Tian Lingxi, Gao Yan, Zi Lihan, Zhe Ruilian, Yang Jun
MOE Key Laboratory of Intelligent Biomanufacturing, School of Bioengineering, Dalian University of Technology, Dalian, 116024, People's Republic of China.
Liaoning Cancer Hospital & Institute, Shenyang, 110000, People's Republic of China.
BMC Cancer. 2025 Apr 15;25(1):695. doi: 10.1186/s12885-025-14116-w.
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.
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.
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.
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.
卵巢癌（OC）作为一种恶性肿瘤，目前缺乏有效的筛查和早期诊断措施。临床生物标志物CA - 125和HE4受假阳性和灵敏度不足的限制。因此，寻找新的生物标志物并构建灵敏的检测方法具有重要意义。
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），并同时提出了一种将逆转录滚环扩增（RT - RCA）和规律成簇间隔短回文重复序列（CRISPR）- Cas12a相结合的策略，用于扩增和检测新型环状RNA生物标志物。该策略使用双Cas12a蛋白（FnCas12a和LbCas12a）和多重crRNA（DCMC - CRISPR）来提高检测灵敏度。详细验证了CRISPR检测环状RNA的灵敏度机制。
DCMC - CRISPR检测方法的检测范围为2000 pM至0.5 fM，检测限低至0.5 fM。DCMC - CRISPR系统的灵敏度比单crRNA CRISPR/Cas12a系统高4 - 11倍。对22例临床OC患者和28例对照的患者外周血RNA提取物进行临床评估表明，DCMC - CRISPR策略优于CA - 125、HE4和ROMA指数。该检测方法与RT - qPCR表现出相当的性能，在该试点队列中具有良好的灵敏度和特异性。
DCMC - CRISPR平台为环状RNA生物标志物筛选和环状RNA诊断提供了一个有前景的解决方案。它突出了扩大其适用性以解决其他癌症疾病的可能性。
