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

项目摘要 人类疟原虫对抗疟原虫药物的抗药性发展 基本上所有常用的抗疟药物都需要加大药物发现的力度。一 更好地了解寄生虫的分子和细胞途径将有助于确定新的 药物靶点尽管寄生虫的基因组在十多年前就已经测序， 超过一半的基因的功能仍然未知。一个主要的瓶颈， 在恶性疟原虫中的功能研究是低的遗传重组效率。在这一建议中，A 将设计一个新的表观遗传基因工程平台， 用于靶向基因工程的CRISPR/Cas9技术。通过融合表观遗传基因 核酸酶缺陷型Cas9酶（dCas 9）的激活子或沉默子，并引导重组体 通过特异性单向导RNA（sgRNA）将dCas 9导入基因的转录起始位点， 已经实现了靶基因的有效上调或下调。优化这个 增强系统的鲁棒性和定时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诱导型模块和 多重gRNA将被整合到该系统中。第二，互补基因 使用dCas 12 a（Cpf 1）的调控系统，dCas 12 a是一种具有遗传学所需特征的新Cas， 在富含AT的基因组中的工程，如恶性疟原虫的基因组，将被工程化和验证。 系统比较这两种dCas基因调控系统的性能， 在不同的发育阶段表达的一组选定的基因， 转录水平将为将来有效使用该系统提供关键指导。是 预计这种基于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具有广泛的应用。

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.