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

Cite this dataset

Hu, Chunyi et al. (2024). Repurposing Type I-A CRISPR-Cas3 for a robust diagnosis of human papillomavirus (HPV) [Dataset]. Dryad. https://doi.org/10.5061/dryad.98sf7m0s8

Abstract

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.

README: Supplementary Material for: Repurposing Type I-A CRISPR-Cas3 for a Robust Diagnosis of human papillomavirus (HPV)

[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

Methods

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 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.

Funding

National University of Singapore, Award: 23-0178-A0001, CRISPR Beyond and Seeking Low-cost Precision Medicine

Ministry of Education, Award: 23-0443-A0001-1, Repurpose Type I-A CRISPR-Cas3 for a Robust HPV Diagnosis Platform