Establishing stable zebrafish mutant lines (Part 1)

The first step in establishing mutant lines is the induction of mutations in wildtype zebrafish. There are a number of ways to create mutations. A highly effective method is CRISPR/Cas9. It involves the injection of guide RNAs, Cas9 protein and stop oligos into embryos at the single-cell stage. CRISPR/Cas9 gives a high level of specificity to mutation induction. A wide variety of mutations are induced; some can be innocous point mutations and others can be insertions and deletions of varying lengths. This injected generation of fish is referred to as G₀. The injected adult fish that develop are mosaic organisms, meaning that different populations of cells have different genotypes (i.e. carry different mutations). To clean this up and have organisms with uniform genotypes, the G₀ adult zebrafish have to be outcrossed with wildtype fish. Ideally, the next generation of zebrafish (F₁) laid should have heterozygotes carrying one wild-type allele and one mutant allele, along with homozygotes carrying only wildtype alleles. Owing to the unpredictability of the process, all the embryos obtained from a cross could be wildtype. This might be an indication that the induced mutation did not go germline. To determine the alleles being carried by the progeny, a process called genotyping must be performed. That will be the discussion for the next segment.