Functional characterization of the multitude of poorly described proteins in the human malarial pathogen, Plasmodium falciparum, requires tools to enable genome-scale perturbation studies. Here, we present GeneTargeter (genetargeter.mit.edu), a software tool for automating the design of homology-directed repair donor vectors to achieve gene knockouts, conditional knockdowns, and epitope tagging of P. falciparum genes. We demonstrate GeneTargeter-facilitated genome-scale design of six different types of knockout and conditional knockdown constructs for the P. falciparum genome and validate the computational design process experimentally with successful donor vector assembly and transfection. The software's modular nature accommodates arbitrary destination vectors and allows customizable designs that extend the genome manipulation outcomes attainable in Plasmodium and other organisms.
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