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

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

作者机构：Key Laboratory of Clinical Laboratory Diagnostics(Ministry of Education)College of Laboratory MedicineChongqing Medical UniversityChongqing 400016China The Cancer Hospital of the University of Chinese Academy of Sciences(Zhejiang Cancer Hospital)Institute of Basic Medicine and Cancer(IBMC)Chinese Academy of SciencesHangzhou 310022China Department of Mechanical Engineering and Applied MechanicsUniversity of PennsylvaniaPhiladelphiaPA 19104United States Department of PathologyNational Cancer Center/National Clinical Research Center for Cancer/Cancer HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100021China Center for Global HealthSchool of Population Medicine and Public HealthChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100730China Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor MicroenvironmentTianjin Lung Cancer InstituteTianjin Medical University General HospitalTianjin 300052China Sichuan Lung Cancer InstituteSichuan Lung Cancer CenterWest China HospitalSichuan UniversityChengdu 610041China 

出 版 物：《Chinese Chemical Letters》 (中国化学快报（英文版）)

年 卷 期：2022年第33卷第8期

页      面：4126-4132页

核心收录：

学科分类：100208[医学-临床检验诊断学] 1002[医学-临床医学] 0703[理学-化学] 10[医学] 

基　　金：supported by China Scholarship Council NIH grant to the University of Pennsylvania(No.K011K01TW011190-01A1) NIH grant to the University of Pennsylvania(No.R21CA228614-01A1) Beijing Hope Run Special Fund from the Cancer Foundation of China(Nos.LC2019L04 and LC2020A36) 

主　　题：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 ***,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 *** 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 *** 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 *** combining CRISPR-Cas9 based cleavage with recombinase polymerase amplification,we converted samples with0.01%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 ***,PASEA s extraordinary efficiency facilitates sensitive real-time detection of somatic mutant alleles at the point of care with custom designed Exo-RPA ***-time PASEA performance was proved equivalent to clinical amplification refractory mutation system(ARMS)-PCR and NGS when testing over hundred cancer patients *** strategy has the potential to reduce the cost and time of cancer screening and genotyping,and to enable targeted therapies in resource-limited settings.