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