Method for rapidly establishing Cas9 dual-element expression carrier library of paired sg RNA

Abstract

The invention discloses a method for rapidly establishing a Cas9 dual-element expression carrier library of paired sg RNA, and relates to a method for rapidly establishing a dual-element carrier in the gene engineering field. According to the method, on the basis of establishing a CRISPR-Cas9 dual-element expression carrier, corresponding positive and negative primers are designed according to a specific gene design target sequence and have two BsaI restriction enzyme cutting sites; through one-step PCR, the target sequence and target segments of two BsaI sites are obtained, and the segments enter the dual-element expression carrier with a Cas9 gene through a simple restriction enzyme cutting and connecting mode to form the Cas9 carrier of the paired sg RNA. A random library and a non-random library can be efficiently established and can be used for large-scale screening of gene functions in plants and meanwhile can be used for establishment and research of a gene interoperation network among multiple genes; it is proved through experimental results that the Cas9 dual-element expression carrier library of the randomly-paired sg RNA of 14 target spots (from 14 genes) is successfullyand efficiently established through the method.

Description

technical field [0001] The invention relates to a method for rapidly constructing a binary vector in the field of genetic engineering, in particular to a method for rapidly constructing a Cas9 binary expression vector library paired with sgRNA. Background technique [0002] Based on the convenience and high efficiency of the CRISPR / Cas9 system, it has been successfully used for genome editing in eukaryotes and prokaryotes, especially in plants and animals. By further improving and optimizing the system, the system has been successfully used in gene transcription regulation, cell tracking, fluorescent labeling, epigenetic modification and single base mutation, etc. When this system is used for gene mutation, large deletion and non-coding gene function research, it is often necessary to express two sgRNAs simultaneously. For large-scale application, it is necessary to construct an expression library of sgRNA. A previous study proposed a method for rapidly constructing a Cas9...

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Problems solved by technology

But so far there is no method for constructing a paired sgRNA Cas9 binary expression vector library for plants

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