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A Nonsequencing Approach for the Rapid Detection of RNA Editing
Published on: April 21, 2022
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GREPore-seq: A Robust Workflow to Detect Changes After Gene Editing Through Long-range PCR and Nanopore Sequencing.
Zi-Jun Quan1, Si-Ang Li1, Zhi-Xue Yang1
1State 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.
Genomics, Proteomics & Bioinformatics
|June 25, 2022
Summary
We developed GREPore-seq, a novel pipeline for analyzing CRISPR/Cas9 gene editing outcomes. This method efficiently detects large insertions and deletions, improving clinical therapy development.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- CRISPR/Cas9 gene editing holds immense clinical potential.
- Comprehensive evaluation of on-target efficiency and unintended edits is crucial.
- Existing workflows lack scalability and cost-effectiveness for detecting large genomic alterations.
Purpose of the Study:
- To establish a pipelined, large-scale, and economical workflow for detecting diverse CRISPR/Cas9 editing outcomes.
- To accurately assess both on-target efficiency and unintended genetic consequences.
- To enable robust analysis of insertions, deletions, and other modifications.
Main Methods:
- Pooled nanopore sequencing of barcoded long-range PCR products.
- Development of the GREPore-seq pipeline to handle nanopore sequencing error rates.
- Detection of nonhomologous end-joining (NHEJ) and homology-directed repair (HDR) mediated edits.
Main Results:
- GREPore-seq accurately quantifies double-stranded oligodeoxynucleotide (dsODN) insertions, comparable to Illumina NGS.
- The method reveals a full spectrum of homology-directed repair (HDR)-mediated large gene knock-ins.
- Discovery of low-level fragmented and full-length plasmid backbone insertions at CRISPR cutting sites.
Conclusions:
- A practical workflow, GREPore-seq, is established for evaluating various CRISPR/Cas9 editing outcomes.
- The workflow quantifies short dsODN insertions, long knock-ins, plasmid insertions, and large fragment deletions.
- GREPore-seq provides a comprehensive tool for assessing gene editing accuracy and safety.
