Selection of Optimal pegRNAs to Enhance Efficiency of Prime Editing in AT-Rich Genome Regions

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Olga V. Volodina^1,a*, Anna G. Demchenko¹, Arina A. Anuchina¹, Oxana P. Ryzhkova¹, Valeriia A. Kovalskaya¹, Ekaterina V. Kondrateva¹, Vyacheslav Y. Tabakov¹, Alexander V. Lavrov¹, and Svetlana A. Smirnikhina¹

¹DIV, Research Centre for Medical Genetics, 115522 Moscow, Russia

^* To whom correspondence should be addressed.

Received: December 27, 2024; Revised: March 18, 2025; Accepted: March 26, 2025

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Prime editing is a highly promising strategy for treating hereditary disorders due to its superior efficiency and safety profile compared to the conventional CRISPR-Cas9 systems. This study is dedicated to development of a causal therapy for cystic fibrosis by targeting the F508del variant of the CFTR gene using prime editing, as this specific deletion accounts for a substantial proportion of cystic fibrosis cases. While prime editing has shown remarkable precision in introducing targeted genetic modifications, its application in AT-rich genomic regions, such as the one containing the F508del variant, remains challenging. To overcome this limitation, we systematically evaluated 24 pegRNAs designed for two distinct prime editing systems, PEmax and PE2-NG. Efficiency of the F508del variant correction reached 2.81% (without normalization for transfection efficiency) in the airway basal cells from the patients with homozygous F508del mutation. However, the average transfection efficiency was only 11.9%, emphasizing critical need for the advancements in delivery methodologies. These findings highlight potential of prime editing as an approach for treating cystic fibrosis, while also underscoring necessity for further optimization of both editing constructs and delivery vectors to achieve clinically relevant correction levels.

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KEY WORDS: prime editing, CFTR, genome editing, pegRNA, F508del

DOI: 10.1134/S0006297924604672

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