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 and Keith Pardee ()
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Margot Karlikow: University of Toronto
Evan Amalfitano: University of Toronto
Xiaolong Yang: University of Toronto
Jennifer Doucet: University of Toronto
Abigail Chapman: University of British Columbia
Peivand Sadat Mousavi: University of Toronto
Paige Homme: University of Toronto
Polina Sutyrina: University of Toronto
Winston Chan: University of Toronto
Sofia Lemak: University of Toronto
Alexander F. Yakunin: University of Toronto
Adam G. Dolezal: University of Illinois at Urbana–Champaign
Shana Kelley: University of Toronto
Leonard J. Foster: University of British Columbia
Brock A. Harpur: Purdue University
Keith Pardee: University of Toronto

Nature Communications, 2023, vol. 14, issue 1, 1-11

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-36874-6

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