Scholarlink

<p>Traditionally perceived as an RNA-specific nuclease, Cas13a has been used primarily for RNA detection. We discover the ability of <i>Leptotrichia buccalis</i> Cas13a (LbuCas13a) to directly target DNA without the restrictions of protospacer flanking sequence and protospacer adjacent motif sequences, coupled with robust <i>trans</i>-cleavage activity. Contrary to conventional understanding, LbuCas13a does not degrade DNA targets. Our study reveals an enhancement in the single-nucleotide specificity of LbuCas13a against DNA compared to RNA. This heightened specificity is attributed to the lower affinity of CRISPR RNA (crRNA) towards DNA, raising the crRNA–DNA binding energy barrier. We introduce a molecular diagnostic platform called superior universal rapid enhanced specificity test with LbuCas13a (SUREST) for high-resolution genotyping. SUREST is capable of detecting DNA concentrations of CYP2C19 (rs4986893) as minute as 0.3 aM (0.18 cps µl<sup>−1</sup>). We also apply SUREST to human genotyping scenarios, indicating that SUREST performs well across a broad range of mutations and sequence contexts. SUREST represents an advancement in real-time nucleic acid detection, making it a useful tool for pathogen identification and mutation analysis in clinical diagnostics.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

LbuCas13a directly targets DNA and elicits strong trans-cleavage activity

  • 期刊论文

  • 发表日期：

    2025年6月20日

查看全文
  • Xiaolong Wu,
  • Siyuan Luo,
  • Chuanghao Guo,
  • Yi Zhao,
  • Jialing Zhong,
  • Ronghuan Hu,
  • Xinyao Yang,
  • Conghui Liu,
  • Qianling Zhang,
  • Songkuan Zhuang,
  • Yong Chen,
  • Yizhen Liu,
  • Xueji Zhang

摘要

Traditionally perceived as an RNA-specific nuclease, Cas13a has been used primarily for RNA detection. We discover the ability of Leptotrichia buccalis Cas13a (LbuCas13a) to directly target DNA without the restrictions of protospacer flanking sequence and protospacer adjacent motif sequences, coupled with robust trans-cleavage activity. Contrary to conventional understanding, LbuCas13a does not degrade DNA targets. Our study reveals an enhancement in the single-nucleotide specificity of LbuCas13a against DNA compared to RNA. This heightened specificity is attributed to the lower affinity of CRISPR RNA (crRNA) towards DNA, raising the crRNA–DNA binding energy barrier. We introduce a molecular diagnostic platform called superior universal rapid enhanced specificity test with LbuCas13a (SUREST) for high-resolution genotyping. SUREST is capable of detecting DNA concentrations of CYP2C19 (rs4986893) as minute as 0.3 aM (0.18 cps µl−1). We also apply SUREST to human genotyping scenarios, indicating that SUREST performs well across a broad range of mutations and sequence contexts. SUREST represents an advancement in real-time nucleic acid detection, making it a useful tool for pathogen identification and mutation analysis in clinical diagnostics.