Towards understanding Trans-cleavage of natural and synthetic nucleic acids by Cas12a for sensitive CRISPR biosensing

Deng, F; Sang, R; Li, Y; Yang, D; Mckinnirey, F; Deng, W; Goldys, EM

Towards understanding Trans-cleavage of natural and synthetic nucleic acids by Cas12a for sensitive CRISPR biosensing

Deng, F Sang, R Li, Y Yang, D Mckinnirey, F Deng, W

Goldys, EM

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Publisher:: Elsevier
Publication Type:: Journal Article
Citation:: Microchemical Journal, 2024, 207, pp. 111850
Issue Date:: 2024-12-01

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Field	Value	Language
dc.contributor.author	Deng, F
dc.contributor.author	Sang, R
dc.contributor.author	Li, Y
dc.contributor.author	Yang, D
dc.contributor.author	Mckinnirey, F
dc.contributor.author	Deng, W https://orcid.org/0000-0002-9413-0978
dc.contributor.author	Goldys, EM
dc.date.accessioned	2025-01-06T04:39:10Z
dc.date.available	2025-01-06T04:39:10Z
dc.date.issued	2024-12-01
dc.identifier.citation	Microchemical Journal, 2024, 207, pp. 111850
dc.identifier.issn	0026-265X
dc.identifier.uri	http://hdl.handle.net/10453/183028
dc.description.abstract	CRISPR/Cas systems have been widely utilized for the development of biosensing platforms for precision molecular diagnostics. Their remarkable biosensing performance critically depends on the efficiency of sequence-independent trans-cleavage in type V and VI Cas effectors. Cas12a, a typical example of type V Cas effector exhibits varying trans-cleavage efficiency on different types of nucleic acids, and also in response to different nucleobase sequences. However, the underlying mechanism of Cas12a's trans-cleavage characteristic remains unclear. To explore this mechanism, we introduced Xeno nucleic acids (XNA) as potential trans-cleavage substrates of Cas12a. XNAs are chemically modified nucleic acid analogues, which originate from chemical modifications of nucleobases, sugar moieties, and the backbone. We observed a progressive decrease in trans-cleavage rates by Cas12a across different types of XNAs, in the following sequence: nucleobase-modified XNA > sugar moiety-modified XNA > backbone-modified XNA. In addition, more complex chemical modifications on either of the three above locations led to the lowering of the trans-cleavage rate of Cas12a. These findings elucidate the mechanism behind Cas12a’ trans-cleavage characteristic, which is attributed to its higher affinity of Cas12a for substrates with simpler chemical structures, leading to increased trans-cleavage efficiency. Based on these findings, we also developed a colorimetric CRISPR/Cas12a biosensing system utilizing XNA for the detection of circulating tumor DNA (ctDNA), with a limit of detection of 10 pM and a 4 logs detection range from 10 pM to 100 nM. These results indicate that XNA can serve as a novel Cas12a trans-cleavage substrate for sensitive biosensing applications.
dc.language	en
dc.publisher	Elsevier
dc.relation.ispartof	Microchemical Journal
dc.relation.isbasedon	10.1016/j.microc.2024.111850
dc.rights	info:eu-repo/semantics/openAccess
dc.subject	0301 Analytical Chemistry
dc.subject.classification	Analytical Chemistry
dc.subject.classification	3401 Analytical chemistry
dc.title	Towards understanding Trans-cleavage of natural and synthetic nucleic acids by Cas12a for sensitive CRISPR biosensing
dc.type	Journal Article
utslib.citation.volume	207
utslib.for	0301 Analytical Chemistry
pubs.organisational-group	University of Technology Sydney
pubs.organisational-group	University of Technology Sydney/Faculty of Engineering and Information Technology
pubs.organisational-group	University of Technology Sydney/Faculty of Engineering and Information Technology/School of Biomedical Engineering
utslib.copyright.status	open_access	*
dc.rights.license	This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0). To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/
dc.date.updated	2025-01-06T04:39:08Z
pubs.publication-status	Published
pubs.volume	207

Abstract:

CRISPR/Cas systems have been widely utilized for the development of biosensing platforms for precision molecular diagnostics. Their remarkable biosensing performance critically depends on the efficiency of sequence-independent trans-cleavage in type V and VI Cas effectors. Cas12a, a typical example of type V Cas effector exhibits varying trans-cleavage efficiency on different types of nucleic acids, and also in response to different nucleobase sequences. However, the underlying mechanism of Cas12a's trans-cleavage characteristic remains unclear. To explore this mechanism, we introduced Xeno nucleic acids (XNA) as potential trans-cleavage substrates of Cas12a. XNAs are chemically modified nucleic acid analogues, which originate from chemical modifications of nucleobases, sugar moieties, and the backbone. We observed a progressive decrease in trans-cleavage rates by Cas12a across different types of XNAs, in the following sequence: nucleobase-modified XNA > sugar moiety-modified XNA > backbone-modified XNA. In addition, more complex chemical modifications on either of the three above locations led to the lowering of the trans-cleavage rate of Cas12a. These findings elucidate the mechanism behind Cas12a’ trans-cleavage characteristic, which is attributed to its higher affinity of Cas12a for substrates with simpler chemical structures, leading to increased trans-cleavage efficiency. Based on these findings, we also developed a colorimetric CRISPR/Cas12a biosensing system utilizing XNA for the detection of circulating tumor DNA (ctDNA), with a limit of detection of 10 pM and a 4 logs detection range from 10 pM to 100 nM. These results indicate that XNA can serve as a novel Cas12a trans-cleavage substrate for sensitive biosensing applications.

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http://hdl.handle.net/10453/183028