CRISPR/Cas9 -mediated gene knockout of Anopheles gambiae FREP1 suppresses malaria parasite infection
(A, B) Percentage of mosquitoes fed on human blood through membrane (A) or naïve mice (B). A chi-squared test was used to compare blood feeding prevalence values. (C) FREP1 gene knockout (KO) larva developed much more slowly than did control larvae. (D) The pupation time of the FREP1 knockout mutants (FREP1-KOs) was significantly slower than that of the wt (X1), Vasa-Cas9 (Cas9), or FREP1-gRNA control lines. The pupation time lagged behind the control time by a median of 2 days. (E) The wing lengths of the FREP1 mutant females or males did not differ from those of the control mosquitoes. (F, G) Life spans of the FREP1 mutants maintained on 10% sucrose solution (F) or after one mouse blood meal (G). The life spans of the mutants were significantly shorter than those of the controls when the mosquitoes were fed on the naïve mice. The pooled values from three replicates are shown, with standard error bars. Survival rates were analyzed by Kaplan-Meier survival analysis. (H) Numbers of eggs laid by female FREP1 heterozygous mutants (FREP1-KO’) or homozygous mutants (FREP1-KOs) were significantly lower than those of the control transgenic lines. Each dot represents the eggs laid by an individual female after a single blood meal on mice. The median values (black horizontal bars) are shown. The p-values were calculated with a Mann-Whitney test. (I) Hatch rates indicate the average percentage of eggs giving rise to 1st and 2nd instar larvae, as determined by two replicates from two consecutive generations. Mean values for hatch rates and standard errors (SE) of replicates are indicated. ****, p<0.0001.