CRISPR/Cas9-Mediated Genome Editing in Zebrafish

Abstract

The CRISPR/Cas9 system is a powerful tool for genome editing in zebrafish. This workflow takes advantage of the genetic tractability of zebrafish and will allow users to edit genomic sites and produce mutant lines using selective breeding. Established lines may then be employed by researchers for downstream genetic and phenotypic analyses.

Keywords: CRISPR/Cas9; Genome editing; Mutagenesis; Zebrafish.

Figure 1:

Genome editing using a one or two sgRNA scheme. (A) Indels are generated through the NHEJ cellular repair process that follows a Cas9-mediated DSB (black arrowhead) at a single exon. (B) sgRNA target sites (black arrowheads) are designed to generate a DSB at the 5’ and 3’ ends of the locus (within or beyond proximal and distal exons). These mutations result in frameshift mutations (A) or large deletions (B), and typically produce null alleles. F₀ and F₁ fish are screened for indels at the target site(s) as described in Figure 3.

Figure 2:

General scheme for knockout line production. (A) 1-cell embryos are injected with Cas9 (mRNA or protein) and sgRNA(s), checked for edits by PCR, and grown to sexual maturity. F₀ adults may then be outcrossed with wild-type individuals, and F₁ offspring screened for germline transmission of mutations. Adults with confirmed genotypes may then be incrossed to generate homozygous mutants for downstream genetic analysis.

Figure 3:

Two primer systems for genotyping Cas9-mediated indels. (A) Indels produced by genome editing of a single target exon. Cas9 generates a double-strand break, which stimulates NHEJ and results in either an insertion or deletion. A single pair of genotyping primers are indicated by horizontal arrows. In the case of a large deletion, genotyping cannot be done with a single set of primers, as for most loci the wild-type allele will be too large for PCR amplification. Primers 1, 2, and 5 constitute the 3-primer system, and primers 1, 3–5 make up the 4-primer system. (B) Digital electrophoresis shows examples of band patterns from edited F₀ alleles, demonstrating mosaic deletions and insertions (WT amplicon = 327 bp). Progeny of injected crispants (F₁) may be screened for specific lesions using the 3- or 4-primer systems. (C) Representative results of a multiplexed reaction with the 3- or 4-primer systems using F₁ progeny of injected crispants, assuming a larger wild type band. Reactions producing an amplicon should be sequenced to confirm the presence of the large deletion.