Key Laboratory of Clinical Laboratory Diagnostics (Chinese Ministry of Education), College of Laboratory Medicine, Chongqing Medical Laboratory Microfluidics and SPRi Engineering Research Center, Chongqing Medical University, Chongqing, 400016, PR China.
The Center for Clinical Molecular Medical Detection, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, PR China.
Talanta. 2023 Aug 15;261:124674. doi: 10.1016/j.talanta.2023.124674. Epub 2023 May 16.
The precise identification of rare single nucleotide variations (SNVs) concomitant with excess wild-type DNA is a valuable method for minimally invasive disease diagnosis and early prediction of drug responsiveness. Selective enrichment of mutant variants via strand displacement reaction offers an ideal approach of SNVs analysis but fails to differentiate wildtype from mutants with variant allele fraction (VAF) < 0.01%. Here, we demonstrate that integration of PAM-less CRISPR-Cas12a and adjacent mutation-enhanced inhibition of wild-type alleles enables highly sensitive measurement of SNVs well below the 0.01% VAF threshold. Raising the reaction temperature to the upper limit of LbaCas12a helps to boost PAM-less activation of collateral DNase activity, which can be further enhanced using PCR additives, leading to ideal discriminative performance for single point mutations. Along with selective inhibitors bearing additional adjacent mutation, it allowed detection of model EGFR L858R mutants down to 0.001% with high sensitivity and specificity. Preliminary investigation on adulterated genomic samples prepared in two different ways also suggests that it can accurately measure ultralow-abundance SNVs extracted directly from clinical samples. We believe that our design, which combines the superior SNV enrichment capability of strand displacement reaction and unparalleled programmability of CRISPR-Cas12a, has the potential to significantly advance current SNV profiling technologies.
精确鉴定伴随过量野生型 DNA 的罕见单核苷酸变异 (SNV) 是一种用于微创疾病诊断和早期预测药物反应性的有价值方法。通过链置换反应选择性富集突变变体提供了 SNV 分析的理想方法，但无法区分具有变异等位基因分数 (VAF) < 0.01%的野生型和突变型。在这里，我们证明了无 PAM 的 CRISPR-Cas12a 的整合和相邻突变增强的野生型等位基因抑制使我们能够在低于 0.01% VAF 阈值的情况下进行高度敏感的 SNV 测量。将反应温度提高到 LbaCas12a 的上限有助于提高无 PAM 的辅激活 collateralDNase 活性，使用 PCR 添加剂可以进一步增强该活性，从而为单点突变提供理想的区分性能。与具有额外相邻突变的选择性抑制剂结合使用，可以以高灵敏度和特异性检测到模型 EGFR L858R 突变体，其浓度低至 0.001%。对两种不同方式制备的掺假基因组样本的初步研究也表明，它可以准确测量直接从临床样本中提取的超低丰度 SNV。我们相信，我们的设计结合了链置换反应优越的 SNV 富集能力和 CRISPR-Cas12a 无与伦比的可编程性，有可能显著推进当前的 SNV 分析技术。
