RT Journal Article
SR Electronic
T1 Biochemically diverse CRISPR-Cas9 orthologs
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.04.29.066654
DO 10.1101/2020.04.29.066654
A1 Giedrius Gasiunas
A1 Joshua K. Young
A1 Tautvydas Karvelis
A1 Darius Kazlauskas
A1 Tomas Urbaitis
A1 Monika Jasnauskaite
A1 Mantvyda Grusyte
A1 Sushmitha Paulraj
A1 Po-Hao Wang
A1 Zhenglin Hou
A1 Shane K. Dooley
A1 Mark Cigan
A1 Clara Alarcon
A1 N. Doane Chilcoat
A1 Greta Bigelyte
A1 Jennifer L. Curcuru
A1 Megumu Mabuchi
A1 Zhiyi Sun
A1 Ryan T. Fuchs
A1 Ezra Schildkraut
A1 Peter R. Weigele
A1 William E. Jack
A1 G. Brett Robb
A1 Česlovas Venclovas
A1 Virginijus Siksnys
YR 2020
UL http://biorxiv.org/content/early/2020/04/30/2020.04.29.066654.abstract
AB CRISPR-Cas9 nucleases are abundant in microbes. To explore this largely uncharacterized diversity, we applied cell-free biochemical screens to rapidly assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of novel Cas9 proteins. This approach permitted the characterization of 79 Cas9 orthologs with at least 7 distinct classes of gRNAs and 50 different PAM sequence requirements. PAM recognition spanned the entire spectrum of T-, A-, C-, and G-rich nucleotides ranging from simple di-nucleotide recognition to complex sequence strings longer than 4. Computational analyses indicated that most of this diversity came from 4 groups of interrelated sequences providing new insight into Cas9 evolution and efforts to engineer PAM recognition. A subset of Cas9 orthologs were purified and their activities examined further exposing additional biochemical diversity. This constituted both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for longer stretches of homology between gRNA and DNA target to function robustly. In all, the diverse collection of Cas9 orthologs presented here sheds light on Cas9 evolution and provides a rich source of PAM recognition and other potentially desirable properties that may be mined to expand the genome editing toolbox with new RNA-programmable nucleases.Competing Interest StatementZ.H., J.K.Y., G.G and V.S. have filed patent applications related to the manuscript. G.G, T.U, M.J. and M.G. are employees of CasZyme. J.K.Y., S.P., Z.H., C.A., and N.D.C. are employees of Corteva Agriscience. J.L.C., M.M., R.T.F, E.S., P.R.W., Z.S., W.E.J. and G.B.R. are employees of NEB. V.S. is a Chairman of CasZyme. V.S. and G.G. have financial interest in CasZyme.