Department of Internal Medicine, Morsani College of Medicine, University of South Floridagrid.170693.a, Tampa, Florida, USA.
Unit of Human Parasite Molecular and Cell Biology, Key Laboratory of Molecular Virology and Immunology, Pasteur Institute of Shanghai, Chinese Academy of Sciences, Shanghai, People's Republic of China.
Microbiol Spectr. 2022 Jun 29;10(3):e0278221. doi: 10.1128/spectrum.02782-21. Epub 2022 May 5.
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 the parasite genome. However, the constitutive expression of dCas9 poses a challenge for studying essential genes, which may lead to adaptive changes in the parasite, masking the true phenotypes. Here, we developed a leak-free inducible CRISPRi/a system by integrating the DiCre/ regulon to allow the expression of dCas9-GCN5/-Sir2a upon transient induction with rapamycin, which allows convenient transcriptional regulation of a gene of interest by introducing a guide RNA targeting its transcription start region. Using eight genes that are either silent or expressed from low to high levels during asexual erythrocytic development, we evaluated the robustness and versatility of this system in the asexual parasites. For most genes analyzed, this inducible CRISPRi/a system led to 1.5- to 3-fold up-or downregulation of the target genes at the mRNA level. Alteration in the expression of and resulted in altered sensitivities to artemisinin. For autophagy-related protein 18, an essential gene related to artemisinin resistance, a >2-fold up- or downregulation was obtained by inducible CRISPRi/a, leading to growth retardation. For the master regulator of gametocytogenesis, , a >10-fold increase of the transcripts was obtained by CRISPRa, resulting in >4-fold higher gametocytemia in the induced parasites. Additionally, inducible CRISPRi/a could also regulate gene expression in gametocytes. This inducible epigenetic regulation system offers a fast way of studying gene functions in Plasmodium falciparum. Understanding the fundamental biology of malaria parasites through functional genetic/genomic studies is critical for identifying novel targets for antimalarial development. Conditional knockout/knockdown systems are required to study essential genes in the haploid blood stages of the parasite. In this study, we developed an inducible CRISPRi/a system via the integration of DiCre/. We evaluated the robustness and versatility of this system by activating or repressing eight selected genes and achieved up- and downregulation of the targeted genes located in both the euchromatin and heterochromatin regions. This system offers the malaria research community another tool for functional genetic studies.
通过将催化失活的 Cas9（dCas9）融合到组蛋白去乙酰化酶（Sir2a）或乙酰转移酶（GCN5）的活性结构域中，该 CRISPR 干扰/激活（CRISPRi/a）系统允许在不引起寄生虫基因组永久变化的情况下在转录水平上进行基因调控。然而，dCas9 的组成型表达对研究必需基因构成了挑战，这可能导致寄生虫发生适应性变化，从而掩盖真实表型。在这里，我们通过整合 DiCre/调控子开发了一种无泄漏的诱导型 CRISPRi/a 系统，该系统允许在短暂诱导雷帕霉素时表达 dCas9-GCN5/-Sir2a，从而通过引入靶向其转录起始区域的向导 RNA 方便地对感兴趣的基因进行转录调控。使用在无性红细胞发育过程中要么沉默要么低表达到高表达的八个基因，我们评估了该系统在无性寄生虫中的稳健性和多功能性。对于大多数分析的基因，该诱导型 CRISPRi/a 系统导致靶基因在 mRNA 水平上的表达上调或下调 1.5 到 3 倍。 和 的表达改变导致对青蒿素的敏感性改变。对于与青蒿素耐药性相关的必需基因自噬相关蛋白 18，通过诱导型 CRISPRi/a 获得了 >2 倍的上调或下调，导致生长迟缓。对于配子发生的主调控因子 ，通过 CRISPRa 获得了 >10 倍的 转录物增加，导致诱导寄生虫中的配子血症增加 >4 倍。此外，诱导型 CRISPRi/a 还可以调节配子细胞中的基因表达。这种诱导型表观遗传调控系统为研究恶性疟原虫中的基因功能提供了一种快速方法。 通过功能遗传/基因组研究了解疟原虫的基本生物学对于确定抗疟药物开发的新靶标至关重要。在寄生虫的单倍体血液阶段，需要条件性敲除/敲低系统来研究必需基因。在这项研究中，我们通过整合 DiCre/开发了一种诱导型 CRISPRi/a 系统。我们通过激活或抑制八个选定的基因来评估该系统的稳健性和多功能性，并实现了靶向位于常染色质和异染色质区域的基因的上调和下调。该系统为疟疾研究社区提供了另一种用于功能遗传研究的工具。
