Harnessing the diversity of Cas9 orthologs for genome editing Tomas Urbaitis1,2, Giedrius Gasiunas1*, Joshua K. Young3*, Darius Kazlauskas2, Monika Jasnauskaite1, Migle Stitilytė1, Mantvyda M. Grusyte1, Sushmitha Paulraj3, Zhenglin Hou3, Clara Alarcon3, N. Doane Chilcoat3, Jennifer L. Curcuru4, Shane K. Dooley5, Megumu Mabuchi4, Ryan T. Fuchs4, Zhiyi Sun4, Ezra Schildkraut4, G. Brett Robb4*, Ceslovas Venclovas2, and Virginijus Siksnys1,2* 1 CasZyme, Vilnius, LT-10257, Lithuania 2 Institute of Biotechnology, Vilnius University, Vilnius, LT-10257, Lithuania 3 Department of Molecular Engineering, Corteva Agriscience™, Johnston, IA 50131, USA 4 New England Biolabs, Ipswich, MA 01938, USA 5 Department of Agricultural and Biosystems Engineering, Iowa State University, Ames, IA 50011, USA * To whom correspondence should be addressed. Email: firstname.lastname@example.org, email@example.com, firstname.lastname@example.org and email@example.com The Cas9 protein from CRISPR-Cas bacterial defense systems has been adopted as a robust and multifaceted genome editing tool. However, for Cas9 to bind a given target, a short nucleotide sequence, termed protospacer adjacent motif (PAM), is required. This PAM constraint as well as insufficient specificity are major obstacles for Cas9 genome editing. Thus, analysis of natural Cas9 orthologs could offer an increased diversity of PAM sequences and biochemical properties which may be beneficial to genome editing applications. We applied a phylogeny-guided bioinformatic approach and developed biochemical screens for the rapid identification and characterization of the PAM and guide RNA requirements of novel Cas9 proteins. This analysis permitted the characterization of 79 Cas9 orthologs with more than 50 distinct PAM sequence requirements and varying guide RNAs which could be classified into seven groups based on sequence and structural homology. A share of orthologs were purified and their activities assessed further revealing additional biochemical diversity in the form of required spacer lengths, tolerance of temperature ranges and generation of distinct cleavage patterns. Our results indicate that the natural diversity of Cas9 orthologs provide a source of PAM recognition and other potentially desirable properties that may be used to expand the genome editing toolbox and permit orthogonal editing approaches as well as provide insight on the fundamental phylogeny of CRISPR-Cas systems.