CRISPR/Cas9-loxP-Mediated Gene Editing as a Novel Site-Specific Genetic Manipulation Tool

Fayu Yang; Changbao Liu; Ding Chen; Mengjun Tu; Haihua Xie; Huihui Sun; Xianglian Ge; Lianchao Tang; Jin Li; Jiayong Zheng; Zongming Song; Jia Qu; Feng Gu

doi:10.1016/j.omtn.2017.04.018

CRISPR/Cas9-loxP-Mediated Gene Editing as a Novel Site-Specific Genetic Manipulation Tool

Mol Ther Nucleic Acids. 2017 Jun 16:7:378-386. doi: 10.1016/j.omtn.2017.04.018. Epub 2017 Apr 25.

Authors

Fayu Yang¹, Changbao Liu², Ding Chen¹, Mengjun Tu¹, Haihua Xie¹, Huihui Sun¹, Xianglian Ge¹, Lianchao Tang¹, Jin Li¹, Jiayong Zheng³, Zongming Song¹, Jia Qu¹, Feng Gu⁴

Affiliations

¹ School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang 325027, China.
² The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325027, China.
³ Department of Gynecology and Obstetrics, People's Hospital of Wenzhou, Wenzhou, Zhejiang 325000, China.
⁴ School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang 325027, China. Electronic address: gufenguw@gmail.com.

Abstract

Cre-loxP, as one of the site-specific genetic manipulation tools, offers a method to study the spatial and temporal regulation of gene expression/inactivation in order to decipher gene function. CRISPR/Cas9-mediated targeted genome engineering technologies are sparking a new revolution in biological research. Whether the traditional site-specific genetic manipulation tool and CRISPR/Cas9 could be combined to create a novel genetic tool for highly specific gene editing is not clear. Here, we successfully generated a CRISPR/Cas9-loxP system to perform gene editing in human cells, providing the proof of principle that these two technologies can be used together for the first time. We also showed that distinct non-homologous end-joining (NHEJ) patterns from CRISPR/Cas9-mediated gene editing of the targeting sequence locates at the level of plasmids (episomal) and chromosomes. Specially, the CRISPR/Cas9-mediated NHEJ pattern in the nuclear genome favors deletions (64%-68% at the human AAVS1 locus versus 4%-28% plasmid DNA). CRISPR/Cas9-loxP, a novel site-specific genetic manipulation tool, offers a platform for the dissection of gene function and molecular insights into DNA-repair pathways.

Keywords: AAVS1; CRISPR/Cas9; NHEJ; gene editing; loxP.