A scalable CRISPR-Cas9 gene editing system facilitates CRISPR screens in the malaria parasite Plasmodium berghei

IF 16.6 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Nucleic Acids Research Pub Date : 2025-01-23 DOI:10.1093/nar/gkaf005
Thorey K Jonsdottir, Martina S Paoletta, Takahiro Ishizaki, Sophia Hernandez, Maria Ivanova, Alicia Herrera Curbelo, Paulina A Saiki, Martin Selinger, Debojyoti Das, Johan Henriksson, Ellen S C Bushell
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Abstract

Many Plasmodium genes remain uncharacterized due to low genetic tractability. Previous large-scale knockout screens have only been able to target about half of the genome in the more genetically tractable rodent malaria parasite Plasmodium berghei. To overcome this limitation, we have developed a scalable CRISPR system called P. berghei high-throughput (PbHiT), which uses a single cloning step to generate targeting vectors with 100-bp homology arms physically linked to a guide RNA (gRNA) that effectively integrate into the target locus. We show that PbHiT coupled with gRNA sequencing robustly recapitulates known knockout mutant phenotypes in pooled transfections. Furthermore, we provide an online resource of knockout and tagging designs to target the entire P. berghei genome and scale-up vector production using a pooled ligation approach. This work presents for the first time a tool for high-throughput CRISPR screens in Plasmodium for studying the parasite’s biology at scale.
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一种可扩展的CRISPR- cas9基因编辑系统促进了疟疾寄生虫伯氏疟原虫的CRISPR筛选
许多疟原虫基因由于遗传易感性低而未被鉴定。以前的大规模基因敲除筛选只能够针对遗传上更容易处理的啮齿动物疟疾寄生虫伯氏疟原虫的大约一半基因组。为了克服这一限制,我们开发了一种可扩展的CRISPR系统,称为P. berghei高通量(PbHiT),该系统使用单个克隆步骤生成具有100 bp同源臂的靶向载体,这些同源臂与有效整合到目标位点的引导RNA (gRNA)物理连接。我们发现,PbHiT与gRNA测序相结合,在混合转染中强有力地概括了已知的敲除突变表型。此外,我们还提供了一个基因敲除和标记设计的在线资源,以整个伯氏线虫基因组为目标,并使用池连接方法扩大载体生产。这项工作首次提出了在疟原虫中进行高通量CRISPR筛选的工具,用于大规模研究寄生虫的生物学。
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来源期刊
Nucleic Acids Research
Nucleic Acids Research 生物-生化与分子生物学
CiteScore
27.10
自引率
4.70%
发文量
1057
审稿时长
2 months
期刊介绍: Nucleic Acids Research (NAR) is a scientific journal that publishes research on various aspects of nucleic acids and proteins involved in nucleic acid metabolism and interactions. It covers areas such as chemistry and synthetic biology, computational biology, gene regulation, chromatin and epigenetics, genome integrity, repair and replication, genomics, molecular biology, nucleic acid enzymes, RNA, and structural biology. The journal also includes a Survey and Summary section for brief reviews. Additionally, each year, the first issue is dedicated to biological databases, and an issue in July focuses on web-based software resources for the biological community. Nucleic Acids Research is indexed by several services including Abstracts on Hygiene and Communicable Diseases, Animal Breeding Abstracts, Agricultural Engineering Abstracts, Agbiotech News and Information, BIOSIS Previews, CAB Abstracts, and EMBASE.
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