| Autor: |
Zi-Jun Quan, Si-Ang Li, Zhi-Xue Yang, Juan-Juan Zhao, Guo-Hua Li, Feng Zhang, Wei Wen, Tao Cheng, Xiao-Bing Zhang |
| Jazyk: |
angličtina |
| Rok vydání: |
2023 |
| Předmět: |
|
| Zdroj: |
Genomics, Proteomics & Bioinformatics, Vol 21, Iss 6, Pp 1221-1236 (2023) |
| Druh dokumentu: |
article |
| ISSN: |
1672-0229 |
| DOI: |
10.1016/j.gpb.2022.06.002 |
| Popis: |
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 comprehensively. 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, plasmid 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). |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
|
