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박준수Joon Soo Park

Johns Hopkins University

ACS Nano, Jun 26 2024, | https://doi.org/10.1021/acsnano.4c05312 Digitized Kinetic Analysis Enhances Genotyping Capacity of CRISPR-Based Biosensing

Joon Soo Park ¹, Patarajarin Akarapipad ¹, Fan-En Chen¹, Fangchi Shao ¹, Heba Mostafa², Kuangwen Hsieh ³, Tza-Huei Wang ^1,3,4

¹Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United States.

²Department of Pathology, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21287, United States.

³Department of Mechanical Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United States.

⁴Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, Maryland 21218, United States.

Corresponding Authors : Kuangwen Hsieh, Tza-Huei Wang

Abstract

CRISPR/Cas systems have been widely employed for nucleic acid biosensing and have been further advanced for mutation detection by virtue of the sequence specificity of crRNA. However, existing CRISPR-based genotyping methods are limited by the mismatch tolerance of Cas effectors, necessitating a comprehensive screening of crRNAs to effectively distinguish between wild-type and point-mutated sequences. To circumvent the limitation of conventional CRISPR-based genotyping, here, we introduce Single-Molecule kinetic Analysis via a Real-Time digital CRISPR/Cas12a-assisted assay (SMART-dCRISPR). SMART-dCRISPR leverages the differential kinetics of the signal increase in CRISPR/Cas systems, which is modulated by the complementarity between crRNA and the target sequence. It employs single-molecule digital measurements to discern mutations based on kinetic profiles that could otherwise be obscured by variations in the target concentrations. We applied SMART-dCRISPR to genotype notable mutations in SARS-CoV-2, point mutation (K417N) and deletion (69/70DEL), successfully distinguishing wild-type, Omicron BA.1, and Omicron BA.2 SARS-CoV-2 strains from clinical nasopharyngeal/nasal swab samples. Additionally, we introduced a portable digital real-time sensing device to streamline SMART-dCRISPR and enhance its practicality for point-of-care settings. The combination of a rapid and sensitive isothermal CRISPR-based assay with single-molecule kinetic analysis in a portable format significantly enhances the versatility of CRISPR-based nucleic acid biosensing and genotyping.

논문정보

형식Research article
게재일2024년 06월 (BRIC 등록일 2024-06-30)
연구진국외 연구진
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