Improving 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 RNAs. 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 transcripts. 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. To study how La affects (e)pegRNAs, we transfected either targeting or nontargeting (e)pegRNA plasmids into K562 PEmax parental cells and La-knockout clonal cell line 4, constructed small RNA libraries from total RNA and performed paired-end sequencing. To study if PE7 affects gene expression as compared to PEmax or PE7 mutant, we transfected K562 cells with plasmids expressing PEmax, PE7 or PE7 mutant and pegRNA plasmid encoding HEK3 1TtoA or PRNP 6GtoT, constructed mRNA libraries from total RNA and performed single end sequencing. Related to GSE255003.