Composing CODAs to cervical cancer screening through an integrated CRISPR and fluorescent nucleic acid approach

摘要 挑战尽管在早期发现时可以治愈，但宫颈癌仍然是第二大导致宫颈癌的原因。 生活在撒哈拉以南非洲的妇女的死亡率。即使有可用的筛查技术（细胞学、视觉 醋酸检查（VIA），有时高风险人乳头瘤病毒[hrHPV]检测），仅 目前，低收入和中等收入国家（LMIC）10 - 20%的合格妇女接受了筛查。列举的障碍 包括技术专门知识、实验室能力、费用和使用机会。大多数发达国家都采用了 基于PCR的HPV筛查，进一步缩小了与中低收入国家的资源差距，80%的癌症死亡发生在中低收入国家。 创新为了应对大流行所揭示的诊断差距，我们小组的团队成员 在人类样本中开发并验证了一种检测核酸靶点的稳健而廉价的技术。 该方法，CRISPR光学检测各向异性或CODA，结合CRISPR/Cas（诺贝尔奖- 具有荧光各向异性（差分旋转运动）的高精度基因编辑的获奖技术 的荧光分子）。自动化核酸读数在<30分钟内生成。我们采取 该技术用于：a）CIN2+的DNA和RNA标志物的检测;和B）临床医生的可靠使用， 临床实验室人员在床旁护理设置。我们对这项提案的计划是将这项技术投入使用， 低资源环境，特别是我们在乌干达和加纳的合作伙伴站点，与LMIC密切合作。然后我们将 利用CODA和其他参数创建一组测试，以最佳方式检测单个 遭遇目的1：构建一个强大的CODA平台，用于HPV的全面筛查。目标2：进一步 检查人体标本上的CODA性能，并为LMIC操作进行优化。目标3：开发一部小说 和快速多模态算法用于LMIC的筛选和治疗。冲击鉴于CODA在核技术方面的核心优势 酸分析（例如，DNA或RNA），我们设想了一种方法，该方法产生高风险的完全定量读数。 HPV DNA和E6、E7和p16 mRNA。由于CODA分析是驱动该提议的所有读数的基础， 对于高危宫颈疾病或侵入性宫颈疾病的最终用户友好、实用和快速分诊， 癌这些好处可以帮助分散和协调筛选工作与这些准则目前 得到了资源丰富国家的认可。

ABSTRACT Challenges. Despite being curable when caught early, cervical cancer remains the second leading cause of death in women living in sub-Saharan Africa. Even with available screening technologies (cytology, visual inspection with acetic acid (VIA), and sometimes high-risk Human Papillomavirus [hrHPV] testing), only 10-20% of eligible women in low-and-middle-income countries (LMIC) are currently screened. Barriers cited include technical expertise, laboratory capacity, cost, and access. Most developed countries have adopted PCR-based HPV screening, furthering the resource divide with LMICs, where 80% of cancer deaths occur. Innovation. In dire response to the diagnostic gaps unveiled by the pandemic, team members within our group developed and validated in human specimens a robust and frugal technology to detect nucleic acid targets. The approach, CRISPR Optical Detection of Anisotropy or CODA, combines CRISPR/Cas (a Nobel Prize- winning technology for highly precise gene editing) with fluorescence anisotropy (differential rotational motion of fluorescent molecules). Automated nucleic acid readouts are generated in < 30 minutes. We have adopted this technology for: a) the detection of DNA and RNA markers of CIN2+; and b) reliable use by clinicians and clinical lab personnel in point-of-care settings. Our plan for this proposal is to operationalize this technology for low resource settings, notably our partner sites in Uganda and Ghana, with close LMIC input. We will then leverage CODA and other parameters to create a panel of tests to optimally detect CIN2+ in a single encounter. Aim 1: Construct a robust CODA platform for comprehensive HPV screening. Aim 2: Further examine CODA performance on human specimens and refine for LMIC operations. Aim 3: Develop a novel and rapid multi-modal algorithm for screen and treat in LMICs. Impact. Given CODA's core strengths in nucleic acid analyses (e.g., DNA or RNA), we envision an approach that yields fully quantitive readouts of high-risk HPV DNA and E6, E7, and p16 mRNA. Since the CODA assay underlies all readouts driving this proposal, a high potential exists for end-user-friendly, practical, and rapid triage of high-risk cervical disease or invasive cancer. These benefits could help decentralize and harmonize screening efforts with those guidelines currently endorsed by resource-rich countries.