Chen Junman, Qiud Tian, Mauk Michael G, Su Zheng, Fan Yaguang, Yuan Dennis J, Zhou Qinghua, Qiao Youlin, Bau Haim H, Ying Jianming, Song Jinzhao
Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, Hangzhou 310022, China.
Chin Chem Lett. 2022 Aug;33(8):4126-4132. doi: 10.1016/j.cclet.2021.11.065. Epub 2021 Nov 26.
Liquid biopsy is a highly promising method for non-invasive detection of tumor-associated nucleic acid fragments in body fluids but is challenged by the low abundance of nucleic acids of clinical interest and their sequence homology with the vast background of nucleic acids from healthy cells. Recently, programmable endonucleases such as clustered regularly interspaced short palindromic repeat (CRISPR) associated protein (Cas) and prokaryotic Argonautes have been successfully used to remove background nucleic acids and enrich mutant allele fractions, enabling their detection with deep next generation sequencing (NGS). However, the enrichment level achievable with these assays is limited by futile binding events and off-target cleavage. To overcome these shortcomings, we conceived a new assay (Programmable Enzyme-Assisted Selective Exponential Amplification, PASEA) that combines the cleavage of wild type alleles with concurrent polymerase amplification. While PASEA increases the numbers of both wild type and mutant alleles, the numbers of mutant alleles increase at much greater rates, allowing PASEA to achieve an unprecedented level of selective enrichment of targeted alleles. By combining CRISPR-Cas9 based cleavage with recombinase polymerase amplification, we converted samples with 0.01% somatic mutant allele fractions (MAFs) to products with 70% MAFs in a single step within 20 min, enabling inexpensive, rapid genotyping with such as Sanger sequencers. Furthermore, PASEA's extraordinary efficiency facilitates sensitive real-time detection of somatic mutant alleles at the point of care with custom designed Exo-RPA probes. Real-time PASEA' performance was proved equivalent to clinical amplification refractory mutation system (ARMS)-PCR and NGS when testing over hundred cancer patients' samples. This strategy has the potential to reduce the cost and time of cancer screening and genotyping, and to enable targeted therapies in resource-limited settings.
液体活检是一种极具前景的非侵入性检测体液中肿瘤相关核酸片段的方法，但面临着临床相关核酸丰度低以及它们与来自健康细胞的大量核酸背景存在序列同源性的挑战。最近，诸如成簇规律间隔短回文重复序列（CRISPR）相关蛋白（Cas）和原核生物Argonaute等可编程核酸内切酶已成功用于去除背景核酸并富集突变等位基因片段，从而能够通过深度下一代测序（NGS）进行检测。然而，这些检测方法可实现的富集水平受到无效结合事件和脱靶切割的限制。为克服这些缺点，我们构思了一种新的检测方法（可编程酶辅助选择性指数扩增，PASEA），该方法将野生型等位基因的切割与同时进行的聚合酶扩增相结合。虽然PASEA增加了野生型和突变等位基因的数量，但突变等位基因数量的增加速率要大得多，使得PASEA能够实现前所未有的靶向等位基因选择性富集水平。通过将基于CRISPR-Cas9的切割与重组酶聚合酶扩增相结合，我们在20分钟内一步将体细胞突变等位基因频率（MAF）为0.01%的样本转化为MAF为70%的产物，从而能够使用如桑格测序仪进行廉价、快速的基因分型。此外，PASEA的非凡效率有助于使用定制设计的Exo-RPA探针在护理点灵敏地实时检测体细胞突变等位基因。在检测超过一百名癌症患者的样本时，实时PASEA的性能被证明与临床扩增不应性突变系统（ARMS）-PCR和NGS相当。这种策略有可能降低癌症筛查和基因分型的成本和时间，并在资源有限的环境中实现靶向治疗。
