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

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• RNA Structure and Function

Background:

• Prime-editing (PE) systems offer precise genome editing but their efficiency relies on guide RNA (pegRNA) stability and structure.
• Structured RNA motifs at the 3' end of pegRNAs are crucial for performance but remain understudied.

Purpose of the Study:

• To introduce PE-PRISM, a high-throughput screening platform for identifying and optimizing 3' RNA motifs in pegRNAs.
• To systematically evaluate diverse RNA motifs, including pseudoknots and G-quadruplexes, for enhancing PE efficiency.

Main Methods:

• Developed PE-PRISM, a pooled screening approach for analyzing thousands of 3' RNA motifs in human cells.
• Employed structure-guided mutagenesis and combinatorial screening to refine promising RNA motifs.
• Tested top-performing motifs, including engineered pseudoknots (tevo2.0, eHAV, eSBRMV1-A), against a panel of pathogenic variants.

Main Results:

• Evaluated 2,858 RNA motifs across four iterative libraries, identifying novel functional elements.
• Optimized motifs demonstrated superior PE efficiency compared to the standard tevopreQ1 motif for over 90% of tested pathogenic variants.
• Enhanced PE efficiency was confirmed in primary human cells and in vivo models (mouse brain and liver).

Conclusions:

• PE-PRISM is an effective tool for discovering and optimizing pegRNA 3' RNA motifs.
• Engineered RNA motifs significantly improve prime-editing efficiency and broaden its applicability for therapeutic variant correction.
• This work advances the engineering of RNA components to enhance gene editing technologies.