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Background <p>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.</p> Methods <p>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.</p> Results <p>The DCMC-CRISPR assay exhibited a broad detection range of 2000&#xa0;pM to 0.5 fM and the limit of detection (LOD) as low as 0.5&#xa0;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.</p> Conclusions <p>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.</p>

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Dual Cas12a and multiplex crRNA CRISPR strategy ultrasensitive detection novel circRNA biomarker for the diagnosis of ovarian cancer

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年4月15日

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  • Lingxi Tian,
  • Yan Gao,
  • Lihan Zi,
  • Ruilian Zhe,
  • Jun Yang

摘要

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.