An extensively debated concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target sites and activity is challenging. We have developed a new protocol for detection of gRNA binding and Cas9 cleavage, based on amplification-free long-read sequencing. This method, which is named “off-target sequencing” (OTS), was assessed using a human cell line that was first re-sequenced using long and highly accurate reads to get a detailed view of all on- and off-target binding regions. The OTS method detected many Cas9 cleavage sites that were not reported by off-target prediction software, including in “dark” regions of the human genome inaccessible by standard short-read sequencing. We are currently investigating whether in vivo off-target editing can occur at these sites.
• Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity. Höijer I, Johansson J, Gudmundsson S, Chin CS, Bunikis I, Häggqvist S, Emmanouilidou A, Wilbe M, den Hoed M, Bondeson ML, Feuk L, Gyllensten U, Ameur A. bioRxiv 2020.02.09.940486; doi: https://doi.org/10.1101/2020.02.09.940486
• Single-Molecule Sequencing: Towards Clinical Applications. Ameur A, Kloosterman WP, Hestand MS. Trends Biotechnol. 2019 Jan;37(1):72-85. https://www.ncbi.nlm.nih.gov/pubmed/30115375
1. Introduction to long-read sequencing technologies
2. New methods study CRISPR-Cas9 off-target activity