TY - JOUR
T1 - Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+
AU - Zou, Roger S.
AU - Liu, Yang
AU - Gaido, Oscar E.Reyes
AU - Konig, Maximilian F.
AU - Mog, Brian J.
AU - Shen, Leo L.
AU - Aviles-Vazquez, Franklin
AU - Marin-Gonzalez, Alberto
AU - Ha, Taekjip
N1 - Publisher Copyright:
© 2023, The Author(s).
PY - 2023/5
Y1 - 2023/5
N2 - Discovery of off-target CRISPR–Cas activity in patient-derived cells and animal models is crucial for genome editing applications, but currently exhibits low sensitivity. We demonstrate that inhibition of DNA-dependent protein kinase catalytic subunit accumulates the repair protein MRE11 at CRISPR–Cas-targeted sites, enabling high-sensitivity mapping of off-target sites to positions of MRE11 binding using chromatin immunoprecipitation followed by sequencing. This technique, termed DISCOVER-Seq+, discovered up to fivefold more CRISPR off-target sites in immortalized cell lines, primary human cells and mice compared with previous methods. We demonstrate applicability to ex vivo knock-in of a cancer-directed transgenic T cell receptor in primary human T cells and in vivo adenovirus knock-out of cardiovascular risk gene PCSK9 in mice. Thus, DISCOVER-Seq+ is, to our knowledge, the most sensitive method to-date for discovering off-target genome editing in vivo.
AB - Discovery of off-target CRISPR–Cas activity in patient-derived cells and animal models is crucial for genome editing applications, but currently exhibits low sensitivity. We demonstrate that inhibition of DNA-dependent protein kinase catalytic subunit accumulates the repair protein MRE11 at CRISPR–Cas-targeted sites, enabling high-sensitivity mapping of off-target sites to positions of MRE11 binding using chromatin immunoprecipitation followed by sequencing. This technique, termed DISCOVER-Seq+, discovered up to fivefold more CRISPR off-target sites in immortalized cell lines, primary human cells and mice compared with previous methods. We demonstrate applicability to ex vivo knock-in of a cancer-directed transgenic T cell receptor in primary human T cells and in vivo adenovirus knock-out of cardiovascular risk gene PCSK9 in mice. Thus, DISCOVER-Seq+ is, to our knowledge, the most sensitive method to-date for discovering off-target genome editing in vivo.
UR - http://www.scopus.com/inward/record.url?scp=85152086129&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85152086129&partnerID=8YFLogxK
U2 - 10.1038/s41592-023-01840-z
DO - 10.1038/s41592-023-01840-z
M3 - Article
C2 - 37024653
AN - SCOPUS:85152086129
SN - 1548-7091
VL - 20
SP - 706
EP - 713
JO - Nature Methods
JF - Nature Methods
IS - 5
ER -