Cas12a RNP-mediated co-transformation enables transgene-free multiplex genome editing, long deletions, and inversions in citrus chromosome.

INTRODUCTION

Citrus canker, caused by subsp. citri (), is a devastating disease worldwide. Previously, we successfully generated canker-resistant cv. Hamlin lines in the T0 generation. This was achieved through the transformation of embryogenic protoplasts using the ribonucleoprotein (RNP) containing Cas12a and one crRNA to edit the canker susceptibility gene, , which led to small indels.

METHODS

Here, we transformed embryogenic protoplasts of Hamlin with RNP containing Cas12a and three crRNAs.

RESULTS

Among the 10 transgene-free genome-edited lines, long deletions were obtained in five lines. Additionally, inversions were observed in three of the five edited lines with long deletions, but not in any edited lines with short indel mutations, suggesting long deletions maybe required for inversions. Biallelic mutations were observed for each of the three target sites in four of the 10 edited lines when three crRNAs were used, demonstrating that transformation of embryogenic citrus protoplasts with Cas12a and three crRNAs RNP can be very efficient for multiplex editing. Our analysis revealed the absence of off-target mutations in the edited lines. These mutant lines were canker- resistant and no canker symptoms were observed after inoculation with and growth was significantly reduced in the mutant lines compared to the wild type plants.

DISCUSSION

Taken together, RNP (Cas12a and three crRNAs) transformation of embryogenic protoplasts of citrus provides a promising solution for transgene-free multiplex genome editing with high efficiency and for deletion of long fragments.