Development of CRISPR/dCas-based epigenetic gene regulation tools in malaria parasite

项目总结 人疟原虫恶性疟原虫对疟疾的抗药性研究进展 基本上，所有常用的抗疟疾药物都需要在药物发现方面做出更多努力。一个 更好地了解寄生虫的分子和细胞途径将有助于识别新的 毒品目标。虽然这种寄生虫的基因组在十多年前就已经测序了， 超过一半的基因的功能仍不清楚。一个主要的瓶颈是 恶性疟原虫的功能研究是遗传重组效率低。在这项提案中， 新的表观遗传基因工程平台将利用 用于靶向基因工程的CRISPR/Cas9技术。通过融合一种表观遗传学 核酸酶缺陷型Cas9酶(DCas9)的激活剂或抑制剂及引导重组 DCas9通过特异的单引导RNA(SgRNA)结合到基因的转录起始点， 已经实现了对目标基因的高效上调或下调。对这一点的优化 系统对提高授时的稳健性和精度是满足要求的关键 研究这种寄生虫的基本基因。首先，TetR和Cre/loxP可诱导模块和 多路传输的gRNA将被集成到这个系统中。第二，互补基因 利用dCas12a(Cpf1)的调控系统，这是一种具有所需遗传特征的新CA 在富含AT的基因组中进行工程设计，如恶性疟原虫，将被设计和验证。 两种DCAS基因调控系统性能的系统比较 一套在不同发育阶段和不同发育阶段表达的基因 转录水平将为今后系统的有效使用提供关键指导。它是 预计这个基于CRISPR/DCAS的多功能表观遗传基因调控系统将 在恶性疟原虫的功能基因组研究中有广泛的应用。

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.