CoHIT: a one-pot ultrasensitive ERA-CRISPR system for detecting multiple same-site indels

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Precision cancer medicine relies heavily on genetic testing.
• Detecting multiple same-site insertions or deletions (indels) presents a significant challenge in molecular diagnostics.

Purpose of the Study:

• To develop a rapid, sensitive, and specific CRISPR-based assay for detecting indels.
• To evaluate the CoHIT (Cas12a-based One-for-all High-speed Isothermal Test) assay for NPM1 gene mutations in acute myeloid leukemia (AML).

Main Methods:

• CoHIT utilizes an engineered AsCas12a variant with high mismatch tolerance and broad PAM scope.
• A single crRNA is employed for detecting multiple NPM1 gene c.863_864 4-bp insertions in a one-pot reaction.
• Optimization of reaction parameters was performed to enhance assay performance.

Main Results:

• CoHIT achieved a detection limit of 0.01% with results available within 30 minutes.
• The assay demonstrated no cross-reactivity with wild-type sequences.
• NPM1 mutations were identified in 30 out of 108 AML patients, with potential for minimal residual disease (MRD) monitoring.

Conclusions:

• CoHIT is a highly efficient and rapid CRISPR-based diagnostic tool for indel detection.
• The assay shows promise for clinical application in AML diagnostics and MRD monitoring.
• CoHIT's adaptability extends to detecting indels in other cancer-related genes (KIT, BRAF, EGFR) and integration with lateral flow strips and microfluidic chips.