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<p>The CRISPR/Cas13a enzyme serves as a powerful tool for RNA detection due to its RNA-targeting capabilities. However, simple and highly sensitive detection using Cas13a faces challenges, such as the need for pre-amplification and elevated reaction temperatures. In this study, we investigate the allosteric regulation mechanism of Cas13a activation by target RNAs with various structures containing the CRISPR anti-tag sequence. We discover that the target RNA secondary structure and anti-tag sequences inhibit the <i>trans</i>-cleavage reaction of Cas13a. By designing and introducing a specific CRISPR anti-tag hairpin, we develop <Emphasis Type="Underline">C</Emphasis>RISPR <Emphasis Type="Underline">A</Emphasis>nti-tag Mediated <Emphasis Type="Underline">R</Emphasis>oom-temperature <Emphasis Type="Underline">R</Emphasis>NA <Emphasis Type="Underline">D</Emphasis>etection (CARRD) using a single CRISPR/Cas13a enzyme. This method enables one-step cascade signal amplification for RNA detection without the need for pre-amplification. We apply the CARRD method to detect human immunodeficiency virus (HIV) and hepatitis C virus (HCV), achieving a detection sensitivity of 10 aM. Furthermore, we validate its clinical feasibility by detecting HIV clinical plasma samples, demonstrating a simple, affordable, and efficient approach for viral RNA detection. Due to its simplicity, sensitivity, and flexible reaction temperature, the CARRD method is expected to have broad applicability, paving the way for the development of field-deployable diagnostic tools.</p>

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CRISPR anti-tag-mediated room-temperature RNA detection using CRISPR/Cas13a

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年10月15日

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  • Jeong Moon,
  • Jiongyu Zhang,
  • Xin Guan,
  • Rui Yang,
  • Chong Guo,
  • Kurt T. Schalper,
  • Lori Avery,
  • David Banach,
  • Rocco LaSala,
  • Ranjit Warrier,
  • Changchun Liu

摘要

The CRISPR/Cas13a enzyme serves as a powerful tool for RNA detection due to its RNA-targeting capabilities. However, simple and highly sensitive detection using Cas13a faces challenges, such as the need for pre-amplification and elevated reaction temperatures. In this study, we investigate the allosteric regulation mechanism of Cas13a activation by target RNAs with various structures containing the CRISPR anti-tag sequence. We discover that the target RNA secondary structure and anti-tag sequences inhibit the trans-cleavage reaction of Cas13a. By designing and introducing a specific CRISPR anti-tag hairpin, we develop CRISPR Anti-tag Mediated Room-temperature RNA Detection (CARRD) using a single CRISPR/Cas13a enzyme. This method enables one-step cascade signal amplification for RNA detection without the need for pre-amplification. We apply the CARRD method to detect human immunodeficiency virus (HIV) and hepatitis C virus (HCV), achieving a detection sensitivity of 10 aM. Furthermore, we validate its clinical feasibility by detecting HIV clinical plasma samples, demonstrating a simple, affordable, and efficient approach for viral RNA detection. Due to its simplicity, sensitivity, and flexible reaction temperature, the CARRD method is expected to have broad applicability, paving the way for the development of field-deployable diagnostic tools.