Mutations in mitochondrial DNA (mtDNA) play critical roles in many human diseases. In vivo visualization of cells bearing mtDNA mutations is important for resolving the complexity of these diseases, which remains challenging. Here we develop an integrated nano Cas12a sensor (InCasor) and show its utility for efficient imaging of mtDNA mutations in live cells and tumor-bearing mouse models. We co-deliver Cas12a/crRNA, fluorophore-quencher reporters and Mg²⁺ into mitochondria. This process enables the activation of Cas12a’s trans-cleavage by targeting mtDNA, which efficiently cleave reporters to generate fluorescent signals for robustly sensing and reporting single-nucleotide variations (SNVs) in cells. Since engineered crRNA significantly increase Cas12a’s sensitivity to mismatches in mtDNA, we can identify tumor tissue and metastases by visualizing cells with mutant mtDNAs in vivo using InCasor. This CRISPR imaging nanoprobe holds potential for applications in mtDNA mutation-related basic research, diagnostics and gene therapies.

线粒体 DNA (mtDNA) 突变在许多人类疾病中发挥着关键作用。携带 mtDNA 突变的细胞的体内可视化对于解决这些疾病的复杂性非常重要，但这仍然具有挑战性。在这里，我们开发了一种集成纳米 Cas12a 传感器 (InCasor)，并展示了其在活细胞和荷瘤小鼠模型中有效成像 mtDNA 突变的实用性。我们将 Cas12a/crRNA、荧光团猝灭剂报告基因和 Mg ²⁺共同递送至线粒体中。该过程通过靶向 mtDNA 来激活 Cas12a 的反式切割，从而有效切割报告基因以生成荧光信号，从而稳健地感知和报告细胞中的单核苷酸变异 (SNV)。由于工程化的 crRNA 显着增加了 Cas12a 对 mtDNA 错配的敏感性，因此我们可以通过使用 InCasor 在体内观察具有突变 mtDNA 的细胞来识别肿瘤组织和转移。这种 CRISPR 成像纳米探针在 mtDNA 突变相关基础研究、诊断和基因治疗方面具有应用潜力。