CRISPR-Cas systems recognize foreign genetic material using CRISPR RNAs (crRNAs). In type II systems, a trans-activating crRNA (tracrRNA) hybridizes to crRNAs to drive their processing and utilization by Cas9. While analyzing Cas9-RNA complexes from Campylobacter jejuni, we discovered tracrRNA hybridizing to cellular RNAs, leading to formation of “noncanonical” crRNAs capable of guiding DNA targeting by Cas9. Our discovery inspired the engineering of reprogrammed tracrRNAs that link the presence of any RNA of interest to DNA targeting with different Cas9 orthologs. This capability became the basis for a multiplexable diagnostic platform termed LEOPARD (leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection). LEOPARD allowed simultaneous detection of RNAs from different viruses in one test and distinguished severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its D614G (Asp⁶¹⁴→Gly) variant with single-base resolution in patient samples.

CRISPR-Cas 系统使用 CRISPR RNA (crRNA) 识别外来遗传物质。在 II 型系统中，反式激活 crRNA (tracrRNA) 与 crRNA 杂交以驱动其被 Cas9 加工和利用。分析空肠弯曲杆菌的Cas9-RNA 复合物时，我们发现 tracrRNA 与细胞 RNA 杂交，导致形成能够引导 Cas9 靶向 DNA 的“非经典”crRNA。我们的发现激发了重新编程 tracrRNA 的工程设计，该工程将任何感兴趣的 RNA 的存在与不同 Cas9 直向同源物的 DNA 靶向联系起来。这种能力成为称为 LEOPARD（利用工程化 tracrRNA 和目标 DNA 进行平行 RNA 检测）的多路复用诊断平台的基础。LEOPARD 允许在一次测试中同时检测来自不同病毒的 RNA，并在患者样本中以单碱基分辨率区分严重急性呼吸系统综合症冠状病毒 2 (SARS-CoV-2) 及其 D614G (Asp ⁶¹⁴ →Gly) 变体。