title: CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity.
abstract: 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 efficacy 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 reaction (PCR)-based method, CRISPR Somatic Tissue Activity Test (CRISPR-STAT), to determine target-specific efficiency of sgRNA. As a proof of principle, we validated our method using 28 sgRNAs with known and varied levels of germline transmission efficiency in zebrafish by analysis of their somatic activity 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 targeting. This method is easy to implement by laboratories with access to a capillary sequencer. Although we validated the method using CRISPR/Cas9 and zebrafish, it can be applied to other model systems and other genome targeting nucleases.
authors.affiliation: Zebrafish Core, Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Blake
authors.initials: B
authors.lastname: Carrington
authors.affiliation: Developmental Genomics Section, Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Gaurav K
authors.initials: GK
authors.lastname: Varshney
authors.affiliation: Developmental Genomics Section, Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Shawn M
authors.initials: SM
authors.lastname: Burgess
authors.affiliation: Zebrafish Core, Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA rsood@mail.nih.gov.
authors.forename: Raman
authors.identifier: http://orcid.org/0000-0001-5565-662X
authors.initials: R
authors.lastname: Sood
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D005453:Fluorescence; D054643:INDEL Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000378:metabolism*; D015027:Zebrafish / Q000235:genetics
chemical_list: D012313:RNA