title: Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency.
abstract: CRISPR-Cas9 is a powerful genome editing technology, yet with off-target effects. Truncated sgRNAs (17nt) have been found to decrease off-target cleavage without affecting on-target disruption in 293T cells. However, the potency of 17nt sgRNAs relative to the full-length 20nt sgRNAs in stem cells, such as human mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), has not been assessed. Using a GFP reporter system, we found that both 17nt and 20nt sgRNAs expressed by lentiviral vectors induce ~95% knockout (KO) in 293T cells, whereas the KO efficiencies are significantly lower in iPSCs (60-70%) and MSCs (65-75%). Furthermore, we observed a decrease of 10-20 percentage points in KO efficiency with 17nt sgRNAs compared to full-length sgRNAs in both iPSCs and MSCs. Off-target cleavage was observed in 17nt sgRNAs with 1-2nt but not 3-4nt mismatches; whereas 20nt sgRNAs with up to 5nt mismatches can still induce off-target mutations. Of interest, we occasionally observed off-target effects induced by the 17nt but not the 20nt sgRNAs. These results indicate the importance of balancing on-target gene cleavage potency with off-target effects: when efficacy is a major concern such as genome editing in stem cells, the use of 20nt sgRNAs is preferable.
authors.affiliation: State Key Laboratory of Experimental Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.
authors.forename: Jian-Ping
authors.initials: JP
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Experimental Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.
authors.forename: Xiao-Lan
authors.initials: XL
authors.lastname: Li
authors.affiliation: Department of Medicine, Loma Linda University, Loma Linda, CA 92350, USA.
authors.forename: Amanda
authors.initials: A
authors.lastname: Neises
authors.affiliation: Department of Medicine, Loma Linda University, Loma Linda, CA 92350, USA.
authors.forename: Wanqiu
authors.initials: W
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Experimental Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.
authors.forename: Lin-Ping
authors.initials: LP
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Experimental Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.
authors.forename: Guang-Zhen
authors.initials: GZ
authors.lastname: Ji
authors.affiliation: State Key Laboratory of Experimental Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.
authors.forename: Jun-Yao
authors.initials: JY
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Experimental Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Experimental Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.
authors.forename: Wei-Ping
authors.initials: WP
authors.lastname: Yuan
authors.affiliation: State Key Laboratory of Experimental Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.|Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Tianjin, China.|Department of Stem Cell &Regenerative Medicine, Peking Union Medical College, Tianjin, China.|Collaborative Innovation Center for Cancer Medicine, Tianjin, China.|Tianjin Key Laboratory of Blood Cell Therapy and Technology, Tianjin, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Cheng
authors.affiliation: State Key Laboratory of Experimental Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.|Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.|Department of Medicine, Loma Linda University, Loma Linda, CA 92350, USA.|Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Tianjin, China.|Department of Stem Cell &Regenerative Medicine, Peking Union Medical College, Tianjin, China.
authors.forename: Xiao-Bing
authors.initials: XB
authors.lastname: Zhang
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D055786:Gene Knockout Techniques / Q000379:methods; D018390:Gene Targeting / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D012313:RNA / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D012313:RNA