Improving prime editing with an endogenous small RNA-binding protein
Prime editing allows precise modification of genomes. 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 (SSB). Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4, PE5), edit types (substitutions, insertions, deletions), guide RNA designs (pegRNAs, epegRNAs), endogenous loci, and cell types, but has no consistent effect on genome editing approaches that rely on standard, unextended guide RNAs. La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts and protects those transcripts from cellular exonucleases. We found that La stabilizes (e)pegRNAs, with particular effect on their 3' extensions, where edits are encoded. Guided by these insights, we developed a prime editing protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed (e)pegRNAs and synthetic pegRNAs optimized for La binding. 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. Overall design: Primary human T cells were electroporated with PE7 or PEmax mRNA and synthetic pegRNAs with different 3' end configurations. Related to GSE253424.