Experimenters may analyze animals at any generation of this process, though caveats apply to each. As CRISPR/Cas is a robust technology that often leads to high mutation rates, the injected (F0) fish often exhibit a phenotype. However, this founder analysis suffers from potential confounds. First, though CRISPR/Cas is reported to have a low rate of off-target gene modification, it may induce phenotypes due to mutation, particularly at homologous sites in the genome. Second, this approach is also prone to false negatives, as some cells of the mosaic animal carry cells with unaltered DNA, or mutations of weak effect. Third, the variety of mutations created lead to reduced reproducibility. Despite these concerns, we regularly analyze injected animals to obtain preliminary data, refine phenotype tests, and observe unanticipated phenotypes. Further crosses are warranted to obtain animals carrying mutations in all cells. One may intercross F0 fish, which will result in offspring carrying mutations in all cells, thereby increasing the likelihood of observing a phenotypic effect.
However, since CRISPR/Cas generates a variety of mutation sizes and sequences even within the germline, each offspring will likely carry a different pair of alleles, complicating interpretations. Furthermore, each parent may contribute mutation(s) in off-target genes to the offspring.
Thus, it is important to outcross F0 animals to wildtypes in order to dilute the effect of off-target effects. Assuming that the on- and off-target loci are unlinked, each generation of outcrossing leads to a reduction by half of the co-inheritance of off-target mutations. Furthermore, unlinked off-target mutations will be inherited by siblings at equivalent rates, providing important controls for effects of this genetic background. Ideally, therefore, crosses of heterozygous F1 (or later) generation fish will yield control genotypes in addition to homozygous animals for analysis, while controlling for off-target effects. We also recommend analyzing ≥2 independent mutant lines for analysis to ensure that phenotypes are reproducible.
We breed biallelic mutants via a combination of incrosses and outcrosses.