title: Polycistronic tRNA and CRISPR guide-RNA enables highly efficient multiplexed genome engineering in human cells.
abstract: CRISPR/Cas9 has been widely used for genomic editing in many organisms. Many human diseases are caused by multiple mutations. The CRISPR/Cas9 system provides a potential tool to introduce multiple mutations in a genome. To mimic complicated genomic variants in human diseases, such as multiple gene deletions or mutations, two or more small guide RNAs (sgRNAs) need to be introduced all together. This can be achieved by separate Pol III promoters in a construct. However, limited enzyme sites and increased insertion size lower the efficiency to make a construct. Here, we report a strategy to quickly assembly multiple sgRNAs in one construct using a polycistronic-tRNA-gRNA (PTG) strategy. Taking advantage of the endogenous tRNA processing system in mammalian cells, we efficiently express multiple sgRNAs driven using only one Pol III promoter. Using an all-in-one construct carrying PTG, we disrupt the deacetylase domain in multiple histone deacetylases (HDACs) in human cells simultaneously. We demonstrate that multiple HDAC deletions significantly affect the activation of the Wnt-signaling pathway. Thus, this method enables to efficiently target multiple genes and provide a useful tool to establish mutated cells mimicking human diseases.
authors.affiliation: Department of Biology, Pennsylvania State University, PA 16802, USA.
authors.forename: Fengping
authors.initials: F
authors.lastname: Dong
authors.affiliation: Department of Plant Pathology and Environmental Microbiology, Pennsylvania State University, PA 16802, USA; College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China.
authors.forename: Kabin
authors.initials: K
authors.lastname: Xie
authors.affiliation: Department of Plant Pathology and Environmental Microbiology, Pennsylvania State University, PA 16802, USA.
authors.forename: Yueying
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Plant Pathology and Environmental Microbiology, Pennsylvania State University, PA 16802, USA. Electronic address: yuy3@psu.edu.
authors.forename: Yinong
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Department of Biology, Pennsylvania State University, PA 16802, USA. Electronic address: yzm1@psu.edu.
authors.forename: Yingwei
authors.initials: Y
authors.lastname: Mao
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D017353:Gene Deletion; D000072669:Gene Editing / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D015894:Genome, Human; D057809:HEK293 Cells; D006655:Histone Deacetylases / Q000235:genetics; D006801:Humans; D009154:Mutation; D011401:Promoter Regions, Genetic; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics*; D060449:Wnt Signaling Pathway
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D012343:RNA, Transfer; D006655:Histone Deacetylases
keywords: Genome editing; HDAC; Polycistronic-tRNA-gRNA; Wnt