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... 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cellular and Molecular Life Sciences Springer Journals

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

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Publisher
Springer International Publishing
Copyright
Copyright © 2017 by The Author(s)
Subject
Life Sciences; Cell Biology; Biomedicine, general; Life Sciences, general; Biochemistry, general
ISSN
1420-682X
eISSN
1420-9071
D.O.I.
10.1007/s00018-017-2524-y
Publisher site
See Article on Publisher Site

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