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SgRNA Expression of CRIPSR-Cas9 System Based on MiRNA Polycistrons as a Versatile Tool to Manipulate Multiple and Tissue-Specific Genome Editing.

Abstract：

:CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications. Typically, sgRNA is constitutively expressed relying on RNA polymerase III promoters. Polymerase II promoters initiate transcription in a flexible manner, but sgRNAs generated by RNA polymerase II promoter lost their nuclease activity. To express sgRNAs in a tissue-specific fashion and endow CRISPR with more versatile function, a novel system was established in a polycistron, where miRNAs (or shRNAs) and sgRNAs alternately emerged and co-expressed under the control of a single polymerase II promoter. Effective expression and further processing of functional miRNAs and sgRNAs were achieved. The redundant nucleotides adjacent to sgRNA were degraded, and 5'- cap structure was responsible for the compromised nuclease capacity of sgRNA: Cas9 complex. Furthermore, this strategy fulfilled conducting multiplex genome editing, as well as executing neural- specific genome editing and enhancing the proportion of homologous recombination via inhibiting NHEJ pathway by shRNA. In summary, we designed a new construction for efficient expression of sgRNAs with miRNAs (shRNAs) by virtue of RNA polymerase II promoters, which will spur the development of safer, more controllable/regulable and powerful CRISPR/Cas9 system-mediated genome editing in a wide variety of further biomedical applications.

journal_name

Sci Rep

journal_title

Scientific reports

authors

Xie C,Chen YL,Wang DF,Wang YL,Zhang TP,Li H,Liang F,Zhao Y,Zhang GY

doi

10.1038/s41598-017-06216-w

subject

Has Abstract

pub_date

2017-07-19 00:00:00

pages

5795

issue

1

issn

2045-2322

pii

10.1038/s41598-017-06216-w

journal_volume

7

pub_type

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