A catalogue of biochemically diverse CRISPR-Cas9 orthologs
Giedrius Gasiūnas, Joshua K. Young, Tautvydas Karvelis, Darius Kazlauskas, Tomas Urbaitis, Monika Jasnauskaite, Mantvyda M. Grusyte, Sushmitha Paulraj, Po-Hao Wang, Zhenglin Hou, Shane K. Dooley, Mark Cigan, Clara M. Alarcón, N. Doane Chilcoat, Greta Bigelyte, Jennifer L. Curcuru, Megumu Mabuchi, Zhiyi Sun, Ryan T. Fuchs, Ezra Schildkraut, Peter Weigele, William E. Jack, G. Brett Robb, Česlovas Venclovas, Virginijus Šikšnys
Abstract
Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.