Staying on target has always been the CRISPR/Cas9 claim to genome editing fame, leading to incredible breakthroughs in the application of this bacterial immune system to mammals and as a cure for inherited disease. But now a sharpshooting team from the University of Virginia have used a clever application of ChIP-Seq to show that sometimes Cas9 can cause off-target effects. Here’s what they scoped out:
- Utilizing antibodies for Cas9, the team used some clever ChIP-seq to map the genomic binding sites with their deactivated version of the Cas9 (dCas9) ‘cleaver’.
- dCas9 was teamed up with one of 12 different ‘target hunting’ single guide RNAs (sgRNAs) to examine the dynamics of the genome editing system.
- The team found dCas9 bound to a number of off-target sites.
- The off-target sites ranged from 10-1,000s, with the sgRNA appearing to be the deciding factor.
- Analyzing the off-target sites, the team found some defining characteristics for missing the mark, including a preference for open chromatin regions.
When the team checked out how catalytically active Cas9 performed, they found that these mismatches weren’t just noise. The off-target binding sites showed indels above the background levels, but thankfully the rates were generally lower than at the desired on-target sites the sgRNAs were ‘gunning’ for. So when it comes down it, these findings lay the groundwork for understanding the deciders of CRISPR/Cas9 targeting while also highlighting the use of ChIP-seq for unbiased detection of both the on and off targets effects of genome editing.
Make sure your genome editing targets line up in Nature Biotechnology, May 2014