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

基于 CRISPR/dCas 的疟疾寄生虫表观遗传基因调控工具的开发

基本信息

批准号：
10084810
负责人：
Jun Miao
金额：
$ 18.69万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-13 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10084810
关键词：
Acetylation Adoption Antimalarials Biology Catalytic Domain Categories Cessation of life Chemicals Clustered Regularly Interspaced Short Palindromic Repeats Communities Cre-LoxP Development Dimerization Down-Regulation Engineered Gene Engineering Enhancers Enzymes Epigenetic Process Essential Genes Evaluation Exons Future Gene Activation Gene Expression Gene Expression Regulation Gene Targeting Genes Genetic Genetic Engineering Genetic Recombination Genetic Transcription Genome Goals Guide RNA Haploidy Histone Acetylation Histone Deacetylase Histone H3 Human Human Biology Intervention Introns Malaria Methodology Methods Molecular Parasites Pathway interactions Performance Plasmodium falciparum Plasmodium falciparum genome Plasmodium genome Proteins Recombinants Regulation Research Resistance development Site Streptococcus pyogenes System Technology Testing Tetanus Helper Peptide Transcription Initiation Site Transcriptional Activation Transfection Untranslated RNA Up-Regulation Validation Virulent Writing base comparative genomics design drug discovery functional genomics gene repression genetic manipulation histone acetyltransferase knock-down knockout gene new therapeutic target novel nuclease parasite genome promoter tool

项目摘要

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.

项目摘要人类疟疾寄生虫疟原虫恶性疟原虫的抗性发展本质上，所有常用的抗疟药都需要增加药物发现的努力。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颖药物靶标。尽管寄生虫的基因组已经在十多年前进行了测序，但超过一半的基因的功能仍然未知。朝向的主要瓶颈恶性疟原虫的功能研究是低遗传重组效率。在此提案中，新的表观遗传基因工程平台将通过利用 CRISPR/CAS9技术用于靶向基因工程。通过融合任何表观遗传学激活剂或消音器进入核酸酶缺陷的CAS9酶（DCAS9），并引导重组 DCAS9通过特定的单个指南RNA（SGRNA）到基因的转录起始位点，已经实现了靶向基因的有效上调或下调。优化增强计时的鲁棒性和精度的系统对于满足要求在此寄生虫中研究基因。首先，TERT和CRE/LOXP诱导模块和多路复用GRNA将集成到该系统中。第二，一个完整的基因使用DCAS12A（CPF1）的调节系统，这是一种具有遗传特征的新CAS 像恶性疟原虫之类的高素质基因组中的工程将进行设计和验证。使用A的这两个DCAS基因调节系统的性能进行系统比较选定基因的套件在不同的开发阶段表达，并且具有不同的转录水平将为系统的未来有效使用提供关键指导。这是预计这种多功能CRISPR/DCAS的表观遗传基因调节系统将在恶性假单胞菌中找到广泛的应用基因组研究。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Plasmodium falciparum GCN5 plays a key role in regulating artemisinin resistance-related stress responses.

DOI：
10.1128/aac.00577-23
发表时间：
2023-10-18
期刊：
Antimicrobial agents and chemotherapy
影响因子：
4.9
作者：
通讯作者：

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

DOI：
10.1128/spectrum.02782-21
发表时间：
2022-06-29
期刊：
MICROBIOLOGY SPECTRUM
影响因子：
3.7
作者：
Liang, Xiaoying;Boonhok, Rachasak;Siddiqui, Faiza Amber;Xiao, Bo;Li, Xiaolian;Qin, Junling;Min, Hui;Jiang, Lubin;Cui, Liwang;Miao, Jun
通讯作者：
Miao, Jun