Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

Biochemically diverse CRISPR-Cas9 orthologs

View ORCID ProfileGiedrius Gasiunas, Joshua K. Young, Tautvydas Karvelis, Darius Kazlauskas, Tomas Urbaitis, Monika Jasnauskaite, Mantvyda Grusyte, Sushmitha Paulraj, Po-Hao Wang, Zhenglin Hou, Shane K. Dooley, Mark Cigan, Clara Alarcon, N. Doane Chilcoat, Greta Bigelyte, Jennifer L. Curcuru, Megumu Mabuchi, Zhiyi Sun, Ryan T. Fuchs, Ezra Schildkraut, Peter R. Weigele, William E. Jack, G. Brett Robb, Česlovas Venclovas, View ORCID ProfileVirginijus Siksnys
doi: https://doi.org/10.1101/2020.04.29.066654
Giedrius Gasiunas
1CasZyme, Vilnius, LT-10257, Lithuania
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Giedrius Gasiunas
Joshua K. Young
2™, Johnston, IA 50131, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: josh.young@corteva.com robb@neb.com siksnys@ibt.lt
Tautvydas Karvelis
3Institute of Biotechnology, Vilnius University, Vilnius, LT-10257, Lithuania
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Darius Kazlauskas
3Institute of Biotechnology, Vilnius University, Vilnius, LT-10257, Lithuania
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Tomas Urbaitis
1CasZyme, Vilnius, LT-10257, Lithuania
3Institute of Biotechnology, Vilnius University, Vilnius, LT-10257, Lithuania
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Monika Jasnauskaite
1CasZyme, Vilnius, LT-10257, Lithuania
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Mantvyda Grusyte
1CasZyme, Vilnius, LT-10257, Lithuania
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Sushmitha Paulraj
2™, Johnston, IA 50131, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Po-Hao Wang
2™, Johnston, IA 50131, USA
6Inari Agriculture, West Lafayette, IN 47906, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Zhenglin Hou
2™, Johnston, IA 50131, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Shane K. Dooley
5Department of Agricultural and Biosystems Engineering, Iowa State University, Ames, IA 50011, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Mark Cigan
2™, Johnston, IA 50131, USA
7Genus plc, Deforest, WI 53532, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Clara Alarcon
2™, Johnston, IA 50131, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
N. Doane Chilcoat
2™, Johnston, IA 50131, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Greta Bigelyte
3Institute of Biotechnology, Vilnius University, Vilnius, LT-10257, Lithuania
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jennifer L. Curcuru
4New England Biolabs, Ipswich, MA 01938, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Megumu Mabuchi
4New England Biolabs, Ipswich, MA 01938, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Zhiyi Sun
4New England Biolabs, Ipswich, MA 01938, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ryan T. Fuchs
4New England Biolabs, Ipswich, MA 01938, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ezra Schildkraut
4New England Biolabs, Ipswich, MA 01938, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Peter R. Weigele
4New England Biolabs, Ipswich, MA 01938, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
William E. Jack
4New England Biolabs, Ipswich, MA 01938, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
G. Brett Robb
4New England Biolabs, Ipswich, MA 01938, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: josh.young@corteva.com robb@neb.com siksnys@ibt.lt
Česlovas Venclovas
3Institute of Biotechnology, Vilnius University, Vilnius, LT-10257, Lithuania
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Virginijus Siksnys
1CasZyme, Vilnius, LT-10257, Lithuania
3Institute of Biotechnology, Vilnius University, Vilnius, LT-10257, Lithuania
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Virginijus Siksnys
  • For correspondence: josh.young@corteva.com robb@neb.com siksnys@ibt.lt
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Supplementary material
  • Preview PDF
Loading

ABSTRACT

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 Statement

Z.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.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
Back to top
PreviousNext
Posted April 30, 2020.
Download PDF

Supplementary Material

Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
Biochemically diverse CRISPR-Cas9 orthologs
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Biochemically diverse CRISPR-Cas9 orthologs
Giedrius Gasiunas, Joshua K. Young, Tautvydas Karvelis, Darius Kazlauskas, Tomas Urbaitis, Monika Jasnauskaite, Mantvyda Grusyte, Sushmitha Paulraj, Po-Hao Wang, Zhenglin Hou, Shane K. Dooley, Mark Cigan, Clara Alarcon, N. Doane Chilcoat, Greta Bigelyte, Jennifer L. Curcuru, Megumu Mabuchi, Zhiyi Sun, Ryan T. Fuchs, Ezra Schildkraut, Peter R. Weigele, William E. Jack, G. Brett Robb, Česlovas Venclovas, Virginijus Siksnys
bioRxiv 2020.04.29.066654; doi: https://doi.org/10.1101/2020.04.29.066654
Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Citation Tools
Biochemically diverse CRISPR-Cas9 orthologs
Giedrius Gasiunas, Joshua K. Young, Tautvydas Karvelis, Darius Kazlauskas, Tomas Urbaitis, Monika Jasnauskaite, Mantvyda Grusyte, Sushmitha Paulraj, Po-Hao Wang, Zhenglin Hou, Shane K. Dooley, Mark Cigan, Clara Alarcon, N. Doane Chilcoat, Greta Bigelyte, Jennifer L. Curcuru, Megumu Mabuchi, Zhiyi Sun, Ryan T. Fuchs, Ezra Schildkraut, Peter R. Weigele, William E. Jack, G. Brett Robb, Česlovas Venclovas, Virginijus Siksnys
bioRxiv 2020.04.29.066654; doi: https://doi.org/10.1101/2020.04.29.066654

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Biochemistry
Subject Areas
All Articles
  • Animal Behavior and Cognition (2441)
  • Biochemistry (4807)
  • Bioengineering (3342)
  • Bioinformatics (14730)
  • Biophysics (6664)
  • Cancer Biology (5194)
  • Cell Biology (7459)
  • Clinical Trials (138)
  • Developmental Biology (4388)
  • Ecology (6906)
  • Epidemiology (2057)
  • Evolutionary Biology (9948)
  • Genetics (7360)
  • Genomics (9555)
  • Immunology (4589)
  • Microbiology (12740)
  • Molecular Biology (4968)
  • Neuroscience (28450)
  • Paleontology (199)
  • Pathology (811)
  • Pharmacology and Toxicology (1400)
  • Physiology (2034)
  • Plant Biology (4528)
  • Scientific Communication and Education (981)
  • Synthetic Biology (1307)
  • Systems Biology (3923)
  • Zoology (731)