CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity

CRISPR/Cas9 has emerged as a versatile genome-engineering tool that relies on a single guide RNA (sgRNA) and the Cas9 enzyme for genome editing. Simple, fast and economical methods to generate sgRNAs have made targeted mutagenesis routine in cultured cells, mice, zebrafish and other model systems. Pre-screening of sgRNAs for target effi-cacy is desirable both for successful mutagenesis and minimizing wasted animal husbandry on targets with poor activity. Here, we describe an easy, quick and cost-effective fluorescent polymerase chain re-action (PCR)-based method, CRISPR Somatic Tissue Activity Test (CRISPR-STAT), to determine target-specific efficiency of sgRNA. As a proof of princi-ple, we validated our method using 28 sgRNAs with known and varied levels of germline transmission ef-ficiency in zebrafish by analysis of their somatic ac-tivity in injected embryos. Our data revealed a strong positive correlation between the fluorescent PCR profiles of the injected embryos and the germline transmission efficiency. Furthermore, the assay was sensitive enough to evaluate multiplex gene target-ing. This method is easy to implement by laborato-ries with access to a capillary sequencer. Although we validated the method using CRISPR/Cas9 and ze-brafish, it can be applied to other model systems and other genome targeting nucleases