Delivery of CRISPR-Cas12a Ribonucleoprotein Complex for Genome Editing in an Embryogenic Citrus Cell Line.

Area of Science:

• Plant Biotechnology
• Genome Editing
• Molecular Biology

Background:

• Clustered regularly interspaced short palindromic repeats (CRISPR) technology is a transformative tool for genome editing.
• The CRISPR-Cas12a system presents advantages over CRISPR-Cas9 for plant applications.
• Traditional plasmid delivery methods in plants raise concerns regarding transgene integration and off-target mutations.

Purpose of the Study:

• To present a detailed protocol for LbCas12a-mediated genome editing in Citrus protoplasts.
• To demonstrate the efficacy of delivering CRISPR-Cas12a components as ribonucleoproteins (RNPs).
• To provide guidelines for assessing genome editing efficiency in citrus.

Main Methods:

• Preparation of CRISPR-Cas12a ribonucleoprotein (RNP) components.
• Assembly and delivery of LbCas12a-RNP complexes into Citrus protoplasts.
• Assessment of genome editing efficiency using molecular techniques.

Main Results:

• Successful application of LbCas12a for genome editing in Citrus protoplasts via RNP delivery.
• Demonstration of RNP delivery as a viable alternative to plasmid-based methods.
• Establishment of a comprehensive protocol for RNP-mediated genome editing in citrus.

Conclusions:

• CRISPR-Cas12a RNP delivery is an effective strategy for genome editing in Citrus protoplasts.
• This method mitigates issues associated with transgene integration and off-target effects.
• The protocol facilitates advancements in citrus breeding and crop improvement.