Protocol for high-precision CRISPR-Cas12a-based SNV detection on synthetic DNA, cell line cfDNA models, and liquid biopsies

Kavish A. V. Kohabir; Jasper Linthorst; Rob M. F. Wolthuis; Erik A. Sistermans

doi:10.1016/j.xpro.2025.103696

Protocol for high-precision CRISPR-Cas12a-based SNV detection on synthetic DNA, cell line cfDNA models, and liquid biopsies

Kavish A. V. Kohabir^*, Jasper Linthorst, Rob M. F. Wolthuis^*, Erik A. Sistermans^*

^*Corresponding author for this work

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Abstract

CRISPR-based diagnostics (CRISPRdx) offer promising tools for rapid and cost-effective genetic testing, but achieving single-nucleotide fidelity remains a challenge. Here, we present a protocol for high-precision detection of single-nucleotide variants (SNVs) using a Cas12a-based approach. We describe how to apply our publicly available ARTEMIS algorithm to identify targetable SNVs, design optimized CRISPR RNAs (crRNAs), and perform fluorescence-based CRISPRdx assays on synthetic DNA, cell line-derived cell-free DNA (cfDNA), and liquid biopsy samples. For complete details on the use and execution of this protocol, please refer to Kohabir et al.1

Original language	English
Article number	103696
Journal	STAR Protocols
Volume	6
Issue number	2
DOIs	https://doi.org/10.1016/j.xpro.2025.103696
Publication status	Published - 20 Jun 2025

Keywords

CRISPR
biotechnology and bioengineering
cancer
cell biology
cell culture
genetics

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.xpro.2025.103696

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title = "Protocol for high-precision CRISPR-Cas12a-based SNV detection on synthetic DNA, cell line cfDNA models, and liquid biopsies",

abstract = "CRISPR-based diagnostics (CRISPRdx) offer promising tools for rapid and cost-effective genetic testing, but achieving single-nucleotide fidelity remains a challenge. Here, we present a protocol for high-precision detection of single-nucleotide variants (SNVs) using a Cas12a-based approach. We describe how to apply our publicly available ARTEMIS algorithm to identify targetable SNVs, design optimized CRISPR RNAs (crRNAs), and perform fluorescence-based CRISPRdx assays on synthetic DNA, cell line-derived cell-free DNA (cfDNA), and liquid biopsy samples. For complete details on the use and execution of this protocol, please refer to Kohabir et al.1",

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AU - Kohabir, Kavish A. V.

AU - Linthorst, Jasper

AU - Wolthuis, Rob M. F.

AU - Sistermans, Erik A.

PY - 2025/6/20

Y1 - 2025/6/20

N2 - CRISPR-based diagnostics (CRISPRdx) offer promising tools for rapid and cost-effective genetic testing, but achieving single-nucleotide fidelity remains a challenge. Here, we present a protocol for high-precision detection of single-nucleotide variants (SNVs) using a Cas12a-based approach. We describe how to apply our publicly available ARTEMIS algorithm to identify targetable SNVs, design optimized CRISPR RNAs (crRNAs), and perform fluorescence-based CRISPRdx assays on synthetic DNA, cell line-derived cell-free DNA (cfDNA), and liquid biopsy samples. For complete details on the use and execution of this protocol, please refer to Kohabir et al.1

AB - CRISPR-based diagnostics (CRISPRdx) offer promising tools for rapid and cost-effective genetic testing, but achieving single-nucleotide fidelity remains a challenge. Here, we present a protocol for high-precision detection of single-nucleotide variants (SNVs) using a Cas12a-based approach. We describe how to apply our publicly available ARTEMIS algorithm to identify targetable SNVs, design optimized CRISPR RNAs (crRNAs), and perform fluorescence-based CRISPRdx assays on synthetic DNA, cell line-derived cell-free DNA (cfDNA), and liquid biopsy samples. For complete details on the use and execution of this protocol, please refer to Kohabir et al.1

KW - CRISPR

KW - biotechnology and bioengineering

KW - cancer

KW - cell biology

KW - cell culture

KW - genetics

UR - https://www.scopus.com/pages/publications/86000781934

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DO - 10.1016/j.xpro.2025.103696

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Protocol for high-precision CRISPR-Cas12a-based SNV detection on synthetic DNA, cell line cfDNA models, and liquid biopsies

Abstract

Keywords

UN SDGs

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