Saturation guide RNA loading overcomes Cas9-induced human cell death. U2OS Cas9

CRISPR-Cas is an RNA-based defense system that enables prokaryotes to recognize invading foreign DNA via cognate crRNA guides and destroy it by CRISPR-associated Cas nucleases. Elucidation of the interference mechanism of the Streptococcus pyogenes Type II CRISPR40 Cas9 system has allowed for the successful repurposing of SpCas9 as a generic genome editing tool, with great promise for human gene therapy3. However, especially for therapeutic applications, caution seems appropriate, because Cas9 systems from some human pathogens might induce a cytotoxic response via an unknown mechanism4. Here we show that when present in human cells, Cas9 nucleases from the pathogenic bacteria Campylobacter jejuni and S. pyogenes have the potential to cause severe DNA damage. In the absence of a CRISPR RNA guide, native Cas9 nucleases from both pathogens enter the host nucleus, where their presence leads to promiscuous double stranded DNA breaks (DSBs) and induction of cell death. DSB induction can be reduced to background levels either by saturation of CjCas9 and SpCas9 with crRNA guides or by disruption of their nuclease activity. Our results demonstrate that Cas9 from bacterial pathogens might play an important role in virulence. Furthermore, we propose that saturating Cas9 with appropriate guide RNAs is crucial for efficient and safe therapeutic applications.