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. [Display omitted] • A miniature CRISPR-Cas3 from N. lactamica confers bacterial immunity • NlaCascade-Cas3 RNP enables high-efficiency, targeted large deletions in human cells • Cas11, a hidden internal translation product, is essential for genome editing • Supplying Cas11 as a framework to harness divergent compact CRISPR-Cas3 editors Tan et al. discover a miniature CRISPR-Cas3 system that can efficiently create targeted large deletions in human genome. An inconspicuous subunit of its Cas machinery, Cas11, is encoded by a hidden ORF embedded in the cas operon. In human cells, Cas11 is the key enabler for compact CRISPR-Cas3 gene editors. [ABSTRACT FROM AUTHOR]