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Modulating the Activity of CRISPR-Cas with Chemical Modifications in Single-Guide RNAs
Poster Title: Modulating the Activity of CRISPR-Cas with Chemical Modifications in Single-Guide RNAs
Submitted on 13 Jan 2021
Author(s): Daniel Ryan, David Taussig, Suhani Thakker, Israel Steinfeld, Ben Lunstad, Rob Kaiser, Ryan McCaffrey, Justin Townsend, Patrick Chaffey, Bo Curry, Doug Dellinger, Laurakay Bruhn
Affiliations: Agilent Technologies
This poster was presented at CSHL Genome Engineering 2020: CRISPR Frontiers
Poster Views: 132
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Poster Information
Abstract: CRISPR is revolutionizing life science and drug development research. Through the targeted application of gene editing, gene knockins, knockouts, or gene expression control, new models and therapeutics are enabled and accelerated. An important consideration in the development of these capabilities is the activity of the CRISPR-Cas system.

With the novel RNA synthesis chemistry outlined in Dellinger et. al. (JACS, 2011) we robustly and routinely synthesize RNA oligos at higher yields per scale than other chemistries. Also, synthesis allows us to site-specifically modify nucleotides in sgRNAs to adjust their longevity and enhance their specificity.

Studies of the stability and activity of multiple guide RNAs demonstrate that different numbers and types of modifications incorporated at their 5’ and 3’ ends can enhance stability of the RNA in cells while maintaining the desired guide RNA functionality.
Summary: Chemical modifications in chemically sgRNAs can provide significant advantages in affecting the duration of sgRNA activity in transfected cells (or in vivo). By introducing M, MS, MP or MSP modifications to the ends of the sgRNA, the half-life can be extended. In addition, incorporation of MP modifications at select positions in the sgRNA sequence can improve the specificity of on-target cleavage while maintaining, or improving, overall efficiency. References: Hendel A, Bak RO, Clark JT, Kennedy AB, Ryan DE, Roy S, Steinfeld I, Lunstad BD, Kaiser RJ, Wilkens AB, Bacchetta R, Tsalenko A, Dellinger D, Bruhn L, Porteus MH. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol. 2015 Sep;33(9):985-989. doi: 10.1038/nbt.3290. Epub 2015 Jun 29. PMID: 26121415; PMCID: PMC4729442.

Ryan DE, Taussig D, Steinfeld I, Phadnis SM, Lunstad BD, Singh M, Vuong X, Okochi KD, McCaffrey R, Olesiak M, Roy S, Yung CW, Curry B, Sampson JR, Bruhn L, Dellinger DJ. Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs. Nucleic Acids Res. 2018 Jan 25;46(2):792-803. doi: 10.1093/nar/gkx1199. PMID: 29216382; PMCID: PMC5778453.

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