Department of Chemical Engineering, University of Florida, Gainesville, FL, USA.
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.
Nat Commun. 2023 Sep 5;14(1):5409. doi: 10.1038/s41467-023-41006-1.
Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage in the presence of split activators. Specifically, the PAM-distal region of the crRNA recognizes RNA targets provided that the PAM-proximal seed region has a DNA target. Our method, Split Activator for Highly Accessible RNA Analysis (SAHARA), detects picomolar concentrations of RNA without sample amplification, reverse-transcription, or strand-displacement by simply supplying a short DNA sequence complementary to the seed region. Beyond RNA detection, SAHARA outperforms wild-type CRISPR-Cas12a in specificity towards point-mutations and can detect multiple RNA and DNA targets in pooled crRNA/Cas12a arrays via distinct PAM-proximal seed DNAs. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.
Cas12a，一种 CRISPR 相关蛋白复合物，具有内在的切割 DNA 底物的能力，并被用于诊断工具来识别 DNA 分子。我们证明了 Cas12a 的多个同源物在分裂激活剂的存在下激活跨切割。具体来说，crRNA 的 PAM 远端区域识别 RNA 靶标，前提是 PAM 近端种子区域具有 DNA 靶标。我们的方法，用于高度可及 RNA 分析的分裂激活剂 (SAHARA)，通过简单地提供与种子区域互补的短 DNA 序列，在没有样品扩增、逆转录或链置换的情况下，检测皮摩尔浓度的 RNA。除了 RNA 检测之外，SAHARA 在针对点突变的特异性方面优于野生型 CRISPR-Cas12a，并且可以通过不同的 PAM 近端种子 DNA 在汇集的 crRNA/Cas12a 阵列中检测多个 RNA 和 DNA 靶标。总之，SAHARA 是一种基于 Cas12a 的简单而强大的核酸检测平台，可进行多重分析，并可能扩展到其他 CRISPR-Cas 酶。
