title: Expression of CRISPR/Cas single guide RNAs using small tRNA promoters.
abstract: The in vivo application of CRISPR/Cas-based DNA editing technology will require the development of efficient delivery methods that likely will be dependent on adeno-associated virus (AAV)-based viral vectors. However, AAV vectors have only a modest, ∼4.7-kb packaging capacity, which will necessitate the identification and characterization of highly active Cas9 proteins that are substantially smaller than the prototypic Streptococcus pyogenes Cas9 protein, which covers ∼4.2 kb of coding sequence, as well as the development of single guide RNA (sgRNA) expression cassettes substantially smaller than the current ∼360 bp size. Here, we report that small, ∼70-bp tRNA promoters can be used to express high levels of tRNA:sgRNA fusion transcripts that are efficiently and precisely cleaved by endogenous tRNase Z to release fully functional sgRNAs. Importantly, cells stably expressing functional tRNA:sgRNA precursors did not show a detectable change in the level of endogenous tRNA expression. This novel sgRNA expression strategy should greatly facilitate the construction of effective AAV-based Cas9/sgRNA vectors for future in vivo use.
authors.affiliation: Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Adam L
authors.initials: AL
authors.lastname: Mefferd
authors.affiliation: Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Anand V R
authors.initials: AV
authors.lastname: Kornepati
authors.affiliation: Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Hal P
authors.initials: HP
authors.lastname: Bogerd
authors.affiliation: Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Edward M
authors.initials: EM
authors.lastname: Kennedy
authors.affiliation: Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Bryan R
authors.initials: BR
authors.lastname: Cullen
mesh_terms: D000818:Animals; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D011401:Promoter Regions, Genetic; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics* / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA
chemical_list: D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D012367:RNA, Viral; D012343:RNA, Transfer
keywords: CRISPR/Cas; gene editing; tRNA processing; tRNase Z