Citation: 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. Programmable endonuclease combined with isothermal polymerase amplification to selectively enrich for rare mutant allele fractions[J]. Chinese Chemical Letters, ;2022, 33(8): 4126-4132. doi: 10.1016/j.cclet.2021.11.065 shu

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

Junman Chen ^{a, b, c, 1} ,
Tian Qiu ^{d, 1} ,
Michael G. Mauk ^c ,
Zheng Su ^e ,
Yaguang Fan ^f ,
Dennis J. Yuan ^b ,
Qinghua Zhou ^g ,
Youlin Qiao ^e ,
Haim H. Bau ^c ,
Jianming Ying ^{d, *,} ,
Jinzhao Song ^{b, c, **,}

a.
Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China
b.
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
c.
Department of Mechanical Engineering and Applied Mechanics, University of Pennsylvania, Philadelphia, PA 19104, United States
d.
Department of Pathology, National Cancer Center / National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China
e.
Center for Global Health, School of Population Medicine and Public Health, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China
f.
Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China
g.
Sichuan Lung Cancer Institute, Sichuan Lung Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, China

^**

songjinzhao@ucas.ac.cn

jmying@cicams.ac.cn

Received Date: 23 September 2021
Revised Date: 18 November 2021
Accepted Date: 23 November 2021
Available Online: 26 November 2021

Figures(4)

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.
- Mutant allele enrichment,
- Programmable endonuclease,
- Liquid biopsy,
- Mutation detection,
- Point-of-care testing,
- CRISPR-Cas9,
- Recombinase polymerase amplification,
- Nucleic acid diagnostics
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沈阳化工大学材料科学与工程学院沈阳 110142