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Use of lbcpf1-rr mutants for CRISPR/cpf1 system in plant gene editing
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A technology of mutants and plants, applied in the fields of applications, plant peptides, plant products, etc., can solve problems such as unfavorable CRISPR/Cpf1 applications
Active Publication Date: 2021-02-19
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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However, the limitation of the recognized PAM site sequence is not conducive to the wider application of CRISPR / Cpf1
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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.
[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 present invention discloses the application of LbCpf1-RR mutant for CRISPR / Cpf1 system in plant gene editing. The present invention uses the OsPDS gene and the OsSBEIIb gene as target genes, constructs a series of vectors targeting a double site of a gene and two genes, and uses the Agrobacterium transformation method to introduce the vector into the rice callus, and utilizes the LbCpf1‑RR mutant The rice plants knocked out of the target gene were successfully obtained. The only difference between the LbCpf1‑RR mutant and the protein LbCpf1 is that the amino acid at position 532 is changed from G to R, and the amino acid at position 595 is changed from K to R. The LbCpf1-RR mutant provided by the present invention expands the editing range of the CRISPR / Cpf1 system in the rice genome due to the expansion of the PAM site sequence it recognizes, which is of great significance for promoting the application of this system in the field of plant genome editing . The invention 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...
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