دورية أكاديمية

Design and Evaluation of a Robust CRISPR Kinetic Assay for Hot-Spot Genotyping.

التفاصيل البيبلوغرافية
العنوان: Design and Evaluation of a Robust CRISPR Kinetic Assay for Hot-Spot Genotyping.
المؤلفون: Blanluet C; CentraleSupelec─Universite Paris-Saclay, 91190 Gif-sur-Yvette, France.; Department of Mechanical Engineering, Stanford University, Stanford, California 94305, United States., Kuo CJ; Department of Medicine, Division of Hematology, Stanford University School of Medicine, Stanford, California 94305, United States., Bhattacharya A; Department of Medicine, Division of Hematology, Stanford University School of Medicine, Stanford, California 94305, United States., Santiago JG; Department of Mechanical Engineering, Stanford University, Stanford, California 94305, United States.
المصدر: Analytical chemistry [Anal Chem] 2024 May 14; Vol. 96 (19), pp. 7444-7451. Date of Electronic Publication: 2024 Apr 29.
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: American Chemical Society Country of Publication: United States NLM ID: 0370536 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1520-6882 (Electronic) Linking ISSN: 00032700 NLM ISO Abbreviation: Anal Chem Subsets: MEDLINE
أسماء مطبوعة: Original Publication: Washington, American Chemical Society.
مواضيع طبية MeSH: Genotyping Techniques*/instrumentation , Genotyping Techniques*/methods , Genotyping Techniques*/standards , Genes, erbB-1*/genetics , Gene Deletion*, Genes, Reporter/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Lung Neoplasms/diagnosis ; Lung Neoplasms/genetics ; Reproducibility of Results
مستخلص: Next-generation sequencing offers highly multiplexed and accurate detection of nucleic acid sequences but at the expense of complex workflows and high input requirements. The ease of use of CRISPR-Cas12 assays is attractive and may enable highly accurate detection of sequences implicated in, for example, cancer pathogenic variants. CRISPR assays often employ end-point measurements of Cas12 trans-cleavage activity after Cas12 activation by the target; however, end point-based methods can be limited in accuracy and robustness by arbitrary experimental choices. To overcome such limitations, we develop and demonstrate here an accurate assay targeting a mutation of the epidermal growth factor gene implicated in lung cancer (exon 19 deletion). The assay is based on characterizing the kinetics of Cas12 trans-cleavage to discriminate the mutant from wild-type targets. We performed extensive experiments (780 reactions) to calibrate key assay design parameters, including the guide RNA sequence, reporter sequence, reporter concentration, enzyme concentration, and DNA target type. Interestingly, we observed a competitive reaction between the target and reporter molecules that has important consequences for the design of CRISPR assays, which use preamplification to improve sensitivity. Finally, we demonstrate the assay on 18 tumor-extracted amplicons and 100 training iterations with 99% accuracy and discuss discrimination parameters and models to improve wild type versus mutant classification.
تواريخ الأحداث: Date Created: 20240429 Date Completed: 20240514 Latest Revision: 20240801
رمز التحديث: 20240802
DOI: 10.1021/acs.analchem.3c05657
PMID: 38684052
قاعدة البيانات: MEDLINE
الوصف
تدمد:1520-6882
DOI:10.1021/acs.analchem.3c05657