Development of Purity Analysis Methods for Synthetic Prime Editing Guide RNAs (pegrnas) with High-Level Secondary Structures for Prime Editing Research Applications

Prime editing (PE) is a novel variation on CRISPR systems that utilizes a Cas9 nickase fused to a reverse transcriptase and a prime editing guide RNA (pegRNA) to achieve high editing efficiency and precision. 1 However, the length requirement of pegRNAs at 120–250 nucleotides (nt) and the potential for secondary structures due to complementary or partially complementary sequences present analytical quality control (QC) challenges for purity analysis of chemically synthesized pegRNA molecules. Here, a method based on capillary gel electrophoresis (CGE) was developed to mitigate the technical challenges by thoroughly disrupting the hydrogen bonds in pegRNA molecules and maintaining their denatured state during analysis. This method can offer high resolution and provide purity quantification of these pegRNAs.