Figure 7.

A general strategy for structure–function analysis. (A) Illustration of the strategy. First, a null mutation is generated by inserting FP–SEC in place of the gene of interest; then, variants are reintroduced into the mutant background. Once the null mutant is made, multiple different variants (as many as desired) can be introduced using the same homology arms and sgRNA for the second HDR step. (B) Genetic scheme for executing this strategy for a nonessential gene. (C) Genetic scheme for executing this strategy for an essential gene. The only additional step is the introduction of a balancer chromosome during isolation of the initial null mutant.