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

Area of Science:

• Molecular Biology
• Genome Engineering
• Biotechnology

Background:

• Prime editing (PE) facilitates precise genome modifications, including base substitutions, insertions, and deletions.
• Engineered pegRNAs (epegRNAs) with RNA pseudoknot sequences enhance PE system stability.
• Existing PE systems often require specific protospacer adjacent motif (PAM) configurations that limit target site accessibility.

Purpose of the Study:

• To compare the efficiency of a wild-type SpCas9-based PE system (PE-wt) and an nSpCas9-NG variant-based PE system (PE-NG) using paired epegRNAs.
• To evaluate PE efficiency across different PAM configurations (NGG, NG) and target site distances.
• To assess the applicability of PE-NG in rice, particularly at NGC-PAM sites.

Main Methods:

• Utilized PE-wt with paired epegRNAs targeting a distal NGG-PAM.
• Employed PE-NG with paired epegRNAs targeting adjacent NG-PAMs.
• Introduced substitution and deletion mutations in target genes using both PE systems.
• Compared editing frequencies between PE-wt and PE-NG across various PAM types (NGG, CCN, NGA, NGT, NGC).

Main Results:

• PE-wt demonstrated efficient genome editing at distal NGG-PAM sites.
• PE-NG showed comparable efficiency to PE-wt when targeting NGA and NGT-PAM sites.
• PE-NG exhibited significantly lower editing efficiency at NGC-PAM sites in rice compared to PE-wt.

Conclusions:

• PE-wt is effective for genome editing even when targeting distal PAM sites.
• PE-NG expands PE applicability but shows reduced efficiency at specific NGC-PAM sites in rice.
• The choice of PE system and PAM compatibility is crucial for optimizing genome editing strategies in plants.