Heritable and Precise Zebrafish Genome Editing Using a CRISPR-Cas System

TLDR

A simple, customizable CRISPR‑Cas9 RNA‑guided nuclease system has been shown to efficiently introduce somatic indel mutations in zebrafish genes. The study aims to extend this system to enable precise sequence edits via ssODNs and to broaden its targeting range from 1 in 128 to 1 in 8 base pairs. This is achieved by combining RGN with single‑stranded oligodeoxynucleotides for precise edits and implementing strategies that increase target density. RGN‑induced mutations are heritable with germline transmission efficiencies up to 100%, demonstrating that the system can produce precise, heritable genome modifications in zebrafish.

Abstract

We have previously reported a simple and customizable CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided Cas9 nuclease (RGN) system that can be used to efficiently and robustly introduce somatic indel mutations in endogenous zebrafish genes. Here we demonstrate that RGN-induced mutations are heritable, with efficiencies of germline transmission reaching as high as 100%. In addition, we extend the power of the RGN system by showing that these nucleases can be used with single-stranded oligodeoxynucleotides (ssODNs) to create precise intended sequence modifications, including single nucleotide substitutions. Finally, we describe and validate simple strategies that improve the targeting range of RGNs from 1 in every 128 basepairs (bps) of random DNA sequence to 1 in every 8 bps. Together, these advances expand the utility of the CRISPR-Cas system in the zebrafish beyond somatic indel formation to heritable and precise genome modifications.