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 report proof-of-principle data evaluating transient expression of a codon optimized CRISPRi/a system for zebrafish across established pigmentary and growth phenotypes. A codon-optimized and catalytically inactive cas9 gene ( dcas9 ) was cloned upstream of codon-optimized Krüppel associated box (KRAB) and methyl-CpG binding protein 2 (MeCP2) for CRISPRi, and VP64 for CRISPRa. 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. CRISPRi-mediated targeting of the promoters or 5'-UTR of transcription factors mitfa and mitfb also results in pronounced hypopigmentation of epidermal melanocytes ( mitfa ), and RPE ( mitfb ). Also, CRISPRi targeting of 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 tested both CRISPRi/a 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 inactivating or activating mrap2a with CRISPRi/a significantly decreases or increases larval body length, respectively. We demonstrate that CRISPRi/a can modulate control of zebrafish gene expression, facilitating efficient assay of candidate gene function and disease relevance.