Chemical Modifications in RNA Interference and CRISPR/Cas Genome Editing Reagents

Chemically modified oligonucleotides (ONs) are routinely used in the laboratory to assess gene function, and clinical advances are rapidly progressing as continual efforts are being made to optimize ON efficacy. Over the years, RNA interference (RNAi) has become one of the main tools used to inhibit RNA expression across a wide variety of species. Efforts have been made to improve the exogenous delivery of the double-stranded RNA components to the endogenous intracellular RNAi machinery to direct efficacious degradation of a user-defined RNA target. More recently, synthetic RNA ONs are being used to mimic the bacterial-derived CRISPR/Cas system to direct specific editing of the mammalian genome. Both of these techniques rely on the use of various chemical modifications to the RNA phosphate backbone or sugar in specific positions throughout the ONs to improve the desired biological outcome. Relevant chemical modifications also include conjugated targeting ligands to assist ON delivery to specific cell types. Chemical modifications are most beneficial for therapeutically relevant ONs, as they serve to enhance target binding, increase drug longevity, facilitate cell-specific targeting, improve internalization into productive intracellular compartments, and mitigate both sequence-specific as well as immune-related off-target effects (OTEs). The knowledge gained from years of optimizing RNAi reagents and characterizing the biochemical and biophysical properties of each chemical modification will hopefully accelerate the CRISPR/Cas technology into the clinic, as well as further expand the use of RNAi to treat currently undruggable diseases. This review discusses the most commonly employed chemical modifications in RNAi reagents and CRISPR/Cas guide RNAs and provides an overview of select publications that have demonstrated success in improving ON efficacy and/or mitigating undesired OTEs.