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J Biol Chem. 2017 Apr 7;292(14):5624-5633. doi: 10.1074/jbc.M116.769034. Epub 2017 Feb 14.

Biallelic insertion of a transcriptional terminator via the CRISPR/Cas9 system efficiently silences expression of protein-coding and non-coding RNA genes.

Liu Y1,2, Han X1,2, Yuan J1,3, Geng T1,2, Chen S1,2, Hu X1,2, Cui IH4, Cui H5,2,6,7.

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From the Institute of Epigenetics and Epigenomics.
College of Animal Science and Technology.
College of Bioscience and Biotechnology.
the Department of Pathology and Laboratory Medicine, New York Presbyterian-Weill Cornell Medicine, New York, New York 10065.
From the Institute of Epigenetics and Epigenomics,
Institute of Comparative Medicine.
Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, Jiangsu 225009, China and.


The type II bacterial CRISPR/Cas9 system is a simple, convenient, and powerful tool for targeted gene editing. Here, we describe a CRISPR/Cas9-based approach for inserting a poly(A) transcriptional terminator into both alleles of a targeted gene to silence protein-coding and non-protein-coding genes, which often play key roles in gene regulation but are difficult to silence via insertion or deletion of short DNA fragments. The integration of 225 bp of bovine growth hormone poly(A) signals into either the first intron or the first exon or behind the promoter of target genes caused efficient termination of expression of PPP1R12C, NSUN2 (protein-coding genes), and MALAT1 (non-protein-coding gene). Both NeoR and PuroR were used as markers in the selection of clonal cell lines with biallelic integration of a poly(A) signal. Genotyping analysis indicated that the cell lines displayed the desired biallelic silencing after a brief selection period. These combined results indicate that this CRISPR/Cas9-based approach offers an easy, convenient, and efficient novel technique for gene silencing in cell lines, especially for those in which gene integration is difficult because of a low efficiency of homology-directed repair.


CRISPR/Cas; gene knockout; gene silencing; long non-coding RNA (long ncRNA, lncRNA); polyadenylation

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