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Nucleic Acids Res. 2016 Nov 2;44(19):e149. Epub 2016 Jul 25.

An easy and efficient inducible CRISPR/Cas9 platform with improved specificity for multiple gene targeting.

Author information

1
Department of Pathology, Yale School of Medicine, New Haven, CT, 06520 USA.
2
Harbin Institute of Technology, Harbin, 150001 China.
3
Department of Oncology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200127 China.
4
Department of Pathology, Yale School of Medicine, New Haven, CT, 06520 USA qin.yan@yale.edu.

Abstract

The CRISPR/Cas9 system is a powerful genome editing tool and has been widely used for biomedical research. However, many challenges, such as off-target effects and lack of easy solutions for multiplex targeting, are still limiting its applications. To overcome these challenges, we first developed a highly efficient doxycycline-inducible Cas9-EGFP vector. This vector allowed us to track the cells for uniform temporal control and efficient gene disruption, even in a polyclonal setting. Furthermore, the inducible CRISPR/Cas9 system dramatically decreased off-target effects with a pulse exposure of the genome to the Cas9/sgRNA complex. To target multiple genes simultaneously, we established simple one-step cloning approaches for expression of multiple sgRNAs with improved vectors. By combining our inducible and multiplex genome editing approaches, we were able to simultaneously delete Lysine Demethylase (KDM) 5A, 5B and 5C efficiently in vitro and in vivo This user friendly and highly efficient toolbox provides a solution for easy genome editing with tight temporal control, minimal off-target effects and multiplex targeting.

PMID:
27458201
PMCID:
PMC5100567
DOI:
10.1093/nar/gkw660
[Indexed for MEDLINE]
Free PMC Article

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