Department of Medical Microbiology and Infectious Diseases, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands.
Laboratory of Microbiology, Wageningen University, Wageningen, Netherlands.
Sci Adv. 2020 Jun 17;6(25):eaaz4849. doi: 10.1126/sciadv.aaz4849. eCollection 2020 Jun.
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, 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 (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.
CRISPR-Cas9 系统在人类致病菌中富集，并通过未知机制与细胞毒性相关。在这里，我们表明，在感染人类细胞后，会将其 Cas9（CjeCas9）核酸酶分泌到细胞质中。接下来，一个天然的核定位信号使 CjeCas9 进入细胞核，在那里它催化金属依赖性的非特异性 DNA 切割，导致细胞死亡。与 CjeCas9 相比， 的天然 Cas9（SpyCas9）更适合指导依赖性编辑。然而，在人类细胞中，天然 SpyCas9 仍可能导致一些 DNA 损伤，这很可能是由于其 ssDNA 切割活性。用适当的向导 RNA 使 SpyCas9 饱和可以完全阻止这种副作用，但对 CjeCas9 只有部分效果。我们得出结论，CjeCas9 在攻击人类细胞方面发挥着积极作用，而不是在病毒防御方面。此外，这些独特的催化特性可能使 CjeCas9 不太适合基因组编辑应用。
