将等位基因特异性 PCR 与 CRISPR-Cas13a 相结合，用于胰腺癌中灵敏的 KRAS 突变检测。
Integrating allele-specific PCR with CRISPR-Cas13a for sensitive KRAS mutation detection in pancreatic cancer.
发表日期：2024 Oct 01
作者：
Samuel Amintas, Grégoire Cullot, Mehdi Boubaddi, Julie Rébillard, Laura Karembe, Béatrice Turcq, Valérie Prouzet-Mauléon, Aurélie Bedel, François Moreau-Gaudry, David Cappellen, Sandrine Dabernat
来源：
Journal of Biological Engineering
摘要：
簇状调控间隔短回文重复序列 (CRISPR)-Cas13a 系统具有高灵敏度检测外源序列的强大潜力。低等位基因频率的 KRASG12 点突变的检测对于胰腺导管腺癌 (PDAC) 的正式诊断可能是有效的。我们对 KRAS 等位基因（野生型和突变型）进行预扩增，以使用 CRISPR-Cas13a 揭示突变型 KRAS 的存在。对于通用 KRAS 预扩增模板，KRASG12D 与 KRASWT 的区分效果很差，并且取决于 crRNA 设计、目标模板的二级结构以及导向器和模板之间不匹配的性质。为了提高特异性，我们使用了称为 CASPER（Cas13a 等位基因特异性 PCR 酶识别）的等位基因特异性 PCR 预扩增方法。 CASPER 能够以低 DNA 输入量对 KRASG12D 进行特异性、灵敏的检测。 CASPER 检测到从患者超声引导下的胰腺细针抽吸液中提取的 DNA 中存在 KRAS 突变。CASPER 易于实施，是一种通用且可靠的方法，几乎适用于任何点突变。© 2024。作者。
The clustered regulatory interspaced short palindromic repeats (CRISPR)-Cas13a system has strong potential for highly sensitive detection of exogenous sequences. The detection of KRASG12 point mutations with low allele frequencies may prove powerful for the formal diagnosis of pancreatic ductal adenocarcinoma (PDAC).We implemented preamplification of KRAS alleles (wild-type and mutant) to reveal the presence of mutant KRAS with CRISPR-Cas13a. The discrimination of KRASG12D from KRASWT was poor for the generic KRAS preamplification templates and depended on the crRNA design, the secondary structure of the target templates, and the nature of the mismatches between the guide and the templates. To improve the specificity, we used an allele-specific PCR preamplification method called CASPER (Cas13a Allele-Specific PCR Enzyme Recognition). CASPER enabled specific and sensitive detection of KRASG12D with low DNA input. CASPER detected KRAS mutations in DNA extracted from patients' pancreatic ultrasound-guided fine-needle aspiration fluid.CASPER is easy to implement and is a versatile and reliable method that is virtually adaptable to any point mutation.© 2024. The Author(s).