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Application for using LbCpf1-RR mutant in CRISPR/Cpf1 system in plant gene editing

A technology of gene editing and mutants, applied in the fields of application, plant peptides, plant products, etc., can solve problems such as unfavorable CRISPR/Cpf1 applications

Active Publication Date: 2018-09-04
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the limitation of the recognized PAM site sequence is not conducive to the wider application of CRISPR / Cpf1

Method used

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  • Application for using LbCpf1-RR mutant in CRISPR/Cpf1 system in plant gene editing
  • Application for using LbCpf1-RR mutant in CRISPR/Cpf1 system in plant gene editing
  • Application for using LbCpf1-RR mutant in CRISPR/Cpf1 system in plant gene editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1, LbCpf1-RR mutants are used in the application of CRISPR / Cpf1 system in plant gene editing

[0069] The target gene, target point name and sequence in this embodiment are shown in Table 2.

[0070] Table 2

[0071]

[0072] 1. Construction of expression vector

[0073] 1. Construction of plasmid pCXUN-LbCpf1(RR)

[0074] (1) The plasmid pCXUN-Cas9 was double digested with restriction endonucleases BamHI and HindIII to obtain a vector backbone 1 of about 9282bp.

[0075] (2) The LbCpf1-OsU6 vector was double-digested with restriction endonucleases BamHI and HindIII to obtain a Ubi-LbCpf1 expression cassette of about 5846 bp.

[0076] (3) Ligate the vector backbone 1 and the Ubi-LbCpf1 expression cassette with T4 ligase to obtain the plasmid pCXUN-LbCpf1.

[0077] (4) Using the plasmid pCXUN-LbCpf1 as a template, use three primer pairs (the first primer pair consists of BstEII-F and LbCpf1(RR)-532-R, the second primer pair consists of LbCpf1(RR)-532- F a...

Embodiment 2

[0144] Example 2, LbCpf1-RVR mutants are used in the application of CRISPR / Cpf1 system in plant gene editing

[0145] The target gene, target point name and sequence in this embodiment are shown in Table 4.

[0146] Table 4

[0147]

[0148] 1. Construction of expression vector

[0149] Artificially synthesized recombinant vector pCXUN-LbCpf1(RVR)-OsU3-RCR1-RCR2(PDS).

[0150] The only difference between the recombinant vector pCXUN-LbCpf1(RVR)-OsU3-RCR1-RCR2(PDS) and the recombinant vector pCXUN-LbCpf1(RR)-OsU3-RCR1-RCR2(PDS) is that the expression of OsU3-RCR1-RCR2(PDS) The cassette was replaced with the expression cassette A, and the nucleotide sequence encoding the LbCpf1-RR mutant was replaced with the nucleotide sequence encoding the LbCpf1-RVR mutant. The nucleotide sequence of the expression cassette A is shown in sequence 5 in the sequence listing.

[0151] The only difference between the recombinant vector pCXUN-LbCpf1(RVR)-OsU3-RCR1-RCR2(SBEIIb) and the recombi...

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Abstract

The invention discloses application for using an LbCpf1 - RR mutant in a CRISPR / Cpf1 system in plant gene editing. The application for using the LbCpf1 - RR mutant in the CRISPR / Cpf1 system in plant gene editing uses an OsPDS gene and an OsSBEIIb gene as target genes, constructs a target double loci of one gene and a series of vectors of two genes, and utilizes an agrobacterium-mediated transformation method to import the vectors into rice callus, and rice plants with the target gene knockout are successfully obtained by using the LbCpf1-RR mutant. The only difference between the LbCpf1-RR mutant and the protein LbCpf1 is that the 532nd amino acid changes from G to R, and the 595th amino acid changes from K to R. The LbCpf1-RR mutant provided by the application for using the LbCpf1 - RR mutant in the CRISPR / Cpf1 system in plant gene editing expands the PAM site sequence identified by the LbCpf1-RR mutant, so that the editing range of the CRISPR / Cpf1 system in rice genome is expanded, great significance for promoting the application of the system in the field of plant genome editing is achieved. The application for using the LbCpf1 - RR mutant in the CRISPR / Cpf1 system in plant geneediting has great application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of LbCpf1-RR mutants in CRISPR / Cpf1 system in plant gene editing. Background technique [0002] CRISPR / Cas9-mediated genome editing technology has become one of the most powerful tools in molecular biology and has been widely used in functional gene improvement of plants and crops. The CRISPR / Cas9 system was first discovered in bacteria and consists of two parts, sgRNA and Cas9 protein (Jinek et al., 2012). Cas9 protein edits any 20bp target sequence immediately following PAM (NGG) through its own endonuclease activity, thereby causing double-strand breaks (double-strand breaks, DSBs) in the genomic DNA sequence of the target site, and then passing Mutations were introduced in two ways: non-homologous end-joining (NHEJ) or homologous recombination-mediated repair (homology-directed repair, HDR). Currently, the commonly used Cas9 protein is SpCas9 and its various mut...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/62A01H5/00A01H6/46
CPCC07K14/415C07K2319/00C12N15/8218C12N2800/80C12N2810/10
Inventor 夏兰琴李少雅赵云德张欣王文生杜文明
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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