Programmable endonuclease combined with isothermal polymerase amplification to selectively enrich for rare mutant allele fractions

作者全名："Chen, Junman; Qiu, Tian; Mauk, Michael G.; Su, Zheng; Fan, Yaguang; Yuan, Dennis J.; Zhou, Qinghua; Qiao, Youlin; Bau, Haim H.; Ying, Jianming; Song, Jinzhao"

作者地址："[Chen, Junman] Chongqing Med Univ, Coll Lab Med, Minist Educ, Key Lab Clin Lab Diagnost, Chongqing 400016, Peoples R China; [Chen, Junman; Yuan, Dennis J.; Song, Jinzhao] Chinese Acad Sci, Univ Chinese Acad Sci, Canc Hosp, Zhejiang Canc Hosp,Inst Basic Med & Canc IBMC, Hangzhou 310022, Peoples R China; [Chen, Junman; Mauk, Michael G.; Bau, Haim H.; Song, Jinzhao] Univ Penn, Dept Mech Engn & Appl Mech, Philadelphia, PA 19104 USA; [Qiu, Tian; Ying, Jianming] Chinese Acad Med Sci & Peking Union Med Coll, Canc Hosp, Natl Clin Res Ctr Canc, Dept Pathol,Natl Canc Ctr, Beijing 100021, Peoples R China; [Su, Zheng; Qiao, Youlin] Chinese Acad Med Sci & Peking Union Med Coll, Ctr Global Hlth, Sch Populat Med & Publ Hlth, Beijing 100730, Peoples R China; [Fan, Yaguang] Tianjin Med Univ Gen Hosp, Tianjin Lung Canc Inst, Tianjin Key Lab Lung Canc Metastasis & Tumor Micr, Tianjin 300052, Peoples R China; [Zhou, Qinghua] Sichuan Univ, West China Hosp, Sichuan Lung Canc Ctr, Sichuan Lung Canc Inst, Chengdu 610041, Peoples R China"

通信作者："Song, JZ (通讯作者)，Chinese Acad Sci, Univ Chinese Acad Sci, Canc Hosp, Zhejiang Canc Hosp,Inst Basic Med & Canc IBMC, Hangzhou 310022, Peoples R China.; Ying, JM (通讯作者)，Chinese Acad Med Sci & Peking Union Med Coll, Canc Hosp, Natl Clin Res Ctr Canc, Dept Pathol,Natl Canc Ctr, Beijing 100021, Peoples R China."

来源：CHINESE CHEMICAL LETTERS

ESI学科分类：CHEMISTRY

WOS号：WOS:000815135400094

JCR分区：Q1

影响因子：9.1

年份：2022

卷号：33

期号：8

开始页：4126

结束页：4132

文献类型：Article

关键词：Mutant allele enrichment; Programmable endonuclease; Liquid biopsy; Mutation detection; Point-of-care testing; CRISPR-Cas9; Recombinase polymerase amplification; Nucleic acid diagnostics

摘要："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 repeats (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. (C) 2022 Published by Elsevier By, on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences."

基金机构："China Scholarship Council; NIH [K01 1K01TW011190-01A1, R21 CA228614-01A1]; Beijing Hope Run Special Fund from the Cancer Foundation of China [LC2019L04, LC2020A36]"

基金资助正文："This work was supported by China Scholarship Council, NIH grant to the University of Pennsylvania (No. K01 1K01TW011190-01A1), NIH grant to the University of Pennsylvania (No. R21 CA228614-01A1), and Beijing Hope Run Special Fund from the Cancer Foundation of China (Nos. LC2019L04 and LC2020A36)."