Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems are an adaptive immune response mechanism in prokaryotes which can target and cleave invading DNA or RNA. The rapid understanding of the type II CRISPR/Cas9 system through biochemical, genetic and structural investigations has contributed to the development of Cas9 for various DNA- and RNA-targeting applications. Recent single-molecule investigations of CRISPR/Cas9 mechanisms have further extended our understanding of target search, binding and cleavage. These investigations are fundamental to the further development of CRISPR/Cas9 tools. This review discusses how single-molecule techniques have illuminated the enzymology of Cas9 endonucleases.
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