Phosphonoacetate Modifications Enhance the Stability and Editing Yields of Guide RNAs for Cas9 Editors
Daniel E. Ryan, Tamar Diamant-Levi, Israel Steinfeld, David Taussig, Savita Visal-Shah, Suhani Thakker, Benjamin D. Lunstad, Robert Kaiser, Ryan McCaffrey, Michael Ortiz, Justin H. Townsend, William R. Welch, Mandeep Singh, Bo Curry, Douglas J. Dellinger, Laurakay Bruhn
Abstract
-methyl-3'-phosphorothioate (MS) modifications at the 3' ends of single-guide RNAs (sgRNAs) to promote high editing yields, in some instances showing an order of magnitude higher editing yield in human cells. MP-modified 3' ends are especially effective at promoting the activity of guide RNAs cotransfected with Cas messenger RNA (mRNA), as the gRNA must persist in cells until the Cas protein is expressed. We demonstrate such an MP enhancement for sgRNAs cotransfected with a BE4 mRNA for cytidine base editing and also demonstrate that MP at the 3' ends of prime editing guide RNAs (pegRNAs) cotransfected with PE2 mRNA can promote maximal prime editing yields. In the presence of serum, sgRNAs with MP-modified 3' ends showed marked improvements in editing efficiency over sgRNAs with MS-modified 3' ends codelivered with Cas9 mRNA and showed more modest improvements at enhancing the activity of transfected ribonucleoprotein (RNP) complexes. Our results suggest that MP should be considered as a performance-enhancing modification for the 3' ends of synthetic gRNAs, especially in situations where the guide RNAs may be susceptible to exonuclease-mediated degradation.