title: Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA.
abstract: Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system has emerged in recent years as a highly efficient RNA-guided gene manipulation platform. Simultaneous editing or transcriptional activation/suppression of different genes becomes feasible with the co-delivery of multiple guide RNAs (gRNAs). Here, we report that multiple gRNAs linked with self-cleaving ribozymes and/or tRNA could be simultaneously expressed from a single U6 promoter to exert genome editing of dystrophin and myosin binding protein C3 in human and mouse cells. Moreover, this strategy allows the expression of multiple gRNAs for synergistic transcription activation of follistatin when used with catalytically inactive dCas9-VP64 or dCas9-p300core fusions. Finally, the gRNAs linked by the self-cleaving ribozymes and tRNA could be expressed from RNA polymerase type II (pol II) promoters such as generic CMV and muscle/heart-specific MHCK7. This is particularly useful for in vivo applications when the packaging capacity of recombinant adeno-associated virus is limited while tissue-specific delivery of gRNAs and Cas9 is desired. Taken together, this study provides a novel strategy to enable tissue-specific expression of more than one gRNAs for multiplex gene editing from a single pol II promoter.
authors: Department of Surgery, Davis Heart and Lung Research Institute, Biomedical Sciences Graduate Program, Biophysics Graduate Program, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA.
authors: Li
authors: L
authors: Xu
authors: Department of Surgery, Davis Heart and Lung Research Institute, Biomedical Sciences Graduate Program, Biophysics Graduate Program, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA.
authors: Lixia
authors: L
authors: Zhao
authors: Department of Surgery, Davis Heart and Lung Research Institute, Biomedical Sciences Graduate Program, Biophysics Graduate Program, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA.
authors: Yandi
authors: Y
authors: Gao
authors: Department of Surgery, Davis Heart and Lung Research Institute, Biomedical Sciences Graduate Program, Biophysics Graduate Program, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA.
authors: Jing
authors: J
authors: Xu
authors: Department of Surgery, Davis Heart and Lung Research Institute, Biomedical Sciences Graduate Program, Biophysics Graduate Program, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA.
authors: Renzhi
authors: R
authors: Han
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics / Q000378:metabolism; D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism; D016189:Dystrophin / Q000235:genetics / Q000378:metabolism; D038681:Follistatin / Q000235:genetics / Q000378:metabolism; D000072669:Gene Editing; D057809:HEK293 Cells; D006801:Humans; D008099:Liver / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018482:Muscle, Skeletal / Q000378:metabolism; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism; D009218:Myosins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics* / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000235:genetics* / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D016189:Dystrophin; D038681:Follistatin; D016337:RNA, Catalytic; D000094704:RNA, Guide, CRISPR-Cas Systems; D012342:RNA, Small Nuclear; D011993:Recombinant Fusion Proteins; C405486:U6 small nuclear RNA; D012343:RNA, Transfer; D004257:DNA Polymerase II; D009218:Myosins