Amplifcation free long read nanopore sequencing for detection of Cas9 off target activity

Ida Höijer (SciLifeLab, Uppsala University) explained that it is well known how CRISPR-Cas9 gene editing can cause off-target effects. These effects are typically predicted using computational tools that assess sequence comparison, which is a quick and easy process, but it can be difficult to model guide RNA (gRNA) binding, and it requires a reference, which may be different to the cell type being studied. Experimental tools are an alternative, which may be more accurate, but rely on PCR and short-read sequencing.

Ida wanted to utilise the strengths of long nanopore reads for off-target identification. She developed the Nano-OTS (off-target sequencing) method, based on the Cas9 enrichment protocol with a few alterations (e.g. only one Cas9 gRNA is used for digestion). As sequencing reads line up at the Cas9 cleavage site, the reads can easily be aligned to the genome, to provide a list of target sequences, which can be used to determine if there was on and/or off-target digestion.

‘We wanted to demonstrate why we believe long-read sequencing is advantageous to detect Cas9 off-targets’; Ida then showed how she has used this method to target so-called ‘dark’ genomic regions (regions identified by Ebbert et al. [2019] as refractory to sequencing with short-read technology). Coverage was demonstrated across the targeted ‘dark’ regions. This demonstrated how Cas9 effects could be detected with Nano-OTS in genomic regions that are difficult to access using short-read methods.

She also explained how this method can be used to identify allele-specific off-target effects, showing an example of how genetic variation can affect gRNA binding in vitro (at the ATXN10 site, in the HEK293 cell line).

To conclude, Ida summarised the reasons for using Nano-OTS for CRISPR-Cas9 off-target detection: the method can measure Cas9 cleavage in vitro at base pair resolution; it can detect Cas9 cleavage in the ‘dark’ genome, ‘which is something that has not been shown before’; gRNAs can be multiplexed at the same time; the protocol is amplification-free so there are no concerns about PCR bias; and  finally, the protocol is quick (requiring one day) and easy, and, with the MinION, portable.