Department of Biology, Philipps-Universität Marburg, Hans-Meerwein-Str. 6, 35043 Marburg, Germany.
Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Strasse 10, 35043 Marburg, Germany.
Nucleic Acids Res. 2024 Nov 11;52(20):12592-12603. doi: 10.1093/nar/gkae879.
Type IV CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) effector complexes are often encoded on plasmids and are proposed to prevent the replication of competing plasmids. The Type IV-A1 CRISPR-Cas system of Pseudomonas oleovorans additionally harbors a CRISPR RNA (crRNA) that tightly regulates the transcript levels of a chromosomal target and represents a natural CRISPR interference (CRISPRi) tool. This study investigates CRISPRi effects of this system using synthetic crRNAs against genome and plasmid sequences. Targeting of reporter genes revealed extended interference in P. oleovorans and Escherichia coli cells producing recombinant CRISPR ribonucleoprotein (crRNP) complexes. RNA sequencing (RNA-seq) analyses of Type IV-A1 CRISPRi-induced transcriptome alterations demonstrated highly effective long-range downregulation of histidine operon expression, whereas CRISPRi effects of dCas9 remained limited to the vicinity of its binding site. Single-molecule microscopy uncovered the localization dynamics of crRNP complexes. The tracks of fluorescently labeled crRNPs co-localized with regions of increased plasmid replication, supporting efficient plasmid targeting. These results identify mechanistic principles that facilitate the application of Type IV-A1 CRISPRi for the regulation of gene expression and plasmid replication.
IV 型 CRISPR-Cas（成簇规律间隔短回文重复序列和 CRISPR 相关蛋白）效应复合物通常编码在质粒上，并被提议用于防止竞争质粒的复制。假单胞菌 oleovorans 的 IV 型-A1 CRISPR-Cas 系统还拥有一个 CRISPR RNA（crRNA），它可以严格调控染色体靶标基因的转录水平，并代表一种天然的 CRISPR 干扰（CRISPRi）工具。本研究使用针对基因组和质粒序列的合成 crRNA 来研究该系统的 CRISPRi 效应。报告基因的靶向研究表明，在产生重组 CRISPR 核糖核蛋白（crRNP）复合物的假单胞菌 oleovorans 和大肠杆菌细胞中，干扰作用得到了扩展。IV 型-A1 CRISPRi 诱导的转录组变化的 RNA 测序（RNA-seq）分析表明，组氨酸操纵子表达的高效远程下调，而 dCas9 的 CRISPRi 效应仍然局限于其结合位点的附近。单分子显微镜揭示了 crRNP 复合物的定位动力学。荧光标记的 crRNP 的轨迹与质粒复制增加的区域共定位，支持有效的质粒靶向。这些结果确定了促进 IV 型-A1 CRISPRi 用于调控基因表达和质粒复制的机制原理。
