Abstract
Sweet orange (Citrus sinensis) is the most economically important species for the citrus industry. However, it is susceptible to many diseases including citrus bacterial canker caused by Xanthomonas citri subsp. citri (Xcc) that triggers devastating effects on citrus production. Conventional breeding has not met the challenge to improve disease resistance of sweet orange due to the long juvenility and other limitations. CRISPR-mediated genome editing has shown promising potentials for genetic improvements of plants. Generation of biallelic/homozygous mutants remains difficult for sweet orange due to low transformation rate, existence of heterozygous alleles for target genes, and low biallelic editing efficacy using the CRISPR technology. Here, we report improvements in the CRISPR/Cas9 system for citrus gene editing. Based on the improvements we made previously [dicot codon optimized Cas9, tRNA for multiplexing, a modified sgRNA scaffold with high efficiency, citrus U6 (CsU6) to drive sgRNA expression], we further improved our CRISPR/Cas9 system by choosing superior promoters [Cestrum yellow leaf curling virus (CmYLCV) or Citrus sinensis ubiquitin (CsUbi) promoter] to drive Cas9 and optimizing culture temperature. This system was able to generate a biallelic mutation rate of up to 89% for Carrizo citrange and 79% for Hamlin sweet orange. Consequently, this system was used to generate canker-resistant Hamlin sweet orange by mutating the effector binding element (EBE) of canker susceptibility gene CsLOB1, which is required for causing canker symptoms by Xcc. Six biallelic Hamlin sweet orange mutant lines in the EBE were generated. The biallelic mutants are resistant to Xcc. Biallelic mutation of the EBE region abolishes the induction of CsLOB1 by Xcc. This study represents a significant improvement in sweet orange gene editing efficacy and generating disease-resistant varieties via CRISPR-mediated genome editing. This improvement in citrus genome editing makes genetic studies and manipulations of sweet orange more feasible.
Highlights
Citrus is one of the most important fruit crops worldwide because of its delightful flavor and scent, as well as its health properties
Citrus production is facing many biotic and abiotic challenges (Li, 2009). Citrus diseases such as citrus canker caused by Xanthomonas citri subsp. citri (Xcc) (Ference et al, 2018) and Huanglongbing (HLB, known as citrus greening) caused by Candidatus Liberibacter asiaticus (Bové, 2006; Wang, 2019; Yuan et al, 2020) are causing devastating effects on the citrus industry
It has been suggested that CRISPR-mediated genome editing will revolutionize the genetic improvements of citrus and other tree crops (Dutt et al, 2020; Wheatley and Yang, 2020)
Summary
Citrus is one of the most important fruit crops worldwide because of its delightful flavor and scent, as well as its health properties. Sweet orange (Citrus sinensis) is the most economically important species for the citrus industry. Citrus diseases such as citrus canker caused by Xanthomonas citri subsp. Citri (Xcc) (Ference et al, 2018) and Huanglongbing (HLB, known as citrus greening) caused by Candidatus Liberibacter asiaticus (Bové, 2006; Wang, 2019; Yuan et al, 2020) are causing devastating effects on the citrus industry. Most elite citrus varieties, including sweet orange and grapefruit varieties, are susceptible to canker and HLB disease. Genetic improvements of citrus via conventional approaches are challenging. It has been suggested that CRISPR-mediated genome editing will revolutionize the genetic improvements of citrus and other tree crops (Dutt et al, 2020; Wheatley and Yang, 2020)
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