Efficient Targeted Mutagenesis Mediated by CRISPR-Cas12a Ribonucleoprotein Complexes in Maize.

Area of Science:

• Plant biotechnology
• Genome editing
• Crop improvement

Background:

• CRISPR-Cas genome editing enables targeted gene modifications in crops.
• CRISPR-Cas12a shows lower editing efficiency from transgenes in maize and soybean.
• Efficient delivery of CRISPR-Cas12a is crucial for crop genetic improvement.

Purpose of the Study:

• To develop efficient CRISPR-Cas12a genome editing methods for maize.
• To improve gene editing efficiency in an elite maize variety using Cas12a ribonucleoprotein complexes (RNPs).
• To establish selection methods for enhancing CRISPR-Cas12a mediated editing.

Main Methods:

• Delivery of Cas12a-gRNA RNP complexes into immature maize embryos via particle bombardment.
• Co-delivery of a PMI selectable marker gene with Cas12a RNP and selection with mannose.
• Utilizing higher activity Cas12a mutants for recalcitrant target sequences.

Main Results:

• Genome edited maize lines were generated at ~7% frequency using biolistic delivery of Cas12a RNP without selection.
• Gene editing rates increased to 60% on average and up to 100% with mannose selection.
• Higher activity Cas12a mutants improved editing efficiency in challenging target sequences.

Conclusions:

• Directly delivering Cas12a RNP complexes via particle bombardment is an effective strategy for maize genome editing.
• Mannose selection significantly enhances CRISPR-Cas12a editing efficiency in maize.
• These advancements provide valuable tools for the genetic improvement of maize.