International Joint Laboratory of Catalytic Chemistry, State Key Laboratory of Advanced Special Steel, Innovation Institute of Carbon Neutrality, College of Sciences, Shanghai University, Shanghai, China.
MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, Guangdong Provincial Key Laboratory of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China.
Nat Commun. 2024 Apr 4;15(1):2901. doi: 10.1038/s41467-024-47281-w.
Simultaneous multi-target detection and multi-site gene editing are two key factors restricting the development of disease diagnostic and treatment technologies. Despite numerous explorations on the source, classification, functional features, crystal structure, applications and engineering of CRISPR-Cas13a, all reports use the contiguous target RNA activation paradigm that only enables single-target detection in vitro and one-site gene editing in vivo. Here we propose a noncontiguous target RNA activation paradigm of Cas13a and establish a CRISPR-Cas13a Gemini System composed of two Cas13a:crRNA binary complexes, which can provide rapid, simultaneous, highly specific and sensitive detection of two RNAs in a single readout, as well as parallel dual transgene knockdown. CRISPR-Cas13a Gemini System are demonstrated in the detection of two miRNAs (miR-155 and miR-375) for breast cancer diagnosis and two small RNAs (EBER-1 and EBER-2) for Epstein-Barr virus diagnosis using multiple diagnostic platforms, including fluorescence and colorimetric-based lateral flow systems. We also show that CRISPR-Cas13a Gemini System can knockdown two foreign genes (EGFP and mCherry transcripts) in mammalian cells simultaneously. These findings suggest the potential of highly effective and simultaneous detection of multiple biomarkers and gene editing of multiple sites.
同时进行多靶点检测和多部位基因编辑是限制疾病诊断和治疗技术发展的两个关键因素。尽管人们对 CRISPR-Cas13a 的来源、分类、功能特征、晶体结构、应用和工程进行了大量探索，但所有报告都使用了连续靶 RNA 激活范例，该范例仅允许在体外进行单靶检测和体内进行单部位基因编辑。在这里，我们提出了 Cas13a 的非连续靶 RNA 激活范例，并建立了由两个 Cas13a:crRNA 二元复合物组成的 CRISPR-Cas13a 双子系统，该系统可以在单个读取中快速、同时、高度特异性和敏感地检测两种 RNA，以及平行双重转基因敲低。CRISPR-Cas13a 双子系统在使用多种诊断平台（包括荧光和比色横向流动系统）检测乳腺癌诊断用的两种 miRNA（miR-155 和 miR-375）和 Epstein-Barr 病毒诊断用的两种小 RNA（EBER-1 和 EBER-2）方面得到了验证。我们还表明，CRISPR-Cas13a 双子系统可以同时敲低哺乳动物细胞中的两个外源基因（EGFP 和 mCherry 转录本）。这些发现表明了同时高效检测多种生物标志物和多部位基因编辑的潜力。
