Fig 1.
Generation of CRISPR/Cas9-mediated FREP1 gene knockout mutants.
(A) Schematic representation of the pDSAT-FREP1-gRNA3 (with blue fluorescence eye reporter gene) transformation plasmid used for the germline transformation of the A. gambiae X1 docking line. Three gRNA target DNA sequences of FREP1 were first independently cloned into gRNA expression template vectors and assembled into a pDSAT vector via the Golden Gate cloning technique using BsaI sites. (B, C) Generation of FREP1-gRNA-expressing (FREP1-gRNA) transgenic line. Fluorescent images of larvae of FREP1-gRNA (blue) transgenic line, along with the wt control (X1, non-fluorescent) strain (B). Fluorescent images of adult transgenic FREP1-gRNA mosquito and wt control (C). (D, E) FREP1-gRNA virgin females were crossed with Vasa-Cas9 (Cas9, YFP, yellow/green fluorescence) males to generate the transheterozygotes (FREP1/Cas9) for producing FREP1 gene knockouts. The transheterozygous progeny has both blue and green fluorescence in larval (D) and adult eyes (E), whereas progeny that did not inherit FREP1-gRNA has only the green fluorescent eye marker. (F) PCR validation of FREP1 gene disruption (sequences available in SI S1 File). 478 bp PCR products from X1, Cas9, FREP1-gRNA, and FREP1/Cas9 transheterozygotes were sequenced (upper panel). PCR showed that the heterozygous FREP1 mutants have both wild type band (478 bp) and the lower band (323 bp) with a 155 bp gene deletion (FREP1-KO’). Homozygous deletion mutant (FREP1-KO) showed only one lower band (SI S1 File).
Fig 2.
Suppression of P. falciparum in FREP1 gene knockout mutants.
(A, B) P. falciparum (NF54) oocyst infection intensities of FREP1 knockout mutants at 8 days post-infection (dpi) when fed on blood with a high (0.1%) (A) or low (0.01%) (B) gametocytemia. P. falciparum oocyst infection intensity at 8 dpi in the wt (X1), Vasa-Cas9 (Cas9), and FREP1-gRNA lines, and FREP1 knockout mutants (FREP1-KOs). The wt (X1), Cas9, and FREP1-gRNA lines served as controls. (C) The prevalence of the oocyst infection was significantly lower in the FREP1 knockout mosquitoes at low infection level. At low infection level, P. falciparum sporozoite loads in the salivary glands (SG) at 14 dpi (D) and the prevalence of the sporozoites (E) was significantly lower in the FREP1 knockout mosquitoes. Assays were performed with at least three biological replicates, except for (A) (two replicates), and equal numbers of parasites from different replicates were pooled for the dot-plots. Each dot represents the number of parasites in an individual midgut or salivary gland, and the horizontal lines (red) indicate the median values. Mann-Whitney and Kruskal-Wallis (KW) tests were used to calculate p-values and determine the significance of parasite numbers. A chi-squared test was used to compare infection prevalence values. Detailed statistical analysis is presented in S2 Table.
Fig 3.
Suppression of P. berghei in CRISPR/Cas9-mediated FREP1 gene knockout mutants.
(A, B) P. berghei (wt, ANKA 2.34) oocyst infection intensities of FREP1 knockout mutants (FREP1-KOs) at 13 days post-infection when fed on P. berghei-infected mice at high (A) or low (B) infection level. wt (X1), Cas9, and FREP1-gRNA lines served as controls. (C) At low infection level the prevalence of the oocyst infection was significantly lower in the FREP1 knockout mosquitoes. At least three biological replicates were included in each assay, and equal numbers of mosquitoes from the different replicates were pooled for the dot-plot analysis. Each dot represents the number of parasites in an individual mosquito, and the horizontal lines (red) indicate the medians. p-values were calculated using Mann-Whitney and Kruskal-Wallis (KW) tests. A chi-squared test was used to compare infection prevalence values. Detailed statistical analysis is presented in S3 Table.
Fig 4.
Fitness effects of FREP1 gene knockout.
(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.