Amintas Samuel, Cullot Grégoire, Boubaddi Mehdi, Rébillard Julie, Karembe Laura, Turcq Béatrice, Prouzet-Mauléon Valérie, Bedel Aurélie, Moreau-Gaudry François, Cappellen David, Dabernat Sandrine
Bordeaux Institute in Oncology - BRIC - BioGo team, Univ. Bordeaux, INSERM U1312, Bordeaux, France.
Department of Tumor Biology and Tumor Library, CHU Bordeaux, Bordeaux, France.
J Biol Eng. 2024 Oct 1;18(1):53. doi: 10.1186/s13036-024-00450-3.
The clustered regulatory interspaced short palindromic repeats (CRISPR)-Cas13a system has strong potential for highly sensitive detection of exogenous sequences. The detection of KRAS 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 KRAS from KRAS 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 KRAS 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.
成簇规律间隔短回文重复序列（CRISPR）-Cas13a系统在对外源序列进行高灵敏度检测方面具有强大潜力。检测低等位基因频率的KRAS点突变对于胰腺导管腺癌（PDAC）的正式诊断可能具有重要作用。
我们对KRAS等位基因（野生型和突变型）进行预扩增，以利用CRISPR-Cas13a揭示突变型KRAS的存在。对于通用的KRAS预扩增模板，KRAS与KRAS之间的区分效果较差，这取决于crRNA设计、靶模板的二级结构以及向导与模板之间错配的性质。为提高特异性，我们使用了一种称为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KRAS进行特异性和灵敏的检测。CASPER检测到了从患者胰腺超声引导下细针穿刺抽吸液中提取的DNA中的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。
