Rapid generation of pigment free, immobile zebrafish embryos and larvae in any genetic background using CRISPR-Cas9 dgRNPs

Abstract

ABSTRACTThe ability to carry out high-resolution, high-magnification optical imaging of living animals is one of the most attractive features of the zebrafish as a model organism. However, formation of obscuring pigmentation as development proceeds and difficulties in maintaining sustained immobilization of healthy, living animals remain challenges that limit the application of live imaging. Chemical treatments can be used to suppress pigment formation and movement, but these treatments can lead to developmental defects. Genetic mutants can also be used to eliminate pigment formation and immobilize animals but maintaining these mutants in lines carrying other combinations of transgenes and mutants is difficult and laborious. Here, we show that CRISPR duplex guide ribonucleoproteins (dgRNPs) targeting the slc45a2 (albino) and chrna1 (nic1) genes can be used to efficiently suppress pigment formation in and immobilize F0 injected animals. CRISPR dgRNPs can be used to generate pigment-free, immobile zebrafish embryos and larvae in any transgenic and/or mutant-carrying background, greatly facilitating high-resolution imaging and analysis of the many transgenic and mutant lines available in the zebrafish.