title: An easy and efficient inducible CRISPR/Cas9 platform with improved specificity for multiple gene targeting.
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.
authors.affiliation: Department of Pathology, Yale School of Medicine, New Haven, CT, 06520 USA.
authors.forename: Jian
authors.initials: J
authors.lastname: Cao
authors.affiliation: Department of Pathology, Yale School of Medicine, New Haven, CT, 06520 USA.
authors.forename: Lizhen
authors.initials: L
authors.lastname: Wu
authors.affiliation: Department of Pathology, Yale School of Medicine, New Haven, CT, 06520 USA.
authors.forename: Shang-Min
authors.initials: SM
authors.lastname: Zhang
authors.affiliation: Department of Pathology, Yale School of Medicine, New Haven, CT, 06520 USA.|Harbin Institute of Technology, Harbin, 150001 China.
authors.forename: Min
authors.initials: M
authors.lastname: Lu
authors.affiliation: Department of Pathology, Yale School of Medicine, New Haven, CT, 06520 USA.
authors.forename: William K C
authors.initials: WK
authors.lastname: Cheung
authors.affiliation: Department of Pathology, Yale School of Medicine, New Haven, CT, 06520 USA.
authors.forename: Wesley
authors.initials: W
authors.lastname: Cai
authors.affiliation: Department of Pathology, Yale School of Medicine, New Haven, CT, 06520 USA.
authors.forename: Molly
authors.initials: M
authors.lastname: Gale
authors.affiliation: Department of Pathology, Yale School of Medicine, New Haven, CT, 06520 USA.|Department of Oncology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200127 China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Xu
authors.affiliation: Department of Pathology, Yale School of Medicine, New Haven, CT, 06520 USA qin.yan@yale.edu.
authors.forename: Qin
authors.initials: Q
authors.lastname: Yan
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000378:metabolism; D015870:Gene Expression; D055786:Gene Knockout Techniques; D023061:Gene Order; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods / Q000592:standards; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D011401:Promoter Regions, Genetic; D000094704:RNA, Guide, CRISPR-Cas Systems; D056485:Retinoblastoma-Binding Protein 2 / Q000172:deficiency
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D056485:Retinoblastoma-Binding Protein 2; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases