Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection

Jie Yang; Yang Song; Xiangyu Deng; Jeffrey A Vanegas; Zheng You; Yuxuan Zhang; Zhengyan Weng; Lori Avery; Kevin D Dieckhaus; Advaith Peddi; Yang Gao; Yi Zhang; Xue Gao

doi:10.1038/s41589-022-01135-y

Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection

Nat Chem Biol. 2023 Jan;19(1):45-54. doi: 10.1038/s41589-022-01135-y. Epub 2022 Sep 22.

Authors

Jie Yang^#¹, Yang Song^#^{2

3}, Xiangyu Deng⁴, Jeffrey A Vanegas¹, Zheng You¹, Yuxuan Zhang^{2

3}, Zhengyan Weng^{2

3}, Lori Avery⁵, Kevin D Dieckhaus⁶, Advaith Peddi⁴, Yang Gao⁷, Yi Zhang^{8

9}, Xue Gao^{10

11

12}

Affiliations

¹ Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX, USA.
² Department of Biomedical Engineering, University of Connecticut, Storrs, CT, USA.
³ Institute of Materials Science, University of Connecticut, Storrs, CT, USA.
⁴ Department of Biosciences, Rice University, Houston, TX, USA.
⁵ Department of Pathology and Laboratory Medicine, UConn Health, Farmington, CT, USA.
⁶ Division of Infectious Diseases, Department of Medicine, UConn Health, Farmington, CT, USA.
⁷ Department of Biosciences, Rice University, Houston, TX, USA. yg60@rice.edu.
⁸ Department of Biomedical Engineering, University of Connecticut, Storrs, CT, USA. yi.5.zhang@uconn.edu.
⁹ Polymer Program, Institute of Materials Science, University of Connecticut, Storrs, CT, USA. yi.5.zhang@uconn.edu.
¹⁰ Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX, USA. xue.gao@rice.edu.
¹¹ Department of Bioengineering, Rice University, Houston, TX, USA. xue.gao@rice.edu.
¹² Department of Chemistry, Rice University, Houston, TX, USA. xue.gao@rice.edu.

^# Contributed equally.

PMID: 36138140
DOI: 10.1038/s41589-022-01135-y

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 13 (Cas13) has been rapidly developed for nucleic-acid-based diagnostics by using its characteristic collateral activity. Despite the recent progress in optimizing the Cas13 system for the detection of nucleic acids, engineering Cas13 protein with enhanced collateral activity has been challenging, mostly because of its complex structural dynamics. Here we successfully employed a novel strategy to engineer the Leptotrichia wadei (Lwa)Cas13a by inserting different RNA-binding domains into a unique active-site-proximal loop within its higher eukaryotes and prokaryotes nucleotide-binding domain. Two LwaCas13a variants showed enhanced collateral activity and improved sensitivity over the wild type in various buffer conditions. By combining with an electrochemical method, our variants detected the SARS-CoV-2 genome at attomolar concentrations from both inactive viral and unextracted clinical samples, without target preamplification. Our engineered LwaCas13a enzymes with enhanced collateral activity are ready to be integrated into other Cas13a-based platforms for ultrasensitive detection of nucleic acids.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

COVID-19*
CRISPR-Cas Systems / genetics
Genome
Humans
Nucleic Acids* / genetics
SARS-CoV-2 / genetics

Substances

Nucleic Acids