Biallelic Insertion of a Transcriptional Terminator via CRISPR/Cas9 Efficiently Silences Expression of Protein-coding and Non-coding RNA Genes [Methods and Resources]

The type II bacterial CRISPR/Cas9 system is a simple, convenient and powerful tool for gene editing that allows for gene targeting. Here, we describe a CRISPR/Cas9-based approach for silencing both protein-coding and non-protein-coding genes by biallelic integration of a PolyA signal. Because of the integration of a BGH PolyA signal into the target genes, gene expression was drastically terminated. Two anti-antibiotic genes were used as markers in the selection of clonal cell lines with biallelic integration of a PolyA signal. Genotyping analysis indicated that 65-93% of the cell lines displayed the desired biallelic silencing after a short time selection. In conclusion, we, for the first time, established an efficient CRISPR/Cas9-based approach to gene silencing using biallelic integration of a PolyA signal with the advantages of double marker selection. It is an easy, convenient and efficient novel technique for gene silencing in cell line. This approach may be especially suitable for cell lines, in which gene integration is difficult due to low efficiency of homology-directed repair.

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