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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 offtarget 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 exonucleasemediated degradation.

A two-step gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE) procedure was devised for the detection of proteinases and the study of proteinase/inhibitor interactions in complex biological extracts. The proteins are first resolved by sodium dodecyl sulfate (SDS)-PAGE under reducing or nonreducing conditions, and electrotransferred into a 0.75 mm-thick accompanying polyacrylamide slab gel containing 0.1% w/v porcine gelatin. The active proteinase bands are developed by a gelatin proteolysis step in the accompanying gel in the presence or absence of diagnostic proteinase inhibitors, allowing the assessment of proteinase classes and the visual discrimination of inhibitor-'sensitive' and -'insensitive' proteinases in complex extracts. Alternatively, protein extracts are preincubated with specific reversible inhibitors before electrophoresis, allowing a rapid discrimination of strong and weak interactions implicating proteinases and reversible inhibitors. In comparison with the standard gelatin/PAGE procedure, that involves copolymerization of gelatin with acrylamide in the resolving gel, this new procedure simplifies proteinase patterns, avoids overestimation of proteinase numbers in complex extracts, and allows in certain conditions the estimation of proteinase molecular weights. Stem bromelain (EC 3.4.22.32), bovine trypsin (EC 3.4.21.4), papain (EC 3.4.22.2), and the extracellular (digestive) cysteine proteinases of five herbivorous pests are used as model enzymes to illustrate the usefulness of this approach in detecting proteinases and in studying their interactions with specific proteinaceous inhibitors potentially useful in biotechnology.