The CRISPR-Cas9 genome editing tool is used to study genomic variants and gene knockouts, and can be combined with transcriptomic analyses to measure the effects of such alterations on gene expression. But how can one be sure that differential gene expression is due to a successful intended edit and not to an off-target event, without performing an often resource-demanding genome-wide sequencing of the edited cell or strain?
To address this question University of Copenhagen researchers developed CRISPRroots: CRISPR-Cas9-mediated edits with accompanying RNA-seq data assessed for on-target and off-target sites. Their method combines Cas9 and guide RNA binding properties, gene expression changes, and sequence variants between edited and non-edited cells to discover potential off-targets. Applied on seven public datasets, CRISPRroots identified critical off-target candidates that were overlooked in all of the corresponding previous studies.
Overview of the CRISPRroots pipeline
The researchers implemented the following main external tools in the seven modules: (1) Cutadapt, Bbduk, FastQC, MultiQC, STAR; (2) Mutect2; (3) RIsearch1; (4) featureCounts, DESeq2; (5) SAMtools; (6) RIsearch2, CRISPRoff; and (7) BEDtools, RIsearch1. The CRISPRroots specific modules are colored in blue. Key input/output files are displayed in dashed boxes. As an option, the off-target search and evaluation (modules 3, 6, 7) can run on a variant-aware version of the genome, generated after discovering germline variants with HaplotypeCaller.
Availability – CRISPRroots is available via https://rth.dk/resources/crispr.