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Chemical modifications of synthetic guide RNA for enhanced RNA stability and reduced cellular toxicity in CRISPR-Cas9 genome editing
Chemical modifications of synthetic guide RNA for enhanced RNA stability and reduced cellular toxicity in CRISPR-Cas9 genome editing
Submitted on 25 Oct 2017

Hidevaldo B. Machado, Megan Basila, Eldon T. Chou, Emily M. Anderson, Melissa L. Kelley, Anja van Brabant Smith
Dharmacon part of Horizon Discovery Group
This poster was presented at GDI conference 2017
Poster Views: 705
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Poster Abstract
Gene disruption and knock-ins can be easily obtained through the formation of precise DNA double-strand breaks with the CRISPR-Cas9 system. In this system, the Cas9 nuclease targets the genomic DNA using a guide RNA, provided as either the native dual-RNA consisting of a DNA-targeting CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), or a chimeric single guide RNA (sgRNA) created through the fusion of crRNA and tracrRNA. DNA-free genome engineering can be achieved by using Cas9 mRNA or Cas9 protein with a guide RNA, such as in vitro transcribed (IVT) sgRNA, synthetic sgRNA or synthetic crRNA:tracrRNA. Synthetic sgRNA or crRNA:tracrRNA offer an advantage over IVT sgRNA by providing little-to-no effect on the cellular immune response in addition to allowing the incorporation of chemical modifications to increase the RNA stability. We have chemically modified both synthetic sgRNA and crRNA:tracrRNA with one to three 2’-O-methyl nucleotides and backbone phosphorothioate linkages (MS) on the 5’ and/or 3’ ends. These modified guide RNAs were delivered into cells alone or with Cas9 mRNA or Cas9 protein using electroporation or lipid-mediated transfection. In electroporation experiments, some modification patterns were found to significantly improve CRISPR-Cas9 gene editing when co-delivered with Cas9 mRNA compared to the unmodified versions, yet most modifications did not significantly increase gene editing when used with Cas9 protein. Lipid-mediated transfection of modified guide RNAs into a Cas9-expressing cell line resulted in similar editing efficiencies as the unmodified synthetic guide RNAs; however, certain modification patterns resulted in increased cellular toxicity. Of the modifications that were nontoxic, some patterns showed modest improvement in editing efficiency when co-transfected with Cas9 mRNA or Cas9 protein. Overall, our results indicate that the placement and number of MS modifications on synthetic guide RNAs are important for increased gene editing by co-electroporation with Cas9 mRNA, but are not necessarily required for lipid-mediated transfection as some chemical modification patterns can increase cellular toxicity and may provide only modest improvement in gene editing efficiency.

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