LbCas12a-D156R Efficiently Edits LOB1 Effector Binding Elements to Generate Canker-Resistant Citrus Plants

Area of Science:

• Plant pathology
• Molecular genetics
• Biotechnology

Background:

• Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a major economic threat to citrus production globally.
• The Xcc effector PthA4 activates the LOB1 gene via effector binding elements (EBEs), leading to canker development.
• Previous Cas9/gRNA editing of EBEs resulted in small indels, potentially allowing pathogen adaptation.

Purpose of the Study:

• To utilize a more efficient CRISPR-Cas12a system for precise genome editing in citrus.
• To develop citrus plants with enhanced resistance to citrus canker by targeting the LOB1 gene's EBE.
• To assess the efficacy and specificity of the ttLbCas12a system in generating stable, resistant citrus lines.

Main Methods:

• Employed a temperature-tolerant LbCas12a variant (ttLbCas12a) for editing the LOB1 EBE region in Pummelo (Citrus maxima).
• Constructed and validated a functional ttLbCas12a:LOBP expression vector via agroinfiltration.
• Generated and analyzed stable transgenic Pummelo lines for EBE mutations, deletions, and canker resistance.

Main Results:

• Successfully generated eight transgenic Pummelo lines expressing ttLbCas12a:LOBP.
• Seven lines exhibited 100% EBE mutation, with one line being homozygous for the edits.
• ttLbCas12a mediated deletions up to 10 bp, conferring significant canker resistance without detectable off-target mutations.

Conclusions:

• The ttLbCas12a system efficiently generates biallelic and homozygous citrus mutants with substantial EBE deletions.
• This approach provides a robust tool for functional genomics studies and breeding of citrus varieties resistant to citrus canker.
• ttLbCas12a offers an improved method for generating durable disease resistance in citrus compared to previous Cas9-based strategies.