I. Phantom: a multiplex CRISPR-Cas9 gRNA toolkit for efficient generation of fully unpigmented F0 zebrafish

Zebrafish are an increasingly popular model organism, valued for their natural transparency during embryonic stages. However, imaging at later developmental stages is affected by several types of pigments distributed over the body surface. These pigments partially or completely absorb or reflect light, and some also exhibit fluorescence, thereby interfering with imaging. Chemical approaches, such as phenylthiourea (PTU), are available to address this problem. However, PTU only inhibits melanin production and can affect development, making its use controversial. Many mutant zebrafish lines lacking single pigments have already been described, and combinations of melanin and iridophore deficient lines such as the casper and crystal fish greatly improved in vivo imaging at late stages. However, breeding multiple recessive mutations with a desired reporter transgene is laborious, and xanthophores remain in these lines. Here, we address these limitations by developing and optimising a multiplex CRISPR-Cas9 gRNA set that simultaneously targets the three major pigment types, while minimising mortality. This approach generates a new phenotype, termed phantom, characterised by the complete absence of visible pigmentation directly in F0 zebrafish larvae, while retaining the ability to survive to adulthood and reproduce.