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[Construction of all-in-one CRISPR/Cas9 vector system targeting miR-101a gene in mouse hepatic cell line AML12].

OBJECTIVE
To develop an all-in-one CRISPR/Cas9 vector system that can efficiently knockdown miR-101a expression in mice.


METHODS
Three sgRNAs targeting mouse miR-101a gene and a small guide (sgRNA) targeting green fluorescent protein gene were designed and constructed into an all-in-one vector system (pENTRY-U6-sgRNA-WT Cas9). Moreover, sgRNA1 and sgRNA3 were selected and constructed into a double-nicking Cas9 vector (pENTRY-U6-sgRNA-U6-sgRNA-Cas9 D10A). The constructed plasmids were transfected into mouse liver AML12 cells for validation by T7 EndoⅠ(T7EⅠ) 72 h after transfection. The pAD vectors were cloned via the Gateway system, and the recombinant adenovirus vectors were packaged in 293A cells. The virus particles were used to infect AML12 cells and the expression levels of mature miR-101a were analyzed to monitor the knockout efficiency after 72 h.


RESULTS
The constructed pENTRY all-in-one vectors were validated by gene sequencing and T7EⅠ assay, which showed CRISPR/Cas9-mediated mismatches at target sites of miR-101a gene. The adenovirus vectors were constructed successfully. The CRISPR/Cas9 containing adenovirus was introduced to AML12 cells and the quantitative real-time PCR assays indicated that the expression level of mature miR-101a was significantly decreased compared with that of the control (all P<0.01).


CONCLUSIONS
We have successfully constructed two "all-in-one" CRISPR/Cas9 vector systems targeting miR-101a gene in mouse liver AML12 cells with high efficiency. It provides experimental basis for research of microRNA, and a reference method for knockout of other miRNAs.