Multiplexed Genome Editing for Efficient Phenotypic Screening in Zebrafish

Zebrafish are widely used to investigate candidate genes for human diseases. While the emergence of CRISPR-Cas9 technology has revolutionized gene editing, the use of individual guide RNAs limits the efficiency and application of this technology in functional genetics research. Multiplexed genome editing significantly enhances the efficiency and scope of gene editing. Herein, we describe an efficient multiplexed genome editing strategy to generate zebrafish mutants. Following behavioural tests and histological examination, we identified one new candidate gene (tmem183a) for hearing loss. This study provides a robust genetic platform to quickly obtain zebrafish mutants and to identify candidate genes by phenotypic readouts.