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

R环触发的I型CRISPR-Cas系统内的旁切单链DNA（single-stranded DNA, ssDNA）核酸酶活性，在核酸检测领域拥有巨大应用潜力。然而，Cas3的过度活跃单链DNA核酸酶（ssDNase）活性会引入不必要的背景噪音与假阳性结果。本研究中，我们从激烈热球菌（Thermococcus siculi）中鉴定出一种新型I-A型Cas3变体，该变体处于自抑制状态，直至被Cascade复合物与R环形成触发。此I-A型CRISPR-Cas3系统不仅展现出扩展的原间隔序列相邻基序（protospacer adjacent motif, PAM）识别能力，还对错配序列表现出显著的不耐受性。此外，它具备双重激活模式——可同时响应DNA与RNA靶标。本研究最终开发出高活性验证体系（Hyper-Active-Verification Establishment, 简称HAVE，即“惠父”），该创新技术可实现临床样本中人乳头瘤病毒（human papillomavirus, HPV）的快速精准诊断，为稳健的分子[……]。解析I-A型系统的PAM偏好性是一项复杂的工作。实验伊始，我们通过精心设计的聚合酶链式反应（Polymerase Chain Reaction, PCR），以PF-161和PR-161为引物，从PAM文库中精准分离得到161 bp的双链DNA。随后，我们采用PF-6-FAM与PR-161作为引物开展另一轮PCR扩增，得到5'端带有6-FAM荧光标记的PCR产物。将终浓度为320 nM的161 bp 6-FAM标记PAM文库DNA分子，与不同浓度（0至2000 nM）的TsiCascade-Cas3复合物进行严格的孵育反应。该精细实验在包含20 mM HEPES（pH 7.5）与100 mM NaCl的缓冲液中进行，反应体系维持37℃孵育1小时。反应后的样品经2%琼脂糖凝胶电泳分离，通过凝胶成像仪（Gel Imager）进行可视化成像。选取低浓度Cascade复合物结合的特异性DNA条带进行后续分析。这些DNA[……]

# 补充材料：重定向I-A型CRISPR-Cas3用于人乳头瘤病毒（HPV）的精准诊断
[[https://datadryad.org/stash/share/fhu-Z4VFqfaAoWTv8emCNsqZH0F9NOCl85XgNhV3ALk](https://datadryad.org/stash/share/fhu-Z4VFqfaAoWTv8emCNsqZH0F9NOCl85XgNhV3ALk)]

## 数据与文件结构说明
• 补充材料
* 桑格测序（Sanger sequencing）结果
• 补充数据
0001_32023050402540_(L4)_[T7].ab1 为测序原始数据；
L4-cexu.dna 为0001_32023050402540_(L4)_[T7].ab1的信号读取结果。
0001_32023050402541_(L5)_[T7].ab1 为测序原始数据；
L4-cexu.dna 为0001_32023050402541_(L5)_[T7].ab1的信号读取结果。
0001_32023050402542_(L6)_[T7].ab1 为测序原始数据；
L4-cexu.dna 为0001_32023050402542_(L6)_[T7].ab1的信号读取结果。
0001_32023050402543_(L7)_[T7].ab1 为测序原始数据；
L4-cexu.dna 为0001_32023050402543_(L7)_[T7].ab1的信号读取结果。