An engineered ScCas9 with broad PAM range and high specificity and activity

Pranam Chatterjee; Noah Jakimo; Jooyoung Lee; Nadia Amrani; Tomás Rodríguez; Sabrina R T Koseki; Emma Tysinger; Rui Qing; Shilei Hao; Erik J Sontheimer; Joseph Jacobson

doi:10.1038/s41587-020-0517-0

An engineered ScCas9 with broad PAM range and high specificity and activity

Nat Biotechnol. 2020 Oct;38(10):1154-1158. doi: 10.1038/s41587-020-0517-0. Epub 2020 May 11.

Authors

Pranam Chatterjee^#^{1

2}, Noah Jakimo^#^{3

4}, Jooyoung Lee⁵, Nadia Amrani⁵, Tomás Rodríguez⁵, Sabrina R T Koseki^{3

4}, Emma Tysinger^{3

4}, Rui Qing⁴, Shilei Hao⁴, Erik J Sontheimer⁵, Joseph Jacobson^{3

4}

Affiliations

¹ MIT Center for Bits and Atoms, Massachusetts Institute of Technology, Cambridge, MA, USA. [email protected].
² MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA, USA. [email protected].
³ MIT Center for Bits and Atoms, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁴ MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁵ RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA.

^# Contributed equally.

PMID: 32393822
DOI: 10.1038/s41587-020-0517-0

Abstract

CRISPR enzymes require a protospacer-adjacent motif (PAM) near the target cleavage site, constraining the sequences accessible for editing. In the present study, we combine protein motifs from several orthologs to engineer two variants of Streptococcus canis Cas9-Sc⁺⁺ and a higher-fidelity mutant HiFi-Sc⁺⁺-that have simultaneously broad 5'-NNG-3' PAM compatibility, robust DNA-cleavage activity and minimal off-target activity. Sc⁺⁺ and HiFi-Sc⁺⁺ extend the use of CRISPR editing for diverse applications.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Motifs / genetics*
CRISPR-Associated Protein 9 / genetics*
CRISPR-Cas Systems / genetics*
Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
DNA Cleavage
Gene Editing / methods
Streptococcus / genetics

Substances

CRISPR-Associated Protein 9

Supplementary concepts

Streptococcus canis

Grants and funding

R01 GM115911/GM/NIGMS NIH HHS/United States