Guide-free Cas9 from pathogenic Campylobacter jejuni bacteria causes severe damage to DNA

Abstract

CRISPR-Cas9 systems are enriched in human pathogenic bacteria and have been linked to cytotoxicity by an unknown mechanism. Here, we show that upon infection of human cells, Campylobacter jejuni secretes its Cas9 (CjeCas9) nuclease into their cytoplasm. Next, a native nuclear localization signal enables CjeCas9 nuclear entry, where it catalyzes metal-dependent nonspecific DNA cleavage leading to cell death. Compared to CjeCas9, native Cas9 of Streptococcus pyogenes (SpyCas9) is more suitable for guide-dependent editing. However, in human cells, native SpyCas9 may still cause some DNA damage, most likely because of its ssDNA cleavage activity. This side effect can be completely prevented by saturation of SpyCas9 with an appropriate guide RNA, which is only partially effective for CjeCas9. We conclude that CjeCas9 plays an active role in attacking human cells rather than in viral defense. Moreover, these unique catalytic features may therefore make CjeCas9 less suitable for genome editing applications.

Campylobacter jejuni Cas9 Modulates the Transcriptome in Caco-2 Intestinal Epithelial Cells

“…To gain insight into the molecular basis behind Caco-2-induced cell damage by CjeCas9-producing C. jejuni [ 14 ], we analyzed the Caco-2 transcriptome at five rationally chosen infection time points obtained after infection. The raw data are publicly available at NCBI Gene Expression Omnibus (GEO), accession series GSE89661.…”

“…Based on our earlier work [ 14 ], it was of interest to us to identify candidate regulators of the cell death and pro-inflammatory pathways that were continuously induced by the wild-type CjeCas9-producing C. jejuni strain. Interestingly, one set of 198 Caco-2 genes was regulated across time by both WT and its isogenic ∆ cas9 mutant ( Figure 3 a and Table S2 ).…”

“…Upon epithelial cell infection, C . jejuni co-localizes with cellular endosomal markers at specific time points [ 13 , 14 ]. This 2–4 h process is a crucial time period, since it determines if C .…”

“…We revealed that the protein encoded by Clustered Regularly Interspaced Palindromic Repeat and associated gene 9 ( cas9 ) of C . jejuni (CjeCas9) is necessary to induce DNA damage and cell death during the infection process in a variety of cell lines [ 14 ]. In C. jejuni -infected cells, the DNA damage markers 53BP1 and y-H2AX are massively activated and contribute to the repair of CjeCas9 related DNA damage [ 14 ].…”

“…In order to pinpoint these processes, we carried out infection assays with Caco-2 intestinal epithelial cells using a wild-type strain and a corresponding isogenic cas9 deletion mutant [ 21 ] and monitored the infection process by extracting total RNA at biologically relevant time points that we earlier determined [ 11 , 12 ] for whole-genome transcriptomics. We intended to validate our hypothesis that CjeCas9 activates the DNA damage and cell fate pathways [ 14 , 21 ].…”