Arifah Adini Q, Vento Justin M, Kurrer Isabella, Achmedov Tatjana, Beisel Chase L
Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Center for Infection Research, 97080 Würzburg, Germany.
Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States.
Microlife. 2025 Jul 3;6:uqaf013. doi: 10.1093/femsml/uqaf013. eCollection 2025.
CRISPR-Cas9 systems are widely used for bacterial genome editing, yet their heterologous expression has been associated with cytotoxicity. The Cas9 nuclease from (SpyCas9) has been one common source, with reports of cytotoxicity with the nuclease alone or in combination with a single-guide RNA observed in some bacteria. However, the potential cytotoxic effects of other components of the CRISPR-Cas9 system remain unknown. Here, we report that expression of the short isoform of the trans-activating CRISPR RNA (tracr-S) from the CRISPR-Cas locus is cytotoxic in , even in the absence of SpyCas9. Deleting a putative transcription regulator in alleviates tracr-S cytotoxicity and leads to expression of the long isoform of the trans-activating CRISPR RNA (tracr-L). Furthermore, cytotoxicity was specific to the tracr-S sequence and was linked to direct interactions with host RNAs. This work thus reveals that additional CRISPR components beyond Cas9 can interfere with the use of heterologous CRISPR-Cas systems in bacteria, with potential implications for the evolution of CRISPR immunity.
CRISPR-Cas9系统被广泛用于细菌基因组编辑，但其异源表达与细胞毒性有关。来自酿脓链球菌的Cas9核酸酶（SpyCas9）是一个常见来源，有报道称在一些细菌中单独的核酸酶或与单向导RNA联合使用时会出现细胞毒性。然而，CRISPR-Cas9系统其他组分的潜在细胞毒性作用仍不清楚。在此，我们报道来自酿脓链球菌CRISPR-Cas位点的反式激活CRISPR RNA短异构体（tracr-S）的表达在大肠杆菌中具有细胞毒性，即使在没有SpyCas9的情况下也是如此。删除大肠杆菌中一个假定的转录调节因子可减轻tracr-S的细胞毒性，并导致反式激活CRISPR RNA长异构体（tracr-L）的表达。此外，细胞毒性对tracr-S序列具有特异性，并且与与宿主RNA的直接相互作用有关。因此，这项工作揭示了除Cas9之外的其他CRISPR组分可干扰细菌中异源CRISPR-Cas系统的使用，这对CRISPR免疫的进化具有潜在影响。
