Ranganathan Vinod, Wahlin Karl, Maruotti Julien, Zack Donald J
Department of Ophthalmology, Wilmer Eye Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.
1] Department of Ophthalmology, Wilmer Eye Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA [2].
Nat Commun. 2014 Aug 8;5:4516. doi: 10.1038/ncomms5516.
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.
重新利用的CRISPR-Cas9系统最近已成为一种革命性的基因组编辑工具。在此，我们报告了靶向所需的引导RNA（gRNA）表达的一种修饰，这极大地扩展了可靶向的基因组。通过常用的U6启动子进行gRNA表达需要鸟苷核苷酸来启动转录，因此将基因组靶向位点限制为GN19NGG。我们证明了使用H1启动子表达的gRNA修饰内源基因的能力，该gRNA可用于靶向AN19NGG和GN19NGG基因组位点。在人类基因组中，AN19NGG位点的出现频率比GN19NGG位点高约15%，并且在人类基因和疾病位点处靶向空间的增加也更为丰富。总之，我们的结果通过将人类基因组和其他真核生物物种内的可靶向位点数量增加一倍以上，增强了CRISPR技术的通用性。
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