Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors

Xiaoling Wang; Yebo Wang; Xiwei Wu; Jinhui Wang; Yingjia Wang; Zhaojun Qiu; Tammy Chang; He Huang; Ren-Jang Lin; Jiing-Kuan Yee

doi:10.1038/nbt.3127

Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors

Nat Biotechnol. 2015 Feb;33(2):175-8. doi: 10.1038/nbt.3127. Epub 2015 Jan 19.

Authors

Affiliations

¹ Department of Virology, Beckman Research Institute of City of Hope, Duarte, California, USA.
² 1] Department of Virology, Beckman Research Institute of City of Hope, Duarte, California, USA. [2] Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University, Hangzhou, Zhejiang, China.
³ Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, California, USA.
⁴ Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University, Hangzhou, Zhejiang, China.

PMID: 25599175
DOI: 10.1038/nbt.3127

Abstract

The utility of CRISPR-Cas9 and TALENs for genome editing may be compromised by their off-target activity. We show that integrase-defective lentiviral vectors (IDLVs) can detect such off-target cleavage with a frequency as low as 1%. In the case of Cas9, we find frequent off-target sites with a one-base bulge or up to 13 mismatches between the single guide RNA (sgRNA) and its genomic target, which refines sgRNA design.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems / genetics*
Genetic Vectors
Genome, Human
HEK293 Cells
High-Throughput Nucleotide Sequencing
Humans
Integrases / genetics
Lentivirus / enzymology
Lentivirus / genetics*
RNA Editing / genetics*

Substances

Integrases