Directed evolution of small RNA-stabilizing motifs that improve prime-editing efficiency

The performance of prime-editing (PE) systems has been improved by systematic engineering of their protein and small RNA components but the structured RNA motifs appended to the 3' end of PE guide RNAs (pegRNAs)-a key determinant of pegRNA stability and editing efficiency-have not been extensively studied. We introduce PE-PRISM, a high-throughput pooled screen to identify and optimize these 3' RNA motifs in human cells. Here, using PE-PRISM, we evaluated 2,858 RNA motifs across four iterative libraries, including natural and engineered pseudoknots, G-quadruplexes and reverse transcriptase recruitment elements. We applied structure-guided mutagenesis and combinatorial variant screening to refine hits, culminating in the engineered and evolved pseudoknot variants tevo2.0, eHAV and eSBRMV1-A. In a screen correcting 847 pathogenic ClinVar variants, the top-performing motifs improved PE efficiency over the widely used tevopreQ₁ motif for >90% of edits. They also increased PE efficiencies for correcting disease-associated mutations in primary human cells and in vivo in mouse brain and liver.