PROJECT SUMMARY The development of resistance in the human malaria parasite Plasmodium falciparum to essentially all commonly used antimalarial drugs demands increased efforts in drug discovery. A better understanding of the parasite’s molecular and cellular pathways will help identify novel drug targets. Although the parasite’s genome has been sequenced more than a decade ago, the functions of more than half of the genes remain unknown. A major bottleneck towards functional studies in P. falciparum is the low genetic recombination efficiency. In this proposal, a new epigenetic gene engineering platform will be designed by taking the advantage of the CRISPR/Cas9 technology for targeted gene engineering. By fusing either an epigenetic activator or silencer to a nuclease-deficient Cas9 enzyme (dCas9) and guiding the recombinant dCas9 to the transcriptional start site of the gene by specific single guide RNA (sgRNA), the efficient up- or down-regulations of the targeted genes have been achieved. Optimization of this system to enhance the robustness and precision of timing is critical to meet the requirements for studying essential genes in this parasite. First, TetR and Cre/loxP inducible modules and multiplexed gRNAs will be integrated into this system. Second, a complementary gene regulation system employing the dCas12a (Cpf1), a new Cas with desired features for genetic engineering in AT-rich genomes like that of P. falciparum, will be engineered and validated. Systematic comparison of the performance of these two dCas gene regulation systems using a suite of selected genes expressed at different development stages and with different transcriptional levels will provide pivotal guidance on future efficient use of the systems. It is anticipated that this versatile CRISPR/dCas-based epigenetic gene regulation system would find broad applications in functional genomic studies in P. falciparum.