Detection of CRISPR-Cas9 off-target activity using amplification-free long read sequencing

A crucial part of any CRISPR-Cas9 genome editing experiment is to design a guide RNA (gRNA) that specifically binds to the target of interest, while not binding to any unintended off-target sites. However, accurate prediction of CRISPR-Cas9 off-target sites is challenging. In this study, we developed new methods for Nanopore (OTS-Nano) and SMRT sequencing (OTS-SMRT) based detection of off-target sites. The methods were applied to study gRNA binding and CRISPR-Cas9 activity in human DNA samples and cells.