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Poster Title: 2'-O-methyl phosphorothioate linkage-modified synthetic guide RNAs for efficient CRISPR-Cas9 genome editing and reduced cellular toxicity
Submitted on 07 Feb 2018
Author(s): Tiana Stastny, Megan Basila, Hidevaldo B. Machado, Emily Anderson, Eldon T. Chou, Melissa L. Kelley, Anja van Brabant Smith
Affiliations: Dharmacon part of Horizon Discovery Group
This poster was presented at 2018 Keystone Conference
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Poster Information
Abstract: The discovery of the bacterial CRISPR-Cas9 system has drastically changed the way we perform genome engineering and significantly shortened the time to results. With this system, a Cas9 endonuclease is targeted to DNA through a complementary guide RNA to cause a double-strand break at the target site. In the native system, a DNA-targeting CRISPR RNA (crRNA) is duplexed with the trans-activating crRNA (tracrRNA) to guide the Cas9 protein; these small RNAs can be chemically synthesized as transfection-ready gene-editing reagents. Alternatively, the crRNA and tracrRNA can be linked together to create a chimeric single guide RNA (sgRNA). For DNA-free editing, sgRNA can be delivered as in vitro transcribed (IVT) RNA or synthetic RNA in combination with Cas9 mRNA or Cas9 protein. An issue arises with IVT sgRNA due to it causing an immune response, which contrasts with synthetic guide RNAs which elicit no immune response. Here, we present chemical modification of both synthetic crRNA:tracrRNA and sgRNA with one to three 2’-O-methyl and phosphorothioates linkages (MS) on the 5' and/or 3' ends of the RNAs. We examined the stability of these RNAs by measuring their gene editing efficiency using an electroporation protocol in which guide RNAs are co-delivered with Cas9 mRNA. Additionally, we assessed modification patterns for their ability to improve gene editing when delivered to cells with electroporation or lipid-mediated transfection along with Cas9 protein, Cas9 mRNA, or into a stably expressing Cas9 cell line. The placement of modifications on guide RNAs must be considered for the respective application. While co-electroporation experiments with Cas9 mRNA require modifications in certain positions on the guide RNA for stability, other applications do not, but in some cases may still show modest increases in gene editing efficiency. Importantly, some modification patterns caused increased cell death. Therefore, we have established a minimal guide RNA modification pattern for efficient gene editing and reduced toxicity in
all applications.
Summary: Chemical modifications to sgRNA & crRNA:tracrRNA have different effects on gene editing efficiency based on delivery method and Cas9 nuclease sourceReferences: M. Basila, M.L. Kelley, A. van Brabant Smith. Minimal 2’-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity. PLoS ONE 12(11): e0188593. (2017). Report abuse »
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