Biallelic insertion of a transcriptional terminator via the CRISPR/Cas9 system efficiently silences expression of protein-coding and non-coding RNA genes
| Autor: | Yangyang Liu, Shihao Chen, Junting Yuan, Hengmi Cui, Tuo-yu Geng, Xu-ming Hu, Isabelle H. Cui, Xiao Han |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Biology Biochemistry 03 medical and health sciences Protein Phosphatase 1 Gene silencing CRISPR Animals Humans Editors' Picks Clustered Regularly Interspaced Short Palindromic Repeats Gene Silencing Molecular Biology Gene Genetics Regulation of gene expression Terminator Regions Genetic CRISPR interference Cas9 Intron Cell Biology Methyltransferases 030104 developmental biology Terminator (genetics) HEK293 Cells Cattle RNA Long Noncoding |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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