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

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Summary

A new CRISPR-based tool, temperature-tolerant LbCas12a (ttLbCas12a), efficiently creates citrus plants resistant to citrus canker by precisely editing the LOB1 gene

Keywords:
LOB1Xanthomonascitruscitrus cancergenome editingttLbCas12a

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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.