AUTHOR=Nishizawa-Yokoi Ayako , Iida Keiko , Mori Akiko , Toki Seiichi 

TITLE=Prime editing using paired pegRNAs targeting NG- or NGG-PAM in rice

JOURNAL=Frontiers in Genome Editing

VOLUME=Volume 7 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/genome-editing/articles/10.3389/fgeed.2025.1550308

DOI=10.3389/fgeed.2025.1550308

ISSN=2673-3439

ABSTRACT=Prime editing (PE) enables precise genome modification, i.e., all 12 types of base substitution, as well as designed insertion and deletion. Previously, we developed an efficient PE system using a pair of engineered pegRNAs (epegRNAs), appending an RNA pseudoknot sequence to the 3′ends of pegRNAs to enhance stability and prevent degradation of the 3′extension. Using a wild-type nSpCas9-based PE system (PE-wt) recognizing an NGG-protospacer adjacent motif (PAM) in this approach, two NGG-PAMs (NGG and CCN) adjacent to the target site are required for targeting by paired pegRNAs; however, this is not the PAM configuration available at most target sites. Using an nSpCas9-NG variant recognizing NG-PAM in PE (PE-NG) can expand applicability. Here, we compare the PE efficiency of PE-wt with paired epegRNAs targeting a distal NGG-PAM versus PE-NG with paired epegRNAs targeting NG-PAMs adjacent to the target site. By introducing substitution and designated deletion mutations into target genes via PE-wt and PE-NG with paired epegRNAs, we demonstrated that PE-wt could edit the target site efficiently despite targeting the distal PAM site when either of the paired epegRNAs for PE-NG targets PGC-PAM. If epegRNAs for PE-NG are designed to recognize NGA and NGT-PAM, there is no significant difference in frequency between PE-NG and PE-wt. These findings indicate that PE efficiency via PE-NG is particularly low at the NGC-PAM in rice.