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Improving the Chemical Synthesis and Performance of Extended-Length Guide RNAs for Genome Editing and Control of Gene Expression
Poster Title: Improving the Chemical Synthesis and Performance of Extended-Length Guide RNAs for Genome Editing and Control of Gene Expression
Submitted on 13 Jan 2021
Author(s): Laurakay Bruhn, Bo Curry, Rob Kaiser, Ben Lunstad, Ryan McCaffrey, Joel Myerson, Subhadeep Roy, Daniel Ryan, Israel Steinfeld, David Taussig, Justin Townsend, Suhani Thakker, Doug Dellinger
Affiliations: Agilent Technologies
This poster was presented at ASGCT 2020
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Poster Information
Abstract: The landscape of CRISPR-based technologies for genome editing and control of gene expression is expanding rapidly and many of these technologies utilize extended-length guide RNAs that are challenging to produce by traditional RNA synthesis chemistries. Two examples include:

(i) the SAM CRISPR-activation system (Konermann, et. al., Nature, 2015) which employs guide RNAs around 160-nt long containing two MS2 RNA aptamers, and

(ii) Prime Editing (Anzalone et. al., Nature, 2019) which employs extensions of the guide RNA to direct the replacement or insertion of new DNA sequences into the genome.

Using a novel RNA synthesis chemistry (Dellinger et. al. JACS, 2011) we find it straightforward to chemically synthesize long RNA oligos in the size range currently utilized for these applications. We are continuing to further develop synthesis and purification methodologies to enable effective synthesis of even longer RNA oligos.

Studies of the stability and activity of 163mer guide RNAs designed for the SAM CRISPR-activation system demonstrate that different numbers and types of modifications incorporated at the 5’ and 3’ ends can enhance their stability in cells while maintaining guide RNA functionality
Summary: Robust chemical synthesis of extended length guide RNAs for emerging applications like CRISPR-activation and Prime Editing affords multiple advantages including:
- increased efficacy of guide RNAs
- robust and scalable production for biotechnological and therapeutic applications
- greater flexibility in the guide RNA design including the ability to incorporate chemical modifications site-specifically to enhance performance
References: Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 2015 Jan 29;517(7536):583-8. doi: 10.1038/nature14136. Epub 2014 Dec 10. PMID: 25494202; PMCID: PMC4420636.

Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019 Dec;576(7785):149-157. doi: 10.1038/s41586-019-1711-4. Epub 2019 Oct 21. PMID: 31634902; PMCID: PMC6907074.

Dellinger et al. “Streamlined Process for the Chemical Synthesis of RNA Using 2’-O-Thionocarbamate-Protected Nucleoside Phosphoramidites in the Solid Phase”, J. Am. Chem. Soc. 133, 11540–11556 (2011).
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