首页|GREPore-seq:A Robust Workflow to Detect Changes After Gene Editing Through Long-range PCR and Nanopore Sequencing

GREPore-seq:A Robust Workflow to Detect Changes After Gene Editing Through Long-range PCR and Nanopore Sequencing

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To achieve the enormous potential of gene-editing technology in clinical therapies,one needs to evaluate both the on-target efficiency and unintended editing consequences comprehen-sively.However,there is a lack of a pipelined,large-scale,and economical workflow for detecting genome editing outcomes,in particular insertion or deletion of a large fragment.Here,we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR/Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR products.Recognizing the high error rates of Oxford nanopore sequencing,we developed a novel pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing(GREPore-seq).GREPore-seq can assess nonhomologous end-joining(NHEJ)-mediated double-stranded oligodeoxynucleotide(dsODN)insertions with comparable accuracy to Illumina next-generation sequencing(NGS).GREPore-seq also reveals a full spectrum of homology-directed repair(HDR)-mediated large gene knock-in,correlating well with the fluorescence-activated cell sorting(FACS)analysis results.Of note,we discovered low-level fragmented and full-length plasmid backbone insertion at the CRISPR cutting site.Therefore,we have established a practical workflow to evaluate various genetic changes,including quantifying insertions of short dsODNs,knock-ins of long pieces,plas-mid insertions,and large fragment deletions after CRISPR/Cas9-mediated editing.GREPore-seq is freely available at GitHub(https://github.com/lisiang/GREPore-seq)and the National Genomics Data Center(NGDC)BioCode(https://ngdc.cncb.ac.cn/biocode/tools/BT007293).

CRISPR/Cas9Genetic changeLong-range PCRNanopore sequencingGREPore-seq

Zi-Jun Quan、Si-Ang Li、Zhi-Xue Yang、Juan-Juan Zhao、Guo-Hua Li、Feng Zhang、Wei Wen、Tao Cheng、Xiao-Bing Zhang

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State Key Laboratory of Experimental Hematology,National Clinical Research Center for Blood Diseases,Haihe Laboratory of Cell Ecosystem,Institute of Hematology & Blood Diseases Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College,Tianjin 300020,China

Center for Stem Cell Medicine,Chinese Academy of Medical Sciences,Tianjin 300020,China

Department of Stem Cell & Regenerative Medicine,Peking Union Medical College,Tianjin 300020,China

National Key Research and Development Program of ChinaNational Key Research and Development Program of ChinaNational Key Research and Development Program of ChinaNational Natural Science Foundation of ChinaNational Natural Science Foundation of ChinaNational Natural Science Foundation of ChinaNational Natural Science Foundation of ChinaNational Natural Science Foundation of ChinaChinese Academy of Medical Sciences(CAMS)Innovation Fund for Medical Sciences(CIFMS)Chinese Academy of Medical Sciences(CAMS)Innovation Fund for Medical Sciences(CIFMS)Chinese Academy of Medical Sciences(CAMS)Innovation Fund for Medical Sciences(CIFMS)Nonprofit Central Research Institute Fund of Chinese Academy of Medical SciencesTianjin Synthetic Biotechnology Innovation Capacity Improvement ProjectCAMS Fundamental Research Funds for Central Research Institutes

2019YFA01108032019YFA01102042021YFA110090081870149820701158189099081730006817701982021-I2M-1-0402021-I2M-1-0412021-12M-1-0012020-PT310-011TSBICIP-KJGG-0173332021093

2023

10.1016/j.gpb.2022.06.002

基因组蛋白质组与生物信息学报(英文版)
中国科学院北京基因组研究所

基因组蛋白质组与生物信息学报(英文版)

CSTPCDCSCD
影响因子：0.495
ISSN：1672-0229
年,卷(期)：2023.6(6)
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