Optimized CRISPR inhibition and activation opens key avenues for systematic biological exploration in zebrafish.

Abstract

The application of CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) technologies in zebrafish has the potential to expand its capacity for the study of gene function significantly. We have developed a codon optimized CRISPRi/a for zebrafish; here we provide proof-of-principle data across established pigmentary and growth phenotypes. We established a zebrafish codon-optimized cas9 gene, harboring mutations D10A and D839A to render the protein catalytically inactive ( dCas9 ). Similarly, codon-optimized Krüppel associated box (KRAB) and methylated CP2 (MeCP2) inactivating domains or VP64 activator domain were cloned downstream from dCas9 for CRISPRi and CRISPRa, respectively. To validate CRISPRi, we targeted key genes in melanocyte differentiation ( sox10, mitfa, and mitfb) ; and melanin production (tyrosinase; tyr ). Microinjection of CRISPRi mRNA and single guide RNAs (sgRNAs) targeting the tyr promoter or 5'-UTR resulted in larvae with hypopigmented epidermal melanocytes. Transcription factors mitfa and mitfb similarly direct differentiation and pigmentation of epidermal melanocytes ( mitfa ) and retinal pigment epithelium (RPE, mitfb ). CRISPRi-mediated targeting of their promoters or 5'-UTR also results in pronounced hypopigmentation of epidermal melanocytes ( mitfa ), and RPE ( mitfb ). So too, targeting CRISPRi to the sox10 promoter results in hypopigmentation of both epidermal melanocytes and RPE consistent with its role upstream of mitfa and mitfb , and tyr . Finally, we asked whether CRISPRi and CRISPRa could be used to modulate a single gene, to yield hypomorphic and hypermorphic effects, selecting mrap2a , as our target. This gene regulates energy homeostasis and somatic growth via inhibition of the melanocortin 4 receptor gene ( mc4r ). We demonstrate that targeting the mrap2a 5'-UTR with CRISPRa or CRISPRi significantly increases or decreases larval body length, respectively. We demonstrate the utility of CRISPRi/a for modulating control of zebrafish gene expression, facilitating efficient assay of candidate gene function and disease relevance.