An efficient method to enrich for knock-out and knock-in cellular clones using the CRISPR/Cas9 system.

An efficient method to enrich for knock-out and knock-in cellular clones using the CRISPR/Cas9 system. Cell Mol Life Sci. 2017 Apr 18;: Authors: Niccheri F, Pecori R, Conticello SG Abstract Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9 nuclease (CRISPR/Cas9) and Transcription Activator-Like Effector Nucleases (TALENs) are versatile tools for genome editing. Here we report a method to increase the frequency of Cas9-targeted cellular clones. Our method is based on a chimeric construct with a Blasticidin S Resistance gene (bsr) placed out-of-frame by a surrogate target sequence. End joining of the CRISPR/Cas9-induced double-strand break on the surrogate target can place the bsr in frame, thus providing temporary resistance to Blasticidin S: this is used to enrich for cells where Cas9 is active. By this approach, in a real experimental setting, we disrupted the Aicda gene in ~70% of clones from CH12F3 lymphoma cells (>40% biallelically). With the same approach we knocked in a single nucleotide to reconstruct the frame of Aicda in these null cells, restoring the function in ~37% of the clones (less than 10% by the standard approach). Targeting of single nucleotide changes in other genes yielded analogous results. These results support our enrichment method as an efficient tool in genome editing. PMID: 28421278 [PubMed - as supplied by publisher]
Source: Cellular and Molecular Life Sciences : CMLS - Category: Cytology Authors: Tags: Cell Mol Life Sci Source Type: research
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