Skip to main content
ARS Home » Research » Publications at this Location » Publication #402790
Research Project: Genetic Improvement of Citrus for Enhanced Resistance to Huanglongbing Disease and Other Stresses

Location: Location not imported yet.

Title: Delivery of CRISPR-Cas12a ribonucleoprotein complex for genome editing in an embryogenic citrus cell line

Author
item FONG, HONG - UNIVERSITY OF MARYLAND
item CULVER, JAMES - UNIVERSITY OF MARYLAND
item Niedz, Randall
item QI, YIPING - UNIVERSITY OF MARYLAND

Submitted to: Methods in Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/24/2023
Publication Date: 3/1/2023
Citation: Fong, H., Culver, J.N., Niedz, R.P., Qi, Y. 2023. Delivery of CRISPR-Cas12a ribonucleoprotein complex for genome editing in an embryogenic citrus cell line. Methods in Molecular Biology. https://doi.org/10.1007/978-1-0716-3131-7_10.
DOI: https://doi.org/10.1007/978-1-0716-3131-7_10

Interpretive Summary: CRISPR technology is a powerful tool that allows scientists to make precise changes to an organism's DNA. This is important because DNA determines how an organism looks and functions, so by changing it, scientists can improve things like crop yields, disease resistance, and nutritional value. The newer CRISPR-Cas12a system is better than the old one for making these changes, especially in plants. Traditional methods of changing plant DNA can be unpredictable and may have unintended consequences but using CRISPR-Cas12a delivered as ribonucleoproteins (RNPs) can help avoid these problems. This protocol provides a detailed guide for how to use this method to make changes to Citrus plants, which could help farmers grow crops that are more resilient, healthier, and better suited to meet the world's food needs.

Technical Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR) technology is a powerful genome 6 editing tool. Recently developed CRISPR-Cas12a system confers several advantages over CRISPR-Cas9, 7 making it ideal for use in plant genome editing and crop improvement. While traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects, CRISPR-Cas12a delivered as ribonucleoproteins (RNPs) can effectively alleviate these potential issues. Here we present a detailed protocol for LbCas12a-mediated genome editing using RNP delivery in Citrus protoplasts. This protocol provides a comprehensive guideline for RNP component preparation, RNP complex assembly and delivery, and editing efficiency assessment.