Molecular basis and engineering of miniature Cas12f with C-rich PAM specificity

CRISPR–Cas12f nucleases are currently one of the smallest genome editors, exhibiting advantages for efficient delivery via cargo-size-limited adeno-associated virus delivery vehicles. Most characterized Cas12f nucleases recognize similar T-rich protospacer adjacent motifs (PAMs) for DNA targeting, substantially restricting their targeting scope. Here we report the cryogenic electron microscopy structure and engineering of a miniature Clostridium novyi Cas12f1 nuclease (CnCas12f1, 497 amino acids) with rare C-rich PAM specificity. Structural characterizations revealed detailed PAM recognition, asymmetric homodimer formation and single guide RNA (sgRNA) association mechanisms. sgRNA engineering transformed CRISPR–CnCas12f1, which initially was incapable of genome targeting in bacteria, into an effective genome editor in human cells. Our results facilitate further understanding of CRISPR–Cas12f1 working mechanism and expand the mini-CRISPR toolbox. Most miniature Cas12f nucleases have T-rich PAM specificity, restricting their targeting scopes. The cryo-EM structure of the Clostridium novyi Cas12f1 reveals the molecular basis for rare C-rich PAM recognition and enables optimization of sgRNA scaffold to improve CnCas12f1 activity.