Low-Cost CRISPR-on-Paper for Cervical Cancer Screening at the Point of Care

摘要 宫颈癌是全球妇女癌症死亡的主要原因之一，特别是在中低位妇女. 收入国家(LMIC)。高危型人乳头瘤病毒(HPV)是宫颈病变的主要病原体 癌症及其前驱病变；因此，世界卫生组织(WHO)建议使用HPV DNA 在LMIC中进行宫颈癌筛查的检测。尽管聚合酶链式反应(PCR)方法已经被 广泛用于HPV DNA检测，由于需要，它们仅限于中央临床实验室 劳动密集型程序和昂贵的设备。在这里，我们建议开发一种简单、快速、高度 灵敏、特定的CRISPR纸上诊断平台，可同时检测多个高危HPV 用于宫颈癌筛查的基因分型。这一创新的诊断系统基于我们的 最近发展起来的AIOD-CRISPR(AIOD-CRISPR)一体式Dual CRISPR-Cas12a检测方法结合了简便性和 等温核酸扩增的高灵敏度与CRISPR检测的高特异性。发展，发展 作为一种低成本的多路分子检测技术，我们将把AIOD-CRISPR分析整合到一个 纸基微流控平台。为了消除对复杂电子仪器的需求，我们将采取 使用放热反应为CRISPR-On-Paper系统产生化学热的优势 一次性暖手器，从而实现了免仪器宫颈癌筛查。检测结果可以是 肉眼阅读或通过编程的智能手机报告，而不需要昂贵的光学 探测器。我们将严格评估和验证我们的CRISPR纸上诊断的临床应用 系统通过与康涅狄格州大学健康与健康中心的临床医生和医护人员合作测试临床样本 赞比亚。如果成功，拟议的项目将通过提供一种简单、 在资源匮乏的环境下，经济实惠、灵敏的宫颈癌快速筛查方法。作为一个平台 技术，所提出的CRISPR-On-Paper诊断系统可以很容易地被改装成检测其他新兴的 病原体。

Abstract Cervical cancer is among the leading causes of cancer death in women worldwide, especially in low- and middle- income countries (LMICs). High-risk human papillomaviruses (HPV) are the main causative agents of cervical cancer and its precursor lesions; therefore, the World Health Organization (WHO) has recommended HPV DNA testing for cervical cancer screening in LMICs. Although polymerase chain reaction (PCR) methods have been widely used for HPV DNA detection, they are restricted to centralized clinical laboratories due to the need for labor-intensive procedures and expensive equipment. Here, we propose to develop a simple, rapid, highly sensitive, and specific CRISPR-on-paper diagnostic platform to simultaneously detect multiple high-risk HPV genotypes for cervical cancer screening at the point of care. This innovative diagnostic system is based on our recently developed all-in-one dual CRISPR-Cas12a (AIOD-CRISPR) assay, which combines the simplicity and high sensitivity of isothermal nucleic acid amplification with the high specificity of CRISPR detection. To develop a low-cost, multiplexed molecular detection technology, we will incorporate the AIOD-CRISPR assay into a paper-based microfluidics platform. To eliminate the need for complex electronic instruments, we will take advantage of an exothermic reaction to generate chemical heat for the CRISPR-on-paper system by using a disposable hand warmer, thus enabling instrument-free cervical cancer screening. The detection results can be read by the naked eye or reported by a programmed smartphone without the need for an expensive optical detector. We will rigorously evaluate and validate the clinical applications of our CRISPR-on-paper diagnostic system by testing clinical samples in collaboration with clinicians and healthcare workers in UConn Health and Zambia. If successful, the proposed project has an important impact on global health by providing a simple, affordable, and sensitive method for rapid screening of cervical cancer in resource-poor settings. As a platform technology, the proposed CRISPR-on-paper diagnostic system can be easily adapted to detect other emerging pathogens.