Abstract To achieve the enormous potential of gene-editing technology in clinical therapies, both the on-target and unintended editing consequences need to be thoroughly evaluated. However, there is a lack of a comprehensive, 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. To overcome the high error rates and indels of nanopore sequencing, we developed a pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing (GREPore-seq). GREPore-seq can detect NHEJ-mediated double-stranded oligodeoxynucleotide (dsODN) insertions with comparable accuracy to Illumina next-generation sequencing (NGS). GREPore-seq also identifies HDR-mediated large gene knock-in, which excellently correlates with FACS analysis data. Low-level plasmid backbone insertion after HDR editing was also detected. We have established a practical workflow to identify genetic changes, including quantifying dsODN insertions, knock-ins, plasmid backbone insertions, and large fragment deletions after CRISPR editing. This toolkit for nanopore sequencing of pooled long amplicons should have broad applications in assessing on-target HDR editing and inadvertent large indels of over 1 kb. GREPore-seq is freely available at GitHub ( https://github.com/lisiang/GREPore-seq ).

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