biostudies-literatureTan RNIGMS NIH HHS852-867.e5https://www.ebi.ac.uk/biostudies/studies/S-EPMC8964063biostudies-literatureUnknown82(4)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.Molecular cellCas11 enables genome engineering in human cells with compact CRISPR-Cas3 systems.PMC8964063R35 GM137883K99 GM117268R00 GM117268R35 GM118174Hou ZGramelspacher MJZhang YXiao YKe AZhou XTan RKrueger RKfalseCas11 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.2022-01-01T00:00:00Z2022 Feb2025-04-20T03:04:12.735Z2025-04-20T03:04:12.735ZS-EPMC89640633505135110.1016/j.molcel.2021.12.032