biostudies-literatureYan JNIDDK NIH HHSNHLBI NIH HHSNHGRI NIH HHSNCI NIH HHSNIGMS NIH HHS639-647https://www.ebi.ac.uk/biostudies/studies/S-EPMC11023932biostudies-literatureUnknown628(8008)Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs¹. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts². We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.NatureImproving prime editing with an endogenous small RNA-binding protein.PMC11023932T32 HG003284K00 CA245718T32 GM007388UM1 HG012660U54 DK106829OT2 CA278665R01 HL150669R35 GM138167DP2 CA239597RM1 HG009490Gilbert LALi HYan JParsons LROyler-Castrillo PSolley SCZhao ASimpson DRavisankar PGoudy LBauer DEChan MMYan WLevesque SJing YSchmidt RWard CCMarson AAdamson BfalseImproving prime editing with an endogenous small RNA-binding protein.Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs¹. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts². We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.2024-01-01T00:00:00Z2024 Apr2026-03-16T09:47:48.387Z2025-08-15T03:07:07.434ZS-EPMC110239323857069110.1038/s41586-024-07259-6