screening positive transgenic plant, more do not contain the positive transgenic plant of external source T-DNA sequence with Basta screening.
The present invention also provides a kind of paddy rice CRY1a gene knockout carrier Oscry1a-sgRNA, according to the sgRNA sequence of paddy rice CRY1a gene design based on CRISPR/Cas9, DNA fragmentation containing the described sgRNA sequence of coding is building up to (DNA fragmentation of described sgRNA sequence of encoding is positioned at U3 promotor downstream) in described Double CRISPR/Cas9 carrier together with U3 promotor, obtains paddy rice CRY1a gene knockout carrier Oscry1a-sgRNA.
Wherein, the nucleotides sequence of sgRNA action site is classified as 5 '-ACAGGCACCTGTCCCAGAACGG-3 '.
The complete sequence of paddy rice CRY1a gene knockout carrier Oscry1a-sgRNA of the present invention is as shown in SEQIDNO:23.
The present invention further provides the construction process of a kind of paddy rice CRY1a homozygous mutation body plant not containing external source T-DNA sequence, adopt agriculture bacillus mediated method, described Oscry1a-sgRNA carrier is proceeded in Rice Callus, transform with the AAM nutrient solution containing inductor and Agrobacterium, material after conversion is through the exercise of Dual culture-screen-break up-take root-transgenic seedling and transplanting, screening transgenic rice plant, first obtain positive transgenic plant with hygromycin B screening, the paddy rice CRY1a homozygous mutation body plant not containing external source T-DNA sequence is obtained again with Basta screening.
Preferably, according to the nucleotide sequence design pair of primers of sgRNA action site, by PCR method qualification rice plant mutational site; Primer sequence is as shown in SEQIDNO:17-18.
The present invention passes through the sequence deriving from multiple different carriers, fragment to cut to connect through enzyme to be inserted in the sequence of pFGC5941, final structure is had the dual anti-carrier of hygromycin B (hygromycin) and Basta resistance simultaneously, utilize hygromycin to screen in conversion process, utilize Basta in Progeny plants, carry out the screening of the progeny transgenic plant not containing external source T-DNA sequence.
Carrier of the present invention imports (Weissbach in vegetable cell by using the standard biologic technological methods such as Ti-plasmids, plant viral vector, directly delivered DNA, microinjection, electroporation, 1998, MethodforPlantMolecularBiologyVIII, AcademyPress, NewYork, 411-463 page; Geiserson and Corey, 1998, PlantMolecularBiology, 2
ndedition).
The present invention utilizes hygromycin and Basta two kinds of resistances respectively as the selection markers of transfer-gen plant first, for the transgenic progeny of screening not containing external source T-DNA sequence provides a kind of feasible pattern, avoid the existence being determined external source T-DNA by the experiment of the molecular Biological Detection of complexity, directly by combining the progeny transgenic plant that can obtain not containing external source T-DNA sequence to the screening of Basta resistance and phenotype.
Accompanying drawing explanation
Fig. 1 is the plasmid map of the Double CRISPR/Cas9 carrier built in the embodiment of the present invention 1.
Fig. 2 is the plasmid map of the paddy rice CRY1a gene knockout carrier Oscry1a-sgRNA built in the embodiment of the present invention 2.
Fig. 3 is the sequencing result of the Oscry1a gene mutation site building the dissimilar Oscry1a mutant of 11 kinds of acquisition in the embodiment of the present invention 3.
Fig. 4 is the result utilizing Basta to screen Oscry1a mutant excised leaf phenotype in the embodiment of the present invention 3, and figure below is the result of being carried out PCR detection by the special primer on T-DNA.
Fig. 5 is the Oscry1a mutant leaf sheath phenotype under blue light not containing external source T-DNA sequence in the embodiment of the present invention 3.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (SambrookJ & RussellDW, Molecularcloning:alaboratorymanual, 2001) condition of, or according to manufacturer's specification sheets advising.
Embodiment 1 has the structure of the Double CRISPR/Cas9 carrier of hygroymcin and Basta
The construction process of Double CRISPR/Cas9 carrier is as follows:
From the carrier containing 35S promoter and NOS terminator sequence, by pcr amplification 35S promoter and NOS terminator sequence, over-lap PCR is utilized 3 × flag sequence of 35S promoter and NOS terminator sequence and external synthesis to be together in series, between EcoRI and HindIII site tandem sequence being inserted into carrier pFGC5941, obtain carrier I; From the carrier containing gypsy insulator sequence, by pcr amplification gypsy insulator sequence, by increasing, the sequence obtained is inserted between EcoRI and the HindIII site of carrier I, obtains carrier II; From the carrier containing NLS-cas9-NLS sequence, by pcr amplification NLS-cas9-NLS sequence, the sequence obtained increasing is inserted into the SpeI site of carrier II, obtains carrier III; Last from the carrier containing 35S:HPT sequence, by pcr amplification 35S:HPT sequence, the sequence obtained increasing is inserted into the XbaI site of carrier III, namely builds and obtains Double CRISPR/Cas9 carrier.
Wherein, 3 × flag sequence is as shown in SEQIDNO:1, and gypsy insulator sequence is as shown in SEQIDNO:2, and NLS-cas9-NLS sequence is as shown in SEQIDNO:3, and 35S:HPT sequence is as shown in SEQIDNO:4.
The primer of amplification 35S promoter and NOS terminator sequence is respectively as shown in SEQIDNO:5-6 and SEQIDNO:7-8; The primer of amplification gypsy insulator sequence is as shown in SEQIDNO:9-10; The primer of amplification NLS-cas9-NLS sequence is as shown in SEQIDNO:11-12; The primer of amplification 35S:HPT sequence is as shown in SEQIDNO:13-14.
The plasmid map building the Double CRISPR/Cas9 carrier obtained is shown in Fig. 1, and the complete sequence of carrier is as shown in SEQIDNO:15.
The structure of embodiment 2 paddy rice CRY1a gene knockout carrier Oscry1a-sgRNA
According to paddy rice CRY1a gene order, by online CRISP-P (http://cbi.hzau.edu.cn/crispr/) design software, the sgRNA sequence that design efficiency is the highest, the nucleotides sequence of sgRNA action site is classified as 5 '-ACAGGCACCTGTCCCAGAACGG-3 '.
From the carrier containing OsU3 promoter sequence, by pcr amplification OsU3 promoter sequence, the primer of amplification OsU3 promoter sequence is as shown in SEQIDNO:19-20.
Then, from the carrier of the DNA fragmentation containing the described sgRNA sequence of coding, the DNA fragmentation of described sgRNA sequence of being encoded by pcr amplification, the primer sequence of use is as shown in SEQIDNO:21-22.
By be inserted into the Double CRISPR/Cas9 carrier of embodiment 1 together containing the coding DNA fragmentation of described sgRNA sequence and OsU3 promotor XbaI and BglII site between (DNA fragmentation of described sgRNA sequence of encoding is positioned at OsU3 promotor downstream), obtain paddy rice CRY1a gene knockout carrier Oscry1a-sgRNA, detected the exactness of insertion point and sequence by order-checking.The plasmid map of carrier Oscry1a-sgRNA is shown in Fig. 2, and the complete sequence of carrier is as shown in SEQIDNO:23.
The structure of the paddy rice CRY1a homozygous mutation body plant of embodiment 3 not containing external source T-DNA sequence
1, the acquisition of transgenic rice plant and phenotype checking
Water intaking rice ' kitaake ' mature seed, artificial or mechanical dejacketing, selects the full bright and clean seed without bacterial plaque and is inoculated on inducing culture after sterilizing and carries out inducing culture.Selection outward appearance is good, the Rice Callus that growing ability is good is acceptor material, agrobacterium-mediated transformation is adopted to proceed in Rice Callus by the carrier Oscry1a-sgRNA of embodiment 2, transform with the AAM conversion fluid being the Agrobacterium of 0.7 containing the Syringylethanone of 100 μMs and O.D. value, the callus soaked by conversion fluid is placed on Dual culture base and carries out Dual culture, 25 DEG C of light culture 3d are placed in screening culture medium and cultivate about 30d, and every 10d subculture once.Then transferred on division culture medium by the kanamycin-resistant callus tissue sifted out and break up about 20d, every 10d subculture once.The kanamycin-resistant callus tissue differentiating green seedling is transferred on root media and takes root, grow hardening after flourishing root system until about 7d, and calculating conversion institute obtains transgenic seedling number.Grown in field is transferred to after hardening 7d.
Wherein, inducing culture based formulas is: a large amount of+B5 trace+NB of N6 is organic+and molysite+copper cobalt mother liquor+2.5mg/L2,4D+0.6g/L acid hydrolyzed casein+2.878g/L proline(Pro)+0.5g/L glutamine+30g/L sucrose, with water preparation, adds plant gel 4g/L after adjusting pH to 5.8 ~ 5.9.
Dual culture based formulas is: a large amount of+B5 trace+NB of N6 is organic+and molysite+2.5mg/L2,4D+0.5g/L glutamine+0.6g/L acid hydrolyzed casein+10g/L glucose+30g/L sucrose, with water preparation, after adjusting pH to 5.2, add plant gel 4g/L.After sterilizing, about 50 DEG C add inductor AS (Syringylethanone) 100 ~ 200 μ g/mL.
Screening and culturing based formulas is: a large amount of+B5 trace+NB of N6 is organic+and molysite+copper cobalt mother liquor+2.5mg/L2,4D+0.6g/L acid hydrolyzed casein+2.878g/L proline(Pro)+0.5g/L glutamine+30g/L sucrose, with water preparation, after adjusting pH to 5.8 ~ 5.9, add plant gel 4g/L.35mg/L Totomycin (purchased from Shanghai Niu Jin Bioisystech Co., Ltd) is added after sterilizing.
Differentiation culture based formulas is: MS inorganic+MS-B5 trace+MS is organic+and molysite+MS-copper cobalt mother liquor+0.05mg/LNAA+2.0mg/LKinetin (kinetin)+30g/L sorbyl alcohol+2g/L caseinhydrolysate+30g/L sucrose, with water preparation, after adjusting pH to 5.8 ~ 5.9, add plant gel 4g/L.
2, the qualification of transgenic positive strain
In order to determine transgenic positive plant, at upstream and downstream design primer (SEQIDNO:17 and 18) of sgRNA action site, pcr amplification also checks order, and by comparing with the sequencing result of wild-type ' kitaake ', determines the mutation type of transgenic line.Because CRISPR/Cas9 leads mutagenic diversity, the Oscry1a mutant that in the present embodiment, common 11 kinds of obtaining are dissimilar.
The sequencing result of the transgenic paddy rice strain containing carrier Oscry1a-sgRNA is shown in Fig. 3.
3, Transgenic Rice is without the screening of T-DNA Insert Fragment
By the transfer-gen plant obtained through above-mentioned screening, take the 3rd leaf of full expand, spread upon on blade with the Basta solution of contain 0.05% polysorbas20 0.3%, pros and cons is smeared evenly.According to the degree of blade by green flavescence, blade is divided into anti-, half anti-and responsive three ranks, within 5 days, observes the color of blade afterwards.The blade of performance sensitive phenotype can be defined as not containing the plant that T-DNA inserts, and be detected object by the U3-sgRNA sequence on T-DNA, Auele Specific Primer (SEQIDNO:24-25) is utilized to carry out PCR detection, whether containing T-DNA sequence (Fig. 4) in DNA electrophoresis determination transfer-gen plant.Oscry1a mutant leaf sheath phenotype under blue light not containing external source T-DNA sequence is shown in Fig. 5.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. Double CRISPR/Cas9 carrier, it is characterized in that, by pcr amplification 35S promoter and NOS terminator sequence, over-lap PCR is utilized 3 × flag sequence of 35S promoter and NOS terminator sequence and external synthesis to be together in series, between EcoRI and HindIII site tandem sequence being inserted into carrier pFGC5941, obtain carrier I; By pcr amplification gypsy insulator sequence, by increasing, the sequence obtained is inserted between EcoRI and the HindIII site of carrier I, obtains carrier II; By pcr amplification NLS-cas9-NLS sequence, the sequence obtained increasing is inserted into the SpeI site of carrier II, obtains carrier III; Finally by pcr amplification 35S:HPT sequence, the sequence obtained increasing is inserted into the XbaI site of carrier III, namely builds and obtains Double CRISPR/Cas9 carrier;
Wherein, 3 × flag sequence is as shown in SEQIDNO:1, and gypsy insulator sequence is as shown in SEQIDNO:2, and NLS-cas9-NLS sequence is as shown in SEQIDNO:3, and 35S:HPT sequence is as shown in SEQIDNO:4.
2. carrier according to claim 1, is characterized in that, the primer of amplification 35S promoter and NOS terminator sequence is respectively as shown in SEQIDNO:5-6 and SEQIDNO:7-8; The primer of amplification gypsy insulator sequence is as shown in SEQIDNO:9-10; The primer of amplification NLS-cas9-NLS sequence is as shown in SEQIDNO:11-12; The primer of amplification 35S:HPT sequence is as shown in SEQIDNO:13-14.
3. carrier according to claim 1 and 2, is characterized in that, the complete sequence of Double CRISPR/Cas9 carrier is as shown in SEQIDNO:15.
4. the engineering bacteria containing carrier described in any one of claim 1-3.
5. carrier described in any one of claim 1-3 is building not containing the application in the transfer-gen plant of external source T-DNA sequence.
6. application according to claim 5, it is characterized in that, for the sgRNA sequence of the target gene design in plant based on CRISPR/Cas9, DNA fragmentation containing the described sgRNA sequence of coding is cloned in carrier described in any one of claim 1-3, conversion of plant, with hygromycin B screening positive transgenic plant, more do not contain the positive transgenic plant of external source T-DNA sequence with Basta screening.
7. paddy rice CRY1a gene knockout carrier Oscry1a-sgRNA, it is characterized in that, according to the sgRNA sequence of paddy rice CRY1a gene design based on CRISPR/Cas9, DNA fragmentation containing the described sgRNA sequence of coding is building up in carrier described in any one of claim 1-3 together with U3 promotor, obtains paddy rice CRY1a gene knockout carrier Oscry1a-sgRNA;
Wherein, the nucleotides sequence of sgRNA action site is classified as 5 '-ACAGGCACCTGTCCCAGAACGG-3 '.
8. carrier according to claim 7, is characterized in that, the complete sequence of carrier Oscry1a-sgRNA is as shown in SEQIDNO:23.
9. the construction process of the paddy rice CRY1a homozygous mutation body plant not containing external source T-DNA sequence, it is characterized in that, adopt agriculture bacillus mediated method, carrier described in claim 7 or 8 is proceeded in Rice Callus, transform with the AAM nutrient solution containing inductor and Agrobacterium, material after conversion is through the exercise of Dual culture-screen-break up-take root-transgenic seedling and transplanting, screening transgenic rice plant, first obtain positive transgenic plant with hygromycin B screening, the paddy rice CRY1a homozygous mutation body plant not containing external source T-DNA sequence is obtained again with Basta screening.
10. method according to claim 9, is characterized in that, according to the nucleotide sequence design pair of primers of sgRNA action site, by PCR method qualification rice plant mutational site; Primer sequence is as shown in SEQIDNO:17-18.
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Cited By (25)
| 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 |
| US9840699B2 (en) | 2013-12-12 | 2017-12-12 | President And Fellows Of Harvard College | Methods for nucleic acid editing |
| 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 |
| CN109402157A (en) * | 2018-12-05 | 2019-03-01 | 四川省农业科学院经济作物育种栽培研究所 | A kind of prokaryotic expression carrier and application with twin antibiotic selection markers |
| US10227581B2 (en) | 2013-08-22 | 2019-03-12 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| 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 |
| 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 |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses 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 |
| WO2023022226A1 (en) * | 2021-08-20 | 2023-02-23 | グランドグリーン株式会社 | Polynucleotide including site-specific nuclease expression cassette |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| 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 |
-
2015
- 2015-12-24 CN CN201510992210.8A patent/CN105543270A/en active Pending
Non-Patent Citations (10)
| Title |
|---|
| HIROSE F ET AL: "Oryza sativa Japonica Group OsCRY1a mRNA for cryptochrome 1a, complete cds", 《GENBANK登录号：AB073546》 * |
| HUI-LI XING ET AL: "A cripsr/cas9 toolkit for multiplex genome editing in plants", 《BMC PLANT BIOLOGY》 * |
| KERSCHEN A ET AL: "Effectiveness of RNA interference in transgenic plants", 《FEBS LETTERS》 * |
| NAPOLI CA ET AL: "Binary vector pFGC5941 phosphinothricin acetyl transferase (BAR) and aminoglycoside", 《GENBANK登录号：AY310901》 * |
| YANG LEI ET AL: "CRISPR-P: a web tool for synthetic single guide RNA design of CRISPR-System in plants", 《MOLECULAR PLANT》 * |
| YU LI ET AL: "Effects of suppressing OsCRY1a gene expression on rice agronomic traits", 《RICE SCIENCE》 * |
| 佘文静: "果蝇gypsy绝缘子有利于拟南芥转基因高效而精确地表达", 《中国优秀硕士学位论文全文数据库 基础科学辑 A006-190》 * |
| 李春艳 等: "多选择标记植物表达载体的构建", 《基因组学与应用生物学》 * |
| 李欣 等: "转bar基因小麦和非转基因小麦抗除草剂鉴定方法比较", 《植物遗传资源学报》 * |
| 韩秀丽 等: "大麦转化中潮霉素和草丁膦的筛选效果研究", 《山东农业大学学报（自然科学版）》 * |
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