Comprehensive analysis and accurate quantification of unintended large gene modifications induced by CRISPR-Cas9 gene editing

Most genome editing analyses to date are based on quantifying small insertions and deletions. Here, we show that CRISPR-Cas9 genome editing can induce large gene modifications, such as deletions, insertions, and complex local rearrangements in different primary cells and cell lines. We analyzed larg...

Full description

Saved in:
Bibliographic Details
Published in:Science advances 2022-10, Vol.8 (42), p.eabo7676-eabo7676
Main Authors: Park, So Hyun, Cao, Mingming, Pan, Yidan, Davis, Timothy H, Saxena, Lavanya, Deshmukh, Harshavardhan, Fu, Yilei, Treangen, Todd, Sheehan, Vivien A, Bao, Gang
Format: Article
Language:English
Subjects:
Biomedicine and Life Sciences
Cell Biology
Cell Line
CRISPR-Cas Systems
Engineering
Gene Editing - methods
Hematopoietic Stem Cells - metabolism
Humans
Programmed Cell Death 1 Receptor - metabolism
SciAdv r-articles
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Most genome editing analyses to date are based on quantifying small insertions and deletions. Here, we show that CRISPR-Cas9 genome editing can induce large gene modifications, such as deletions, insertions, and complex local rearrangements in different primary cells and cell lines. We analyzed large deletion events in hematopoietic stem and progenitor cells (HSPCs) using different methods, including clonal genotyping, droplet digital polymerase chain reaction, single-molecule real-time sequencing with unique molecular identifier, and long-amplicon sequencing assay. Our results show that large deletions of up to several thousand bases occur with high frequencies at the Cas9 on-target cut sites on the (11.7 to 35.4%), (14.3%), and (13.2%) genes in HSPCs and the (15.2%) gene in T cells. Our findings have important implications to advancing genome editing technologies for treating human diseases, because unintended large gene modifications may persist, thus altering the biological functions and reducing the available therapeutic alleles.
ISSN:2375-2548
2375-2548
DOI:10.1126/sciadv.abo7676