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

Author

Listed:
  • Vinod Ranganathan

    (Wilmer Eye Institute, The Johns Hopkins University School of Medicine)

  • Karl Wahlin

    (Wilmer Eye Institute, The Johns Hopkins University School of Medicine)

  • Julien Maruotti

    (Wilmer Eye Institute, The Johns Hopkins University School of Medicine)

  • Donald J. Zack

    (Wilmer Eye Institute, The Johns Hopkins University School of Medicine
    The Johns Hopkins University School of Medicine
    The Johns Hopkins University School of Medicine
    Institute of Genetic Medicine, The Johns Hopkins University School of Medicine)

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.

Suggested Citation

  • Vinod Ranganathan & Karl Wahlin & Julien Maruotti & Donald J. Zack, 2014. "Expansion of the CRISPR–Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs," Nature Communications, Nature, vol. 5(1), pages 1-8, December.
    • Handle: RePEc:nat:natcom:v:5:y:2014:i:1:d:10.1038_ncomms5516
    DOI: 10.1038/ncomms5516
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