Background: Ovarian cancer (OC), as a malignant tumor, currently lacks effective screening early diagnosis measures. Clinical biomarkers CA-125 and HE4 are limited by false positives and insufficient sensitivity. Therefore, it's of great significance to search for new biomarker and construct sensitive detection method.

Methods: We found a novel circRNA biomarker (hsa_circ_0049101) by RNA sequencing, and simultaneously propose a strategy, which integrates reverse transcription rolling circle amplification (RT-RCA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a to amplify and detect novel circRNA biomarker. This strategy use Dual Cas12a protein (FnCas12a and LbCas12a) and Multiplex CrRNA (DCMC-CRISPR) to enhance detection sensitivity. The sensitivity mechanism of CRISPR to detect circRNA was verified in detail.

Results: The DCMC-CRISPR assay exhibited a broad detection range of 2000 pM to 0.5 fM and the limit of detection (LOD) as low as 0.5 fM. The DCMC-CRISPR system has 4-11 times higher sensitivity than single-crRNA CRISPR/Cas12a system. Clinical assessment of RNA extracts from patient's peripheral blood of 22 clinical OC patients and 28 controls demonstrates the DCMC-CRISPR strategy outperformed CA-125, HE4, and the ROMA index. The assay demonstrated comparable performance to RT-qPCR, exhibiting favorable sensitivity and specificity in this pilot cohort.

Conclusions: The DCMC-CRISPR platform offers a promising solution for circRNA biomarker screening and circRNA diagnostic. It highlights the possibility of expanding its applicability to address other cancer diseases.

Keywords: CRISPR/Cas12a; CircRNA biomarker; Dual Cas12a Multiplex crRNA; Early diagnostic; Ovarian cancer.

© 2025. The Author(s).

背景： 卵巢癌（OC）作为一种恶性肿瘤，目前缺乏有效的早期筛查和诊断措施。临床生物标志物CA-125和HE4由于假阳性和灵敏度不足而受到限制。因此，寻找新的生物标志物并构建敏感的检测方法具有重要意义。

方法： 我们通过RNA测序发现了一种新型环状RNA生物标志物（hsa_circ_0049101），同时提出一种结合逆转录滚环扩增（RT-RCA）和CRISPR-Cas12a来放大和检测新环状RNA生物标志物的策略。该策略使用双重Cas12a蛋白（FnCas12a和LbCas12a）以及多路crRNA（DCMC-CRISPR）来增强检测灵敏度。详细验证了CRISPR检测circRNA的灵敏性机制。

结果： DCMC-CRISPR检测试剂盒表现出从2000 pM到0.5 fM的广泛检测范围，其最低检测限（LOD）低至0.5 fM。DCMC-CRISPR系统比单一crRNA CRISPR/Cas12a系统的灵敏度高出4-11倍。对22例临床卵巢癌患者和28名对照组患者的血液RNA提取物进行的临床评估表明，该策略优于CA-125、HE4以及ROMA指数。在这一试点队列中，检测方法表现出与RT-qPCR相当的性能，并具有良好的敏感性和特异性。

结论： DCMC-CRISPR平台为环状RNA生物标志物筛查和诊断提供了有前途的解决方案，强调了将其适用性扩展到其他癌症疾病的可能性。

关键词： CRISPR/Cas12a；环状RNA生物标志物；双重Cas12a多路crRNA；早期诊断；卵巢癌。