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
Monday, April 21, 2014
Quantifying Genome-Editing Outcomes with SMRT Sequencing tinyurl.com/nx9l6sb #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:
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