CRISPR-associated (Cas) endonucleases specifically target and cleave RNA or DNA based on complementarity to a guide RNA. Cas endonucleases – including Cas9, Cas12a, and Cas13 – have been adopted for a wide array of biotechnological tools, including gene editing, transcriptional modulation, and diagnostics. These tools are facilitated by ready reprogramming of guide RNA sequences and the varied nucleic acid binding and cleavage activities observed across diverse Cas endonucleases. However, the large size of most Cas endonucleases (950–1400 amino acids) can restrict applications. The recent discovery of miniature Cas endonucleases (400–800 amino acids) provides the potential to overcome this limitation. Here, we review recent advances in understanding the structural mechanisms of two miniature Cas endonucleases, Cas12f and Cas12j.

Clustered regularly interspaced short palindromic repeats

Protospacer adjacent motif

Target nucleic acid binding

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