A Leak-Free Inducible CRISPRi/a System for Gene Functional Studies in Plasmodium falciparum

• DOI: 10.1128/spectrum.02782-21
• Publication Date: 2022-05-05T13:00:56Z
• Authors: Xiaoying Liang; Rachasak Boonhok; Faiza Amber Siddiqui; Bo Xiao; Xiaolian Li; Junling Qin; Hui Min; Lubin Jiang; Liwang Cui; Jun Miao
• Keywords: Conditional gene knockout; Gene knockout

By fusing catalytically dead Cas9 (dCas9) to active domains of histone deacetylase (Sir2a) or acetyltransferase (GCN5), this CRISPR interference/activation (CRISPRi/a) system allows gene regulation at the transcriptional level without causing permanent changes in parasite genome. However, constitutive expression dCas9 poses a challenge for studying essential genes, which may lead adaptive parasite, masking true phenotypes. Here, we developed leak-free inducible CRISPRi/a by integrating DiCre/loxP regulon allow dCas9-GCN5/-Sir2a upon transient induction with rapamycin, convenient interest introducing guide RNA targeting its transcription start region. Using eight genes that are either silent expressed from low high levels during asexual erythrocytic development, evaluated robustness and versatility parasites. For most analyzed, led 1.5- 3-fold up-or downregulation target mRNA level. Alteration PfK13 PfMYST resulted altered sensitivities artemisinin. autophagy-related protein 18, an related artemisinin resistance, >2-fold up- was obtained CRISPRi/a, leading growth retardation. master regulator gametocytogenesis, PfAP2-G, >10-fold increase PfAP2-G transcripts CRISPRa, resulting >4-fold higher gametocytemia induced Additionally, could also regulate gametocytes. This epigenetic offers fast way functions Plasmodium falciparum. IMPORTANCE Understanding fundamental biology malaria parasites through functional genetic/genomic studies is critical identifying novel targets antimalarial development. Conditional knockout/knockdown systems required study haploid blood stages parasite. In study, via integration DiCre/loxP. We activating repressing selected achieved targeted located both euchromatin heterochromatin regions. research community another tool genetic studies.