ClockWork: Genotyping of frameshift mutations induced by CRISPR-Cas9

Abstract

Here we present Clockwork, a high-throughput bioinformatics pipeline for genotyping CRISPR-Cas9 induced frameshift mutations in single neurons. Clockwork is part of a project that aims to elucidate the molecular mechanisms of replication-independent mutations associated with the mutational signature SBS5. In this project key components involved in DNA repair and mutagenesis are mutated by CRISPR-Cas9, introducing frameshift mutations and subsequently studying its effect on the mutational rate, pattern and load. However, the success rate of CRISPR-Cas9 is highly dependent on the local sequence context and guide sequences, with samples frequently having no mutations. Therefore Clockwork addresses a need for genotyping successful frameshifts, saving valuable resources by avoiding whole genome sequencing of unedited samples. Clockwork’s efficacy is interrogated with a proof-of-concept dataset consisting of hybrid Mus musculus and Mus spretus embryonic stem cells as well as human retinal pigment epithelium cells.