CRISPR-Cas12a 利用 dsDNA 报告分子识别 DNA 靶配方。

Recognition of DNA Target Formulations by CRISPR-Cas12a Using a dsDNA Reporter.

机构信息

Department of Chemistry, University at Albany, State University of New York, 1400 Washington Avenue, Albany, New York 12222, United States.

The RNA Institute, University at Albany, State University of New York, 1400 Washington Avenue, Albany, New York 12222, United States.

出版信息

ACS Synth Biol. 2021 Jul 16;10(7):1785-1791. doi: 10.1021/acssynbio.1c00204. Epub 2021 Jun 18.

Abstract

CRISPR-Cas12a is a powerful platform for DNA-based diagnostics. The detection scheme relies on unselective shredding of a fluorescent ssDNA reporter upon target DNA recognition. To extend the reporter library beyond ssDNAs, we discovered a fluorescent reporter type using a dsDNA template. In this design, the fluorescence of the dsDNA reporter is quenched contact-quenching mechanism. Upon detection, the quenched fluorescence recovers with the activation Cas12a complex. Here, we compared the probing performance of two dsDNA reporters with two ssDNA reporters. The rate of the Cas12a trans-cleavage reaction was studied using one of the dsDNA reporters under different settings. The detection of different sizes of dsDNA or ssDNA targets was studied systematically under three different temperatures. Lower thresholds for ssDNA and dsDNA target size were identified. The mismatch tolerance and target specificity were examined for both ssDNA and dsDNA targets, separately. The probing performance of the dsDNA reporter was evaluated in a random DNA pool with and without target strands. We report that dsDNA can serve as a tunable fluorescence reporter template expanding the toolbox for Cas12a-based diagnostics.

摘要

CRISPR-Cas12a 是一个强大的基于 DNA 的诊断平台。该检测方案依赖于在靶 DNA 识别后对荧光 ssDNA 报告分子的非选择性粉碎。为了将报告分子库扩展到 ssDNA 以外,我们发现了一种使用 dsDNA 模板的荧光报告分子类型。在这种设计中,dsDNA 报告分子的荧光被猝灭,猝灭机制为接触猝灭机制。在检测时,猝灭的荧光随着 Cas12a 复合物的激活而恢复。在这里,我们比较了两种 dsDNA 报告分子与两种 ssDNA 报告分子的探测性能。使用其中一种 dsDNA 报告分子在不同条件下研究了 Cas12a 反式切割反应的速率。在三种不同温度下系统地研究了不同大小的 dsDNA 或 ssDNA 靶标的检测。确定了 ssDNA 和 dsDNA 靶标大小的更低阈值。分别检查了 ssDNA 和 dsDNA 靶标对错配容忍度和靶标特异性。在有和没有靶链的随机 DNA 池中评估了 dsDNA 报告分子的探测性能。我们报告 dsDNA 可以作为一种可调谐的荧光报告分子模板,扩展了基于 Cas12a 的诊断工具包。

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