Improving prime editing with an endogenous small RNA-binding protein

• 成果类型: Article
• 署名作者: Yan, Jun; Oyler-Castrillo, Paul; Ravisankar, Purnima; Ward, Carl C.; Levesque, Sebastien; Jing, Yangwode; Simpson, Danny; Zhao, Anqi; Li, Hui; Yan, Weihao; Goudy, Laine; Schmidt, Ralf; Solley, Sabrina C.; Gilbert, Luke A.; Chan, Michelle M.; Bauer, Daniel E.; Marson, Alexander; Parsons, Lance R.; Adamson, Britt
• 署名单位: Princeton University; Princeton University; Harvard University; Harvard University Medical Affiliates; Boston Children's Hospital; Harvard University; Harvard University Medical Affiliates; Dana-Farber Cancer Institute; Harvard University; Harvard University; Massachusetts Institute of Technology (MIT); Broad Institute; Harvard University; Harvard Medical School; Princeton University; University of California System; University of California San Francisco; Medical University of Vienna; University of California System; University of California San Francisco; University of California System; University of California San Francisco; UCSF Medical Center; UCSF Helen Diller Family Comprehensive Cancer Center; University of California System; University of California Berkeley; University of California System; University of California San Francisco; Cornell University
• 刊物名称: Nature
• ISSN/ISSBN: 0028-4527
• DOI: 10.1038/s41586-024-07259-6
• 发表日期: 2024-04-18
• 页码: 639-647
• 关键词: human-cells; la; transcription; expression; antigen

摘要

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 polymeraseIII transcripts(2). 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.