DOI: 10.5061/dryad.98sf7m0s8
title: Repurposing Type I-A CRISPR-Cas3 for a robust diagnosis of human papillomavirus (HPV)

description: 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 molecular diagnostic
 tool based on the Type I-A CRISPR-Cas3 system. Our findings contribute to
 understanding type I-A CRISPR-Cas3 system regulation and facilitate the
 creation of advanced diagnostic solutions with broad clinical
 applicability.
description: 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
 <em>Tsi</em>Cascade-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 sequences were meticulously
 extracted and subjected to PCR analysis, employing PF-161 and PR-161 as
 primers, for further scrutiny. To delve deeper into PAM
 preferences, the preceding assay was complemented by a nuanced examination
 of positions 2 and 3. This examination involved the synthesis of 16 pairs
 of single-stranded DNAs, each spanning 59 nt and featuring adenine at
 position 1 in the PAM sequence. These oligonucleotides were thoughtfully
 annealed, and their subsequent processing involved two rounds of PCR
 reactions, masterfully conducted with PF-97/PR-97 and PF-97/PR-CY5
 primers. The outcome was the production of 3'-CY5 labeled 97 bp
 ANN-PAM DNAs, each with a distinct PAM sequence. These PCR products, now
 bearing various PAM sequences, underwent an intricate incubation process
 with an escalating concentration of the Cascade complex, meticulously
 ranging from 0 to 2000 nM. The entire process unfolded at a controlled
 temperature of 25°C for a duration of one hour. The DNA-Cascade complex
 samples, resulting from this process, were directly loaded onto a native
 polyacrylamide gel. They subsequently underwent electrophoresis in 1× TBE
 at 4°C, with the efficiency of DNA-Cascade complex binding thoughtfully
 evaluated by visualizing the bands using the LI-COR Odyssey
 system.
description: # Supplementary Material for: Repurposing Type I-A CRISPR-Cas3 for a
 Robust Diagnosis of human papillomavirus (HPV)
 \[[https://datadryad.org/stash/share/fhu-Z4VFqfaAoWTv8emCNsqZH0F9NOCl85XgNhV3ALk](https://datadryad.org/stash/share/fhu-Z4VFqfaAoWTv8emCNsqZH0F9NOCl85XgNhV3ALk)] ## 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