Expansion of the CRISPR–Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs

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 g...

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Bibliographic Details
Published in:	Nature communications Vol. 5; no. 1; p. 4516
Main Authors:	Ranganathan, Vinod, Wahlin, Karl, Maruotti, Julien, Zack, Donald J.
Format:	Journal Article
Language:	English
Published:	London Nature Publishing Group UK 08-08-2014 Nature Publishing Group
Subjects:	13/100 13/109 13/44 14/63 38/22 38/23 42 631/1647/1513/1967/3196 631/61 Animals Bacterial Proteins - genetics Base Sequence Binding Sites Cattle Chickens Clustered Regularly Interspaced Short Palindromic Repeats Computational Biology Embryonic Stem Cells - cytology Genetic Engineering - methods Genome, Human Green Fluorescent Proteins - metabolism HEK293 Cells Humanities and Social Sciences Humans Mice Molecular Sequence Data multidisciplinary Mutation Nucleotides - chemistry Plasmids - metabolism Promoter Regions, Genetic Rats RNA, Guide, CRISPR-Cas Systems - genetics Science Science (multidisciplinary) Streptococcus pyogenes Zebrafish
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Summary:	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 GN 19 NGG. We demonstrate the ability to modify endogenous genes using H1 promoter-expressed gRNAs, which can be used to target both AN 19 NGG and GN 19 NGG genomic sites. AN 19 NGG sites occur ~15% more frequently than GN 19 NGG 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. Current CRISPR-mediated genome-editing methods are limited by the requirement for a specific +1 nucleotide when using the U6 promoter to drive guide RNA synthesis. Now, Ranganathan et al. report a modification of the CRISPR–Cas9 system that more than doubles the number of targetable CRISPR sites within the human genome.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 These authors contributed equally to this work.
ISSN:	2041-1723 2041-1723
DOI:	10.1038/ncomms5516