A CRISPR/NCas9-mediated site-directed base replacement in plants

Abstract

A CRISPR / nCas9 mediated site-directed base substitution applied to plants. The site-directed base substitution is achieved by a system of site-directed editing plant genomes, and the system comprises a BE3 plant expression vector expressing a fusion protein composed of nCas9 (D10A), deaminase, and a uracil DNA glycosylase inhibitory protein, and the system was verified by using rice OsPDS and OsSBEIIb as target genes. The results showed that among three selected target sites, expected site-directed mutant plants were all obtained, accurate site mutation of the base in rice was achieved, and the highest efficiency reached about 20%, thus providing a feasible and effective method of base substitution for crop breeding. The method has strong potential for application in agricultural breeding and provides a basis for rapidly improving important agronomic traits of crops.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the application of a 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. It was first discovered in bacteria and consists of two parts, sgRNA and Cas9 (Jinek et al., 2012). CRISPR / Cas9 induces double-strand breaks (DSBs) in the target DNA sequence through its own endonuclease activity, followed by non-homologous end joining (NHEJ) or homologous recombination. There are two ways to introduce mutations by homology-directed repair (HDR). Most of the mutations induced by the NHEJ pathway are nucleotide insertions or deletions, resulting in frameshift mutations, while HDR is mediated by homologous donor DNA mediated fragment insertion or nucleotide correction (Jinek et al., 2012). The recognition of the target site by the CRISPR / Cas9 s...

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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