![]() ![]() ![]() Further aggravating this process, the circular plasmids required for stable retention in transfected parasites tend to concatemerize under drug selection, reducing the likelihood of integration and complicating interpretation of some phenotypes. Historically, these experiments relied on infrequent, coincidental genome breaks at or near the target site, often necessitating many months of continuous cultivation and drug-cycling to obtain detectable levels of integration. Gene manipulation in this system requires single or double crossover homologous recombination into the haploid genome as the parasite lacks the enzymes to carryout non-homologous end joining. Because transfection efficiencies are low, selectable markers are required the available markers have facilitated functional analyses, but their relatively small number adds to the difficulties faced in molecular and biochemical studies of this pathogen. These goals depend on transfection studies in cultured P. Since Plasmodium falciparum, a virulent human malaria parasite, acquires resistance to most antimalarial drugs quickly, new drug targets and a better understanding of resistance mechanisms are needed to sustain advances in global health. The advantages and limitations of various strategies for endogenous gene editing are also discussed.ĭespite advances from combination therapies and public health measures such as bednets, more than 400,000 people still die of malaria annually. 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. 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 recently devised a new application of the Bxb1 integrase strategy to meet this need through an intronic attB sequence within the gene of interest. While various enabling technologies have been introduced over the past two decades, facile and broad-range modification of essential genes remains challenging. It is, however, aggravated by a low transfection efficiency, a paucity of selectable markers and a biased A/T-rich genome. Genetic manipulation of the human malaria parasite Plasmodium falciparum is needed to explore pathogen biology and evaluate antimalarial targets. ![]()
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