Nanopore off-target sequencing (Nano-OTS) reveals unforeseen CRISPR-Cas9 activity
About Adam Ameur
Adam Ameur is associate professor and senior bioinformatician at the SciLifeLab National Genomics Infrastructure in Sweden. His work is focused on technology development and novel sequencing applications for the study of human health and disease. Ongoing activities include the construction of a whole-genome reference dataset for genetic variation in the Swedish population, as well as introduction of long-read single molecule sequencing into clinical routine. Since 2017 he is also an adjunct researcher at Monash University in Melbourne, Australia.
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 Oxford Nanopore sequencing (Nano-OTS). The Nano-OTS method was assessed using a human cell line, which was first re-sequenced using long reads to get a detailed view of all on- and off-target binding regions. We then applied Nano-OTS to investigate the specificity of three different gRNAs, resulting in a set of 55 high-confidence gRNA binding sites, many of which were not reported by off-target prediction software. We are currently investigating whether in vivo off-target editing can occur at these sites.