High-throughput generation and phenotypic characterization of zebrafish CRISPR mutants of DNA repair genes

Abstract

A systematic knowledge of the roles of DNA repair genes at the level of the organism has been limited largely due to the lack of appropriate animal models and experimental techniques. Zebrafish has become a powerful vertebrate genetic model system with availability due to the ease of genome editing and large-scale phenotype screening. Here, we generated zebrafish loss-of-function mutants for 33 DNA repair and replication genes through multiplexed CRISPR/Cas9-mediated mutagenesis. High-throughput phenotypic characterization of our mutant collection revealed that four genes (atad5a, ddb1, pcid2, pcna) are essential for proper embryonic development and hematopoiesis; seven genes (apex1, atrip, ino80, mre11a, shfm1, telo2, wrn) are required for growth and development during juvenile stage and six genes (blm, brca2, fanci, rad51, rad54l, rtel1) play critical roles in sex development. Furthermore, mutation in seven genes (atad5a, brca2, pcid2, polk, rad51, shfm1, xrcc1) displayed hypersensitivity to DNA damage agents. Further characterization of atad5a-/- mutants demonstrated that Atad5a is required for normal brain development and that its loss induces ATM-p53 dependent apoptosis in the brain. Our zebrafish mutant collection provides a unique resource for understanding of the roles of DNA repair genes at the organismal level.