CRISPR-induced DNA reorganization for multiplexed nucleic acid detection

Margot Karlikow; Evan Amalfitano; Xiaolong Yang; Jennifer Doucet; Abigail Chapman; Peivand Sadat Mousavi; Paige Homme; Polina Sutyrina; Winston Chan; Sofia Lemak; Alexander F Yakunin; Adam G Dolezal; Shana Kelley; Leonard J Foster; Brock A Harpur; Keith Pardee

doi:10.1038/s41467-023-36874-6

CRISPR-induced DNA reorganization for multiplexed nucleic acid detection

Nat Commun. 2023 Mar 17;14(1):1505. doi: 10.1038/s41467-023-36874-6.

Authors

Margot Karlikow¹, Evan Amalfitano², Xiaolong Yang², Jennifer Doucet², Abigail Chapman³, Peivand Sadat Mousavi², Paige Homme², Polina Sutyrina², Winston Chan², Sofia Lemak⁴, Alexander F Yakunin^{4

5}, Adam G Dolezal⁶, Shana Kelley^{2

7

8

9

10

11}, Leonard J Foster³, Brock A Harpur¹², Keith Pardee^{13

14}

Affiliations

¹ Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, M5S 3M2, Canada. margot.karlikow@gmail.com.
² Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, M5S 3M2, Canada.
³ Department of Biochemistry & Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.
⁴ Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
⁵ Centre for Environmental Biotechnology, School of Natural Sciences, Bangor University, Bangor, Gwynedd, LL57 2UW, UK.
⁶ Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.
⁷ Institute of Biomedical Engineering, University of Toronto, Toronto, ON, M5S 3G9, Canada.
⁸ Department of Chemistry, Faculty of Arts and Science, University of Toronto, Toronto, ON, M5S 3H4, Canada.
⁹ Department of Biochemistry and Molecular Genetics, Northwestern University, Chicago, IL, 60611, USA.
¹⁰ Department of Chemistry, Northwestern University, Evanston, IL, 60208, USA.
¹¹ Department of Biomedical Engineering, Northwestern University, Evanston, IL, 60208, USA.
¹² Department of Entomology, Purdue University, 901 W State Street, West Lafayette, IN, 47907, USA.
¹³ Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, M5S 3M2, Canada. keith.pardee@utoronto.ca.
¹⁴ Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, ON, M5S 1A1, Canada. keith.pardee@utoronto.ca.

Abstract

Nucleic acid sensing powered by the sequence recognition of CRIPSR technologies has enabled major advancement toward rapid, accurate and deployable diagnostics. While exciting, there are still many challenges facing their practical implementation, such as the widespread need for a PAM sequence in the targeted nucleic acid, labile RNA inputs, and limited multiplexing. Here we report FACT (Functionalized Amplification CRISPR Tracing), a CRISPR-based nucleic acid barcoding technology compatible with Cas12a and Cas13a, enabling diagnostic outputs based on cis- and trans-cleavage from any sequence. Furthermore, we link the activation of CRISPR-Cas12a to the expression of proteins through a Reprogrammable PAIRing system (RePAIR). We then combine FACT and RePAIR to create FACTOR (FACT on RePAIR), a CRISPR-based diagnostic, that we use to detect infectious disease in an agricultural use case: honey bee viral infection. With high specificity and accuracy, we demonstrate the potential of FACTOR to be applied to the sensing of any nucleic acid of interest.

Publication types

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

MeSH terms

Agriculture
Animals
Biosensing Techniques*
CRISPR-Cas Systems / genetics
DNA / genetics
Head
Nucleic Acid Amplification Techniques
Nucleic Acids*
RNA / genetics

Substances

DNA
Nucleic Acids
RNA