Cas11 enables genome engineering in human cells with compact CRISPR-Cas3 systems

Leading CRISPR-Cas technologies employ Cas9 and Cas12 enzymes that generate RNA-guided dsDNA breaks. Yet, the most abundant microbial adaptive immune systems, Type I CRISPRs, are under-exploited for eukaryotic applications. Here, we report the adoption of a minimal CRISPR-Cas3 from Neisseria lactamica (Nla) type I-C system to create targeted large deletions in the human genome. RNP delivery of its processive Cas3 nuclease and target recognition complex Cascade can confer-95% editing efficiency. Unexpectedly, NlaCascade assembly in bacteria requires internal translation of a hidden component Cas11 from within the cas8 gene. Furthermore, expressing a separately encoded NlaCas11 is the key to enable plasmid-and mRNA-based editing in human cells. Finally, we demonstrate that supplying cas11 is a universal strategy to systematically implement divergent I-C, I-D, and I-B CRISPR-Cas3 editors with compact sizes, distinct PAM preferences, and guide orthogonality. These findings greatly expand our ability to engineer long-range genome edits.

Automated summary

This study reports a new CRISPR-Cas3 system that can create targeted large deletions in the human genome. RNP delivery of Cas3 nuclease and target recognition complex can achieve editing efficiency of up to 95%. Furthermore, the study demonstrates that supplying cas11 is a universal strategy to enable different types of CRISPR-Cas3 editors and expands our ability to engineer long-range genome edits.