Cas9 protein binary expression vector as well as construction method, application and transformation system thereof

Abstract

The invention discloses a Cas9 protein binary expression vector as well as a construction method, application and a transformation system thereof, a binary expression vector GPiE-egfp is taken as a basis, and an eGFP (enhanced green fluorescent protein) coding gene of the binary expression vector GPiE-egfp is replaced by an optimized Cas9 protein coding gene to obtain the Cas9 protein binary expression vector. The invention relates to a Cas9 protein coding gene promoter, which is provided with an element capable of being copied and amplified in escherichia coli and agrobacterium, LB T-DNA and RB T-DNA capable of carrying exogenous genes, a promoter Glgpdp of a constitutive expression gene gpd in ganoderma lucidum, and an optimized Cas9 protein coding gene. The N terminal of the optimized cas9 gene comprises the first intron of Glgpd and the first three amino acid coding sequences of the first exon, totally 76 basic groups and a 3*FLAG tag, and at the same time, a nuclear localization signal coding sequence NLS is added at each of the N terminal and the C terminal of the cas9 gene. The codon-optimized Cas9 protein binary expression vector disclosed by the invention is suitable for expressing Cas9 protein in filamentous fungus mushroom.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and in particular relates to a Cas9 protein binary expression vector capable of overexpressing the Cas9 protein in higher filamentous fungus Lentinus edodes and its construction method, application and transformation system. Background technique [0002] CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) system is the third-generation gene editing technology following ZNF and TALENS technology, and is widely used in prokaryotic and eukaryotic cell level and individual level gene editing cell lines. [0003] Compared with other gene editing technologies, such as new finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), CRISPR / Cas9 is simpler to operate and easier to target multiple loci in the same cell at the same time , the system could become a new, safer and less toxic alternative to current genome engineering methods. [0004] So far, ...

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Problems solved by technology

[0006] The present invention provides an optimized binary expression vector capable of overexpressing Cas9 protein in higher filamentous fungus Lentinus edodes, and solves the technical problem that Cas9 protein cannot be expressed in Lentinus edodes

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