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Application of CRISPR/Cas12a gene editing system in physcomitrella patens gene editing

A technology of Physcomitrella patens and gene editing, applied in the field of genetic engineering, can solve the problems that the efficiency of gene knockout mutants is less than 20%, and the acquisition of multi-gene knockout mutants is limited, and the probability of off-target is small and high Effects of Multiple Gene Editing Efficiency

Active Publication Date: 2019-05-31
KUNMING INST OF BOTANY - CHINESE ACAD OF SCI
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  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, the gene knockout method for Physcomitrella patens mainly uses homologous recombination, but the efficiency of this method to obtain gene knockout mutants is less than 20%.
However, multiple gene knockout requires different resistance combinations, which limits the acquisition of multiple gene knockout mutants.

Method used

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  • Application of CRISPR/Cas12a gene editing system in physcomitrella patens gene editing

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Embodiment 1

[0070] Example 1 Application of CRISPR / Cas12a gene editing system in gene editing of Physcomitrella patens

[0071] 1. Cultivation method of Physcomitrella patens

[0072] BCDAT medium was used to cultivate moss, and the material of Physcomitrella patens was polished and subcultured under the photoperiod of 16h light / 8h dark, 80μmol photons m -2 the s -1 Under the condition of light intensity of 25°C, cultured for 5 days, uniform young protonema were formed.

[0073] 2. Construction of Cas12a protease expression vector

[0074] A nuclear localization signal (Nucleus Location Signal, NLS) is added to both ends of the nucleic acid sequence encoding Cas12a, and the Cas12a nucleotide sequence with nuclear localization signals at both ends is synthesized by Shanghai Jierui Bioengineering Co., Ltd. The Cas12a fragment of the nuclear localization signal was ligated to the plasmid pAct-Cas9 by enzyme cleavage ligation, the enzyme cleavage sites were NcoI and NcoI, and the expressio...

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Abstract

The invention provides application of a CRISPR / Cas12a gene editing system in physcomitrella patens gene editing, and belongs to the technical field of gene engineering. The application comprises the following steps that 1, a Cas12a protease expression vector is constructed; 2, a gRNA expression vector is constructed; 3, the Cas12a protease expression vector, the gRNA expression vector and resistance expression plasmids are used for converting physcomitrella patens, and resistance screening is carried out to obtain mutant plants. The CRISPR / Cas12a gene editing system applied to physcomitrella patens gene editing has the advantages of being high in gene editing efficiency, small in target missing probability and especially capable of achieving efficient editing of multiple targets.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the application of the CRISPR / Cas12a gene editing system in gene editing of Physcomitrella patens. Background technique [0002] Physcomitrella patens (scientific name: Physcomitrella patens) belongs to the family Cucurbitaceae and the genus Physcomitrella patens. It is distributed in Europe, Asia, Africa and Oceania, and is distributed in Zhangjiajie, Hunan Province, China. [0003] The growth of Physcomitrella patens requires simple nutrients and is easy to cultivate; its gametophyte is dominant in its life cycle, and the phenotype of its mutants can be directly studied; its nuclear genome is prone to high-frequency occurrence of exogenous DNA with homologous fragments Homologous recombination makes precise gene disruption and gene knockout possible, and provides good materials for the study of gene function. Physcomitrella patens has many similar characteristics w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N15/82A01H5/00
CPCC12N9/22C12N15/8213C12N2310/20C12N15/11C12N15/8202
Inventor 刘莉普晓俊刘丽娜李萍杨红
Owner KUNMING INST OF BOTANY - CHINESE ACAD OF SCI
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