Diverse target gene modifications in Plasmodium falciparum using Bxb1 integrase and an intronic attB

Genetic manipulation of the human malaria parasite Plasmodium falciparum is needed to explore pathogen biology and evaluate antimalarial targets. It is, however, aggravated by a low transfection efficiency, a paucity of selectable markers and a biased A/T-rich genome. While various enabling technologies have been introduced over the past two decades, facile and broad-range modification of essential genes remains challenging. We recently devised a new application of the Bxb1 integrase strategy to meet this need through an intronic attB sequence within the gene of interest. Although this attB is silent and without effect on intron splicing or protein translation and function, it allows efficient gene modification with minimal risk of unwanted changes at other genomic sites. We describe the range of applications for this new method as well as specific cases where it is preferred over CRISPR-Cas9 and other technologies. The advantages and limitations of various strategies for endogenous gene editing are also discussed.