title: Biallelic insertion of a transcriptional terminator via the CRISPR/Cas9 system efficiently silences expression of protein-coding and non-coding RNA genes.
abstract: 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 ofPPP1R12C,NSUN2(protein-coding genes), andMALAT1(non-protein-coding gene). BothNeoRandPuroRwere 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.
authors.affiliation: From the Institute of Epigenetics and Epigenomics.|College of Animal Science and Technology.
authors.forename: Yangyang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: From the Institute of Epigenetics and Epigenomics.|College of Animal Science and Technology.
authors.forename: Xiao
authors.initials: X
authors.lastname: Han
authors.affiliation: From the Institute of Epigenetics and Epigenomics.|College of Bioscience and Biotechnology.
authors.forename: Junting
authors.initials: J
authors.lastname: Yuan
authors.affiliation: From the Institute of Epigenetics and Epigenomics.|College of Animal Science and Technology.
authors.forename: Tuoyu
authors.initials: T
authors.lastname: Geng
authors.affiliation: From the Institute of Epigenetics and Epigenomics.|College of Animal Science and Technology.
authors.forename: Shihao
authors.initials: S
authors.lastname: Chen
authors.affiliation: From the Institute of Epigenetics and Epigenomics.|College of Animal Science and Technology.
authors.forename: Xuming
authors.initials: X
authors.lastname: Hu
authors.affiliation: the Department of Pathology and Laboratory Medicine, New York Presbyterian-Weill Cornell Medicine, New York, New York 10065.
authors.forename: Isabelle H
authors.initials: IH
authors.lastname: Cui
authors.affiliation: From the Institute of Epigenetics and Epigenomics, hmcui@yzu.edu.cn.|College of Animal Science and Technology.|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.
authors.forename: Hengmi
authors.initials: H
authors.lastname: Cui
mesh_terms: D000818:Animals; D002417:Cattle; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D020868:Gene Silencing; D057809:HEK293 Cells; D006801:Humans; D008780:Methyltransferases / Q000096:biosynthesis* / Q000235:genetics; D054645:Protein Phosphatase 1 / Q000096:biosynthesis* / Q000235:genetics; D062085:RNA, Long Noncoding / Q000096:biosynthesis* / Q000235:genetics; D013728:Terminator Regions, Genetic
chemical_list: C557041:MALAT1 long non-coding RNA, human; D062085:RNA, Long Noncoding; D008780:Methyltransferases; C515610:NSUN2 protein, human; C540874:PPP1R12C protein, human; D054645:Protein Phosphatase 1
keywords: CRISPR/Cas; gene knockout; gene silencing; long non-coding RNA (long ncRNA, lncRNA); polyadenylation