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

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 autonomous entry of the guide-free CjeCas9 into the nucleus, where it catalyzes metal-dependent non-specific nicking of double stranded DNA, eventually leading to cell death. Compared to CjeCas9, we confirmed that Cas9 of Streptococcus pyogenes (SpyCas9) is more efficient for guide-dependent editing, and that in the absence of a guide it does not affect double stranded DNA. Although transduction of the SpyCas9 protein to human cells still may cause some DNA damage, most likely due to its ssDNA cleavage activity, this problem can be completely prevented by saturation with an appropriate guide, whereas this is only partly true for CjeCas9. Overall, we conclude that CjeCas9 has evolved towards an active role in the attack of human cells rather than the canonical defense against viruses and that the unique catalytic features of this nuclease may make it therefore less suitable for genome editing applications