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Nanopore off-target sequencing (Nano-OTS) reveals unforeseen CRISPR-Cas9 activity


A much-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.

Adam Ameur presents an amplification-free long-read sequencing protocol (Nano-OTS) for detection of gRNA binding and Cas9 cleavage, based on Nanopore sequencing. The Nano-OTS method was assessed using the human cell line HEK293, which was first re-sequenced using long reads to get a detailed view of all on- and off-target binding regions.

Nano-OTS was then used to investigate the specificity of three different gRNAs, resulting in a set of 55 high-confidence gRNA binding sites. Twenty-five (45%) of these sites were not reported by off-target prediction software.

Finally, by performing a de novo genome assembly of the long-read data, we were able to re-discover 98.7% of the gRNA binding sites without any prior information about the human reference genome suggesting that CRISPR-Cas9 off-target sites can be efficiently mapped also in organisms where the genome sequence is unknown.

Authors: Adam Ameur

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