New developments in CRISPR technology, with a focus on mouse and human cell applications.
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: