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

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 (AT) rich Plasmodium DNA in E. coli. We overcome these roadblocks by demonstrating that a high integrity library of P. berghei genomic DNA (>77% AT) in a bacteriophage N15-based vector can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating Plasmodium 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 10-fold over conventional designs. The feasibility of efficient genetic modification at scale will stimulate collaborative, genome-wide knockout and tagging programs for P. berghei.