Application of CRISPR/nCas9 mediated site-directed base substitution in plant

Abstract

The invention discloses application of CRISPR / nCas9 mediated site-directed base substitution in a plant. The invention provides a plant genome site-directed edition system. The system comprises a BE3 plant expression carrier (expressing a fusion protein composed of nCas9(D10A), deaminase and a uracil DNA glycosylase inhibitory protein), and rice OsPDS and OsSBEIIb are taken as target genes for verifying the system. Results show that an expected site-directed mutant plant is respectively obtained in the three selected target spots, accurate site mutation of a base is realized in rice, and the highest efficiency reaches about 20%, so that a feasible and effective base substitution method is provided for crop breeding, the method has strong application potential in the aspect of agricultural breeding, and a foundation is provided for rapidly improving important agronomic traits of crops.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an application of CRISPR / nCas9-mediated site-directed base replacement in plants. Background technique [0002] CRISPR / Cas9-mediated genome editing technology has become one of the most powerful tools in molecular biology. Found in bacteria for the first time, it consists of two parts, sgRNA and Cas9 (Jinek et al., 2012). CRISPR / Cas9 causes double-strand breaks (double-strand breaks, DSBs) at the target site through its own endonuclease activity, and then through non-homologous end joining (NHEJ) or homologous recombination Homology-directed repair (HDR) introduces mutations in two ways. Most of the mutations induced by the NHEJ pathway are insertions or deletions of nucleotides, resulting in frameshift mutations, while in HDR, fragment insertions or nucleotide corrections are mediated by homologous donor DNA (Jinek et al., 2012). The recognition of target sites by the...

AI Technical Summary

Problems solved by technology

A large number of agronomic traits in crops are caused by mutations of single bases. After the introduction of DSB by traditional CRISPR / Cas9 technology, HDR always occurs at a rather low frequency compared with the random process of non-homologous end joining. Only a few reports show that CRISPR / Cas9-mediated HDR is feasible in crops (Li et al., 2015; Svitashev et al., 2015; Endo et al., 2016; Shi et al., 2016; Sun et al., 2016), making a large number of Agronomic traits cannot be improved quickly

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