title: CRISPR/Cas9 with single guide RNA expression driven by small tRNA promoters showed reduced editing efficiency compared to a U6 promoter.
abstract: Multiplex genome engineering in vivo with CRISPR/Cas9 shows great promise as a potential therapeutic approach. The ability to incorporate multiple single guide RNA (sgRNA) cassettes together with Cas9 gene expression in one AAV vector could greatly enhance the efficiency. In a recent Method article, Mefferd and coworkers indicated that small tRNA promoters could be used to drive sgRNA expression to facilitate the construction of a more effective AAV vector. In contrast, we found that when targeting endogenous genomic loci, CRISPR/Cas9 with tRNA promoter-driven sgRNA expression showed much reduced genome editing activity, compared with significant cleavage with U6 promoter-driven sgRNA expression. Though the underlying mechanisms are still under investigation, our study suggests that the CRISPR/Cas9 system with tRNA promoter-driven sgRNA expression needs to be reevaluated before it can be used for therapeutic genome editing.
authors.affiliation: CAS Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.|University of Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Yuda
authors.initials: Y
authors.lastname: Wei
authors.affiliation: CAS Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.|University of Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Qiu
authors.affiliation: CAS Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.|University of Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Yanhao
authors.initials: Y
authors.lastname: Chen
authors.affiliation: CAS Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.|University of Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Gaigai
authors.initials: G
authors.lastname: Liu
authors.affiliation: CAS Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.|University of Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Yongxian
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Taizhou Hospital of Traditional Chinese Medicine, Taizhou 225300, China.
authors.forename: Luwei
authors.initials: L
authors.lastname: Xu
authors.affiliation: CAS Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.|University of Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Qiurong
authors.initials: Q
authors.lastname: Ding
mesh_terms: D064113:CRISPR-Cas Systems; D005095:Exoribonucleases / Q000235:genetics*; D000072669:Gene Editing / Q000379:methods*; D015870:Gene Expression; D011401:Promoter Regions, Genetic; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D012343:RNA, Transfer; D005095:Exoribonucleases; C103661:U6 nuclease
keywords: CRISPR/Cas9; genome editing; tRNA promoter