Phosphonoacetate Modifications Enhance the Stability and Editing Yields of Guide RNAs for Cas9 Editors

CRISPR gene editing
and control systems continue to emerge and
inspire novel research and clinical applications. Advances in CRISPR
performance such as optimizing the duration of activity in cells,
tissues, and organisms, as well as limiting off-target activities,
have been extremely important for expanding the utility of CRISPR-based
systems. By investigating the effects of various chemical modifications
in guide RNAs (gRNAs) at defined positions and combinations, we find
that 2′-O-methyl-3′-phosphonoacetate
(MP) modifications can be substantially more effective than 2′-O-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.