Repurposing Type I-A CRISPR-Cas3 for a robust diagnosis of human papillomavirus (HPV)

R-loop-triggered collateral single-stranded DNA (ssDNA) nuclease activity within Class 1 Type I CRISPR-Cas systems holds immense potential for nucleic acid detection. However, the hyperactive ssDNase activity of Cas3 introduces unwanted noise and false-positive results. In this study, we identified a novel Type I-A Cas3 variant derived from Thermococcus siculi, which remains in an auto-inhibited state until it is triggered by Cascade complex and R-loop formation. This Type I-A CRISPR-Cas3 system not only exhibits an expanded protospacer adjacent motif (PAM) recognition capability but also demonstrates remarkable intolerance towards mismatched sequences. Furthermore, it exhibits dual activation modes—responding to both DNA and RNA targets. The culmination of our research efforts has led to the development of the Hyper-Active-Verification Establishment (HAVE, 惠父). This innovation enables swift and precise human papillomavirus (HPV) diagnosis in clinical samples, providing a robust molecul..., The elucidation of PAM preferences within the Type I-A system was an intricate process. To commence, 161 bp double-stranded DNAs were diligently isolated from the PAM library through a judiciously planned PCR reaction, employing PF-161 and PR-161 as primers. Another noteworthy PCR amplification ensued, this time featuring PF-6-FAM and PR-161 primers, thus engendering 5'-6-FAM labeled PCR products. The ensuing 161 bp 6-FAM-PAM library DNA molecules, valuing at 320 nM, were subjected to a rigorous incubation with varying concentrations of the TsiCascade-Cas3 complex (ranging from 0 to 2000 nM). This meticulous procedure unfolded in a buffer comprising 20 mM HEPES (pH 7.5) and 100 mM NaCl, all maintained at a temperature of 37°C for an hour. The samples that emerged from this process underwent electrophoresis on a 2% agarose gel, their visualization achieved through a Gel Imager. Specific bands, indicative of DNA binding with low Cascade complex concentrations, were singled out. These DNA ..., , # Supplementary Material for: Repurposing Type I-A CRISPR-Cas3 for a Robust Diagnosis of human papillomavirus (HPV)

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## Description of the data and file structure

● Supplementary Material

* Sanger sequencing results

● Supplementary Data

0001_32023050402540_(L4)_[T7].ab1 is the raw data from sequencing;

L4-cexu.dna is the signal read out result for 0001_32023050402540_(L4)_[T7].ab1

0001_32023050402541_(L5)_[T7].ab1 is the raw data from sequencing;

L4-cexu.dna is the signal read out result for 0001_32023050402541_(L5)_[T7].ab1

0001_32023050402542_(L6)_[T7].ab1 is the raw data from sequencing;

L4-cexu.dna is the signal read out result for 0001_32023050402542_(L6)_[T7].ab1

0001_32023050402543_(L7)_[T7].ab1 is the raw data from sequencing;

L4-cexu.dna is the signal read out result for 0001_32023050402543_(L7)_[T7].ab1