Development of Plant Prime-Editing Systems for Precise Genome Editing

Area of Science:

• Molecular Biology
• Plant Biotechnology
• Genome Engineering

Background:

• Prime editing is a powerful genome editing technology for precise DNA modifications.
• Existing prime editing systems are primarily optimized for human cells.
• Developing efficient prime editing tools for plants is crucial for crop improvement.

Purpose of the Study:

• To develop and characterize a plant-adapted prime editor 2 (pPE2) system for rice.
• To evaluate the editing efficiency of pPE2 and optimized variants (pPE3, pPE3b) in rice.
• To enhance prime editing efficiency through a surrogate system for cell enrichment.

Main Methods:

• Development of a plant prime editor 2 (pPE2) system.
• Testing pPE2 activity by introducing targeted mutations in an HPT-ATG reporter in rice.
• Optimization using PE3 and PE3b strategies with modified guide RNAs.
• Creation of a surrogate pPE2 system with an HPT-ATG reporter for enriched screening.

Main Results:

• The pPE2 system demonstrated programmable editing at various rice genome sites.
• Editing frequencies in transgenic T0 plants ranged from 0% to 31.3%, varying by genomic site and guide RNA structure.
• pPE3 systems did not significantly improve editing frequencies compared to pPE2 at tested sites.
• The surrogate pPE2 system facilitated easier detection of nucleotide editing in enriched, resistant calli.

Conclusions:

• The developed plant prime editing systems offer versatile and flexible genome editing in rice.
• The pPE2 system is a viable tool for precise genetic modifications in plants.
• Further optimization may be needed to enhance editing efficiency across diverse genomic contexts.