A scalable pipeline for highly effective genetic modification of a malaria parasite

Abstract

In malaria parasites, the systematic experimental validation of drug and vaccine targets by reverse genetics is constrained by the inefficiency of homologous recombination and by the difficulty of manipulating adenine and thymine (A+T)-rich DNA of most Plasmodium species in Escherichia coli. We overcame these roadblocks by creating a high-integrity library of Plasmodium berghei genomic DNA (>77% A+T content) in a bacteriophage N15-based vector that can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating P. berghei genetic modification vectors at genome scale in serial liquid cultures on 96-well plates. Vectors have long homology arms, which increase recombination frequency up to tenfold over conventional designs. The feasibility of efficient genetic modification at scale will stimulate collaborative, genome-wide knockout and tagging programs for P. berghei.

Figure 1. Characterisation of the P. berghei large insert genomic DNA library PbG01

(a) Schematic of the phage N15-derived pJAZZ vector used to generate the genomic library, showing hairpin telomeres (black), telomerase gene (TelN), replication factor and origin (repA), and kanamycin resistance gene (aph). (b) Distribution of insert sizes. (c) PbG01 inserts mapped on 65 kbp of chromosome 9 illustrates typical coverage. (d) Observed genome coverage by actual library inserts is compared with modelled coverage by random inserts. Percentage of genes covered to at least 50% is shown.

Figure 2. Modification of PbG01 inserts in E. coli by lambda Red recombineering and site specific recombinase

(a) A 2-stage strategy for gene deletion. Primer extensions homologous to 3′ and 5′ P. berghei target sequence are shown in magenta and green. (b) The strategy for 3′ tagging. (c) Step-by-step verification of vector product by PCR genotyping. See panels (a) and (b) for typical primer locations.

Figure 3. Knock out vector production in 96 parallel liquid cultures

Steps 1-3 and 5 take place in E. coli, steps 4 and 6 use purified vector in vitro. Cloning and genotyping is deferred until step 6. Following introduction of the recombinase plasmid, bacteria are cultured at a permissive temperature of 30 °C (step 1). Recombinase expression is induced by arabinose (step 2), and bacteria are electroporated with PCR products containing the zeo-PheS cassette flanked by 50 base pairs homologous to the chosen target locus (step 3). An in vitro Gateway reaction (step 4) switches the bacterial marker to one for P. berghei. Plasmids are retransformed into E. coli and plated on p-chlorophenylalanine (YEG-Cl) to select for recombination products lacking pheS (step 5). Colonies are picked for PCR verification (step 6). Percentages shown in red give average efficiencies of individual steps. See also Supplementary Protocol 1.

Figure 4. Validation of recombineered vectors in P. berghei ANKA

(a) Primary genotyping of resistant parasite pools by Southern hybridisation of separated chromosomes. The probe recognises two copies of the dhfr-ts 3′UTR in the targeting vector (variable band) and additionally highlights chromosome 7 (endogenous dhfr-ts gene), and chromosome 3 (gfp transgene integrated into the p230p locus, PBANKA_030600). The expected chromosomal location of target genes is given by the first two digits of the gene ID. * = recombinant genotype is not in the majority, as judged by band intensity. (b) Western blot analysis showing expression of HA-tagged proteins in lysates from schizonts and gametocytes. (c) Immunolocalisation of HA-tagged proteins showing localisation to the cytosol (PBANKA_082340, PGK), or a peripheral staining pattern consistent with localisation to the inner membrane complex (PBANKA_143660, alveolin 3, IMC1h). Fixed and permeabilised ookinetes were counter stained with Hoechst for DNA and with a monoclonal antibody against the major surface protein P28. Scale bar = 10 μm.

Figure 5. Effect of homology arm length on targeting frequency

(a) A panel of deletion vectors for the pdeδ gene. The restriction enzymes shown were used to modify lengths of homology arms. (b) Transfection efficiency is plotted against the sum of both homology arms. Error bars show standard deviations from three transfections.