Amplification-free long-read nanopore sequencing for detection of Cas9 off-target activity
About Ida Höijer
Ida Höijer is a Ph.D. candidate from SciLifeLab, Uppsala University in Sweden. Prior to starting her Ph.D., Ida worked for the National Genomics Infrastructure (NGI), which is a Swedish national center for massively parallel sequencing and genotyping. At NGI she gained a lot of experience with NGS, with a focus on long-read sequencing. Ida’s Ph.D. work is centered around targeted long-read sequencing methods and their applications in medical and clinical genomics.
CRISPR-Cas9 induced off-target effects is a heavily debated concern while performing genome editing, and the prediction and detection of these are challenging. To address this issue, we developed an amplification-free long-read sequencing method for detection of Cas9 off-target activity in vitro. We call this method Nanopore off-target sequencing (Nano-OTS). Nano-OTS can detect Cas9 cleavage sites for one or several gRNAs in individual DNA samples, and our results show that a number of these sites are not reported by Cas9 off-target prediction software. Importantly, Nano-OTS can also detect Cas9 cleavage sites in “dark” genomic regions, where short-reads fail to align.