title: Expansion of the CRISPR-Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs.
abstract: The repurposed CRISPR-Cas9 system has recently emerged as a revolutionary genome-editing tool. Here we report a modification in the expression of the guide RNA (gRNA) required for targeting that greatly expands the targetable genome. gRNA expression through the commonly used U6 promoter requires a guanosine nucleotide to initiate transcription, thus constraining genomic-targeting sites to GN19NGG. We demonstrate the ability to modify endogenous genes using H1 promoter-expressed gRNAs, which can be used to target both AN19NGG and GN19NGG genomic sites. AN19NGG sites occur ~15% more frequently than GN19NGG sites in the human genome and the increase in targeting space is also enriched at human genes and disease loci. Together, our results enhance the versatility of the CRISPR technology by more than doubling the number of targetable sites within the human genome and other eukaryotic species.
authors.affiliation: Department of Ophthalmology, Wilmer Eye Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.
authors.forename: Vinod
authors.initials: V
authors.lastname: Ranganathan
authors.affiliation: 1] Department of Ophthalmology, Wilmer Eye Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA [2].
authors.forename: Karl
authors.initials: K
authors.lastname: Wahlin
authors.affiliation: 1] Department of Ophthalmology, Wilmer Eye Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA [2].
authors.forename: Julien
authors.initials: J
authors.lastname: Maruotti
authors.affiliation: 1] Department of Ophthalmology, Wilmer Eye Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA [2] Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA [3] Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA [4] Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA [5] Department of Genetics, Institut de la Vision, 17 rue Moreau, 75012 Paris, France.
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Zack
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002645:Chickens; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D019295:Computational Biology; D053595:Embryonic Stem Cells / Q000166:cytology; D005818:Genetic Engineering / Q000379:methods; D015894:Genome, Human; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D051381:Rats; D013297:Streptococcus pyogenes; D015027:Zebrafish
chemical_list: D001426:Bacterial Proteins; D009711:Nucleotides; D000094704:RNA, Guide, CRISPR-Cas Systems; D049452:Green Fluorescent Proteins
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