Improving prime editing with an endogenous small RNA-binding protein

Jun Yan, Paul Oyler-Castrillo, Purnima Ravisankar, Carl C. Ward, Sébastien Levesque, Yangwode Jing, Danny Simpson, Anqi Zhao, Hui Li, Weihao Yan, Laine Goudy, Ralf Schmidt, Sabrina C. Solley, Luke A. Gilbert, Michelle M. Chan, Daniel E. Bauer, Alexander Marson, Lance R. Parsons and Britt Adamson ()
Additional contact information
Jun Yan: Princeton University
Paul Oyler-Castrillo: Princeton University
Purnima Ravisankar: Princeton University
Carl C. Ward: Gladstone–UCSF Institute of Genomic Immunology
Sébastien Levesque: Boston Children’s Hospital
Yangwode Jing: Princeton University
Danny Simpson: Princeton University
Anqi Zhao: Princeton University
Hui Li: Princeton University
Weihao Yan: Princeton University
Laine Goudy: Gladstone–UCSF Institute of Genomic Immunology
Ralf Schmidt: Gladstone–UCSF Institute of Genomic Immunology
Sabrina C. Solley: Princeton University
Luke A. Gilbert: Arc Institute
Michelle M. Chan: Princeton University
Daniel E. Bauer: Boston Children’s Hospital
Alexander Marson: Gladstone–UCSF Institute of Genomic Immunology
Lance R. Parsons: Princeton University
Britt Adamson: Princeton University

Nature, 2024, vol. 628, issue 8008, 639-647

Abstract: Abstract 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.

Date: 2024
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DOI: 10.1038/s41586-024-07259-6

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