Monday, April 21, 2014

Quantifying Genome-Editing Outcomes with SMRT Sequencing #CRISPR

Been thinking recently about the most practical way to thoroughly assess off-target mutagenesis… Seems like a good job for a medium-throughput-scale next-gen sequencing.   The workflow could be 1. identify the off-targets, to a depth of ~ 4 mismatches; these will number in the dozens at least.  2. PCR all off-targets from CRISPR'd sample.  3. NGS, but we probably won't need millions of reads; thousands will do, especially for live founder mice and certainly for testing their progeny.   This recent paper is a good example of quantifying editing at the on-target site, but could obviously be adapted to analyzing off-targets too:

Ayal Hendel, Eric J. Kildebeck,  Eli J. Fine, Joseph T. Clark, Niraj Punjya, Vittorio Sebastiano,Gang Bao, Matthew H. Porteus
Cell Reports, Volume 7, Issue 1, p293–305, 10 April 2014

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