Fig. 1: Workflow of the CoHIT system.

a By the introduction of amino acid substitutions, the engineered AsCas12a protein was endowed with stronger mismatch tolerance than the WT AsCas12a protein and was able to recognize dsDNA with a few mismatched bases relative to the crRNA. b Genomic DNA of a patient is extracted from the cell sample and detected using the one-pot ERA-Cas12a reaction. In addition to the WT sequence, the target DNA site can contain indels with several possible variants (e.g. Variants 1 ~ 3 with 4-bp insertions). The reaction mixture contains ERA reagents, a pair of primers, FAM-ssDNA-BHQ1 reporters, the engineered AsCas12a protein enAsU-R, and a single crRNA. During the isothermal amplification reaction at 39 °C, both the WT and Variants 1 ~ 3 are rapidly amplified. At the same time, the Cas12a/crRNA complex captures the Variant 1 amplicons through sequence complementation, as well as the Variant 2 and 3 amplicons due to the mismatch tolerance of the engineered AsCas12a protein, and then, green fluorescence signals are emitted. However, WT amplicons are ignored because of the quite difference from the crRNA recognition sequence, and no cross signal is observed. The detection can be completed within 30 min using only a portable isothermal fluorescence detector. Source data are provided as a Source Data file.