CRISPR/Cas9 single transcription unit directionally modified backbone vector and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and concretely relates to a CRISPR / Cas9 single transcription unit directionally modified backbone vector and an application thereof. Technical problems to be solved in the invention comprise low species versatility and difficult realization of Cas9 protein expression and gRNA transcription cooperation of present CRISPR / Cas9 genome editing systems. A technical scheme in the invention is characterized in that a CRISPR / Cas9 single transcription unit backbone vector is constructed, and transcription of Cas9 and a guide RNA core unit are regulated by a promoter. The invention also provides a method for constructing a target site specifically modified Cas9-gRNA recombinant vector by adopting the CRISPR / Cas9 single transcription unit backbone vector. The efficient CRISPR / Cas9 single transcription unit backbone vector can effectively realize Pol II promoter driving-based Cas9 and gRNA unit cooperative transcription, and also can realize simple, fast and efficient genome directional heredity modification of various eukaryotes.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a CRISPR / Cas9 single transcription unit directional modification backbone carrier and application thereof. Background technique [0002] In recent years, with the advancement of model animal and plant genome sequencing projects and the improvement of related gene manipulation technologies, sequence-specific nucleases (zinc-fingernuclease, ZFN; transcription activator-like effectors nuclease, TALEN; clustered regularly interspaced short palindromic repeats / CRISPR- associatedprotein-9, CRISPR / Cas9), which can cause DSBs at specific sites in the genome of the target organism, and under the action of the endogenous DNA repair system of the organism, different types of directional genetic modification of the target genome can be achieved. [0003] In the CRISPR / Cas9 genome editing system, the specific cleavage of the genome target sequence by CRISPR / Cas9 mainly...

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Problems solved by technology

[0005] However, in the above CRISPR / Cas9 genome editing system, there are inherent defects in the design of the independent Cas9 protein expression unit and gRNA transcription unit, and it is difficult to achieve synergy between Cas9 protein expression and gRNA transcription

At the same time, since the transcription of gRNA units basically depends on PolIII-type promoters such as U6 and U3 with strong species preference, it is necessary to screen specific PolIII-type small RNA transcription promoters for different target organisms, and it is also difficult to achieve spatiotemporal specificity and induce transcriptional regulation , which greatly limits the working efficiency and application scope of CRISPR / Cas9 in targeted genome modification

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