Efficient generation of transgene-free canker-resistantCitrus sinensisusing Cas12a/crRNA ribonucleoprotein

Abstract

Citrus is one of the top three tree crops in the world and its production faces many devastating disease challenges such as citrus canker caused by Xanthomonas citri subsp. citri (Xcc). Genetic improvement of citrus via traditional approaches is a lengthy (approximately 20 years) and laborious process. Biotechnological approaches including CRISPR genome editing technologies have shown promise. However, none of the citrus plants generated by biotechnological approaches have been commercialized, which primarily resulted from the transgenic nature of the genetically modified plants. Here, we successfully developed transgene-free canker-resistant Citrus sinensis lines in the T0 generation within 10 months through transformation of embryogenic citrus protoplasts with Cas12a/crRNA ribonucleoprotein (RNP) to edit the canker susceptibility gene CsLOB1. Among the 39 regenerated lines, 38 are biallelic/homozygous mutants based on Sanger sequencing analysis of targeting sites and whole genome sequencing, demonstrating a 97.4% biallelic/homozygous mutation rate. The edited lines do not contain off-target mutations. The CsLOB1 edited C. sinensis lines demonstrate no differences from wild type plants except canker resistance. Importantly, the transgene-free canker-resistant C. sinensis lines have received regulatory approval by USDA APHIS. This study presents an efficient genome editing approach for citrus using Cas12a/crRNA RNP, which has a broad impact on genetic improvement of elite citrus varieties and potentially other tree crops and their genetic study. This study represents a breakthrough by generating the first transgene-free canker-resistant C. sinensis lines that provide a sustainable and efficient solution to control citrus canker.

Mechanisms underneathXanthomonas citrisubsp.citripromoting ofAgrobacterium-mediated transient expression in citrus

“…Intriguingly, the plant cell wall degrading enzymes encoded by Xcc has neglectable effect on its promotion of Agrobacterium transformation as demonstrated by the xpsD mutant. Xcc activation of plant cell wall degrading enzymes is through PthA-LOB1 (de Souza-Neto et al ., 2023; Su et al ., 2023; Zhang et al ., 2017; Zou et al ., 2021) which likely contributes to the loss of the promoting effect of the pthA4 mutant on Agrobacterium -mediated TE.…”

Mind the (CRISPR) gaps

“…This is especially relevant for introducing into elite cultivars traits that are not present in the wild germplasm, as well as for improving so-called orphan crops by de novo introduction of key domestication traits. CRISPR significantly accelerates the genetic improvement of various crops, such as, for example, Citrus, where its application enabled the generation of canker-resistant lines in less than a year (Su et al, 2023).…”

Generation of transgene-free canker-resistant Citrus sinensis cv. Hamlin in the T0 generation through Cas12a/CBE co-editing

“…CRISPR/Cas mediated genome editing is deemed the most promising approach to breed new citrus cultivars, owing to its short time requirement, precise genetic improvement and predictable results (Chen et al, 2019;Gao, 2021;Cao et al, 2022;Huang et al, 2022a). To date, SpCas9/gRNA from Streptococcus pyogenes, SaCas9/gRNA from Staphylococcus aureus, LbCas12a/crRNA from Lachnospiraceae bacterium and base editor derived from SpCas9/gRNA have been successfully adapted to modify citrus genomes for gene function study and new citrus cultivar breeding (Jia and Wang, 2014a;Jia et al, 2016;Peng et al, 2017;Zhang et al, 2017;Jia et al, 2017a, Jia et al, 2017bLeBlanc et al, 2018;Zhu et al, 2019;Jia et al, 2019a;Dutt et al, 2020;Huang et al, 2020;Jia and Wang, 2020;Huang and Wang, 2021;Jia et al, 2021;Alquezar et al, 2022;Jia et al, 2022;Mahmoud et al, 2022;Parajuli et al, 2022;Yang et al, 2022;Huang et al, 2022bHuang et al, , 2023Su et al, 2023).…”

“…Multiple strategies have been developed to produce transgene-free plants after target-gene editing with CRISPR/Cas (Kocsisova and Coneva, 2023). Some examples include delivering DNA-free gene editing reagents such as ribonucleoproteins (Woo et al, 2015;Malnoy et al, 2016;Subburaj et al, 2016;Svitashev et al, 2016;Liang et al, 2017;Andersson et al, 2018;Su et al, 2023), novel delivery vectors such as viruses (Mei et al, 2019;Ma et al, 2020;Liu et al, 2023), unconventional selection methods to bypass integration of transgenes (Veillet et al, 2019;Alquezar et al, 2022;Huang et al, 2022bHuang et al, , 2023Wei et al, 2023), graft-mobile editing systems (Yang et al, 2023), and so on. Initially, Alquezar et al and Huang et al independently developed transgene-free citrus using CBE-mediated editing (Alquezar et al, 2022;Huang et al, 2022b).…”