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

Yin Liu, Xinyi Liu, Dongyi Wei, Lu Dang, Xiaoran Xu, Shisheng Huang, Liwen Li, Sanyun Wu, Jinxian Wu, Xiaoyan Liu, Wenjun Sun, Wanyu Tao, Yongchang Wei, Xingxu Huang, Kui Li (), Xinjie Wang () and Fuling Zhou ()
Additional contact information
Yin Liu: Wuhan University
Xinyi Liu: Chinese Academy of Agricultural Sciences
Dongyi Wei: Wuhan University
Lu Dang: Third Affiliated Hospital of Guangzhou Medical University
Xiaoran Xu: Wuhan University
Shisheng Huang: Zhejiang Lab
Liwen Li: Chinese Academy of Agricultural Sciences
Sanyun Wu: Wuhan University
Jinxian Wu: Wuhan University
Xiaoyan Liu: Wuhan University
Wenjun Sun: ShanghaiTech University
Wanyu Tao: ShanghaiTech University
Yongchang Wei: Wuhan University
Xingxu Huang: ShanghaiTech University
Kui Li: Chinese Academy of Agricultural Sciences
Xinjie Wang: Chinese Academy of Agricultural Sciences
Fuling Zhou: Wuhan University

Nature Communications, 2024, vol. 15, issue 1, 1-14

Abstract: Abstract Genetic testing is crucial for precision cancer medicine. However, detecting multiple same-site insertions or deletions (indels) is challenging. Here, we introduce CoHIT (Cas12a-based One-for-all High-speed Isothermal Test), a one-pot CRISPR-based assay for indel detection. Leveraging an engineered AsCas12a protein variant with high mismatch tolerance and broad PAM scope, CoHIT can use a single crRNA to detect multiple NPM1 gene c.863_864 4-bp insertions in acute myeloid leukemia (AML). After optimizing multiple parameters, CoHIT achieves a detection limit of 0.01% and rapid results within 30 minutes, without wild-type cross-reactivity. It successfully identifies NPM1 mutations in 30 out of 108 AML patients and demonstrates potential in monitoring minimal residual disease (MRD) through continuous sample analysis from three patients. The CoHIT method is also competent for detecting indels of KIT, BRAF, and EGFR genes. Integration with lateral flow test strips and microfluidic chips highlights CoHIT’s adaptability and multiplexing capability, promising significant advancements in clinical cancer diagnostics.

Date: 2024
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DOI: 10.1038/s41467-024-49414-7

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