Comprehensive analysis of prime editing outcomes in human embryonic stem cells

TLDR

Prime editing is a precise genome‑editing technique that copies desired modifications into DNA without donor templates, offering promise for gene function studies, disease modeling, and correcting pathogenic mutations in human pluripotent stem cells. The study aimed to comprehensively test prime editing in human pluripotent stem cells. The authors created a doxycycline‑inducible prime editing platform to conduct these tests. Prime editing in hPSCs induced all substitution and small indel types, matched base‑editing performance in correcting an α‑1‑antitrypsin mutation, and whole‑genome sequencing revealed no guide‑RNA‑independent off‑target mutations, demonstrating its potential to complement existing CRISPR tools.

Abstract

Prime editing is a versatile and precise genome editing technique that can directly copy desired genetic modifications into target DNA sites without the need for donor DNA. This technique holds great promise for the analysis of gene function, disease modeling, and the correction of pathogenic mutations in clinically relevant cells such as human pluripotent stem cells (hPSCs). Here, we comprehensively tested prime editing in hPSCs by generating a doxycycline-inducible prime editing platform. Prime editing successfully induced all types of nucleotide substitutions and small insertions and deletions, similar to observations in other human cell types. Moreover, we compared prime editing and base editing for correcting a disease-related mutation in induced pluripotent stem cells derived form a patient with α 1-antitrypsin (A1AT) deficiency. Finally, whole-genome sequencing showed that, unlike the cytidine deaminase domain of cytosine base editors, the reverse transcriptase domain of a prime editor does not lead to guide RNA-independent off-target mutations in the genome. Our results demonstrate that prime editing in hPSCs has great potential for complementing previously developed CRISPR genome editing tools.