Type I-Fv and engineered type IV-A1 CRISPR-Cas effectors facilitate genome reduction in Escherichia coli.

Class 1 CRISPR-Cas systems utilize multi-subunit effector ribonucleoprotein complexes to identify and target DNA. Upon recognition, type I systems recruit the helicase/nuclease Cas3 for DNA degradation, while type IV-A systems use the helicase CasDinG for transcriptional repression. Here, we developed two recombinant class 1 CRISPR-Cas genome editing tools for inducing large genomic deletions: the compact type I-Fv (also termed I-F2) system from Shewanella putrefaciens and the type IV-A1 system from Pseudomonas oleovorans. In the latter, CasDinG was engineered to include a C-terminal HNH nuclease domain, conferring DNA cleavage activity and enabling analysis of CasDinG processivity. Whole-genome sequencing of Escherichia coli BL21-AI was used to monitor genome reduction and DNA repair mechanisms in response to CRISPR-Cas-induced damage. Small deletions were flanked by microhomologies, consistent with repair via alternative end joining, whereas deletions larger than 10 kb consistently terminated at nearby IS1 elements, implicating these sequences in the repair process. This study introduces compact type I and engineered type IV-A genome editing tools with distinct protospacer-adjacent motif requirements and provides new insights into CasDinG evolution and the DNA repair pathways engaged during CRISPR-Cas-mediated genome editing.