Efficient CRISPR-Cas9-mediated genome editing in Plasmodium falciparum

Abstract

Malaria is a major cause of global morbidity and mortality, and new strategies for treating and preventing this disease are needed. Here we show that the Streptococcus pyogenes Cas9 DNA endonuclease and single guide RNAs (sgRNAs) produced using T7 RNA polymerase (T7 RNAP) efficiently edit the Plasmodium falciparum genome. Targeting the genes encoding native knob-associated histidine-rich protein (kahrp) and erythrocyte binding antigen 175 (eba-175), we achieved high (≥ 50-100%) gene disruption frequencies within the usual time frame for generating transgenic parasites.

Figure 1

Validating T7 RNA polymerase functions in P. falciparum. (a) Schematic of the Cas9, sgRNA and target DNA interaction. The sgRNA base pairs with a 20 nucleotide target DNA region defined by an NGG protospacer adjacent motif (PAM). Cas9 induces double strand DNA cleavage (arrow heads) 3 nucleotides upstream of the PAM site. (b) Plasmids used to test T7 RNAP expression and activity. In the reporter plasmid, pT7 RL1, the Renilla luciferase (RL) gene is expressed from a T7 promoter-T7 terminator cassette. The pT7 RL2 control plasmid contains the RL gene and T7 terminator, but no T7 promoter. A second control plasmid, pΔT7, lacks the T7 promoter-RL gene-T7 terminator cassette. (c) Western blot analysis of T7 RNAP protein production in parasites. Blots were probed with anti-T7 RNAP and anti-NPTII (loading control) antibodies. (d) Quantitative RT-PCR analysis of normalized RL transcript levels produced after the indicated transfections. Levels are above background only for the pT7 RNAP plus pT7 RL1 case. Data shown are mean ± s.d. (n = 3 technical replicates). (e) Northern blot analysis for RL transcript production, with the bsd selection marker transcript probed as a control. (f) Relative parasite growth over four successive generations both in the presence or absence of T7 RNAP and a T7 promoter-driven expression cassette. Data are shown as mean ± s.d. (n = 3 biological replicates) and analyzed for significance by One-way ANOVA.

Figure 2

CRISPR/Cas9-mediated disruption of the P. falciparum kahrp locus. (a) Generalized schematic of the Cas9 and T7 promoter-driven sgRNA-T plasmid used for genome editing. For homology directed repair of the induced double strand break, pT7 RNAP is modified to include homologous regions flanking a T2a-RL gene. Successful repair eliminates the original sgRNA target site, and creates a translational fusion between the upstream fragment of the disrupted target and the T2a-RL genes. (b) Measurement of RL expression in parasite population when the kahrp locus is targeted by a kahrp-sgRNA-T or pUC19-sgRNA-T control in the presence of a suitable donor plasmid. (c) PCR primers to specifically detect homology-directed repair at a target cut site in the kahrp locus amplify products of the expected size for kahrp-sgRNA-T but not pUC19-sgRNA-T transfected parasite population. (d) Southern and (e) Western blot analyses of parasite populations obtained after transfection with the pUC19- and kahrp- sgRNA-Ts. (f) SEM imaging analysis of parasite populations obtained after transfection with a no sgRNA-T control and a kahrp- sgRNA-T. (g, h) PCR and Western blot analyses as in (c) and (e), respectively, of cloned parasites derived from the kahrp-sgRNA-T edited pool. The unedited pUC19-sgRNA-T pool with an intact native kahrp locus is used as a positive control.

Figure 3

Assessing the fate of kahrp-sgRNA-T-induced cleavage of the kahrp locus in the absence of a homologous donor plasmid. (a) Schematic of the validated pT7 RNAP and pCas9-kahrp sgRNA-T plasmids used in this experiment. (b) Deep sequencing analysis of the region targeted for cleavage by kahrp-sgRNA-T. A representative set (1 of 3 independent experiments) of twenty bacterial clones analyzed by Sanger sequencing is illustrated.