CRISPR-LbCas12a-mediated modification of citrus

Area of Science:

• Plant biotechnology
• Genome editing
• Molecular biology

Background:

• CRISPR-Cas12a (Cpf1) systems have been engineered for plant genome modification.
• Citrus canker, caused by Xanthomonas citri, poses a significant threat to citrus production.
• The CsLOB1 gene, induced by PthA4, is a key susceptibility factor in citrus canker.

Purpose of the Study:

• To evaluate the efficacy of CRISPR-LbCas12a for editing the citrus genome.
• To target and modify the CsLOB1 gene, a susceptibility factor for citrus canker.
• To assess the potential of CRISPR-LbCas12a in developing disease-resistant citrus varieties.

Main Methods:

• Employed CRISPR-LbCas12a derived from Lachnospiraceae bacterium ND2006 for citrus genome editing.
• Utilized Xcc-facilitated agroinfiltration to deliver CRISPR-LbCas12a components into Duncan grapefruit.
• Targeted the CsPDS gene and the PthA4 effector binding elements (EBE_PthA4) in the CsLOB1 promoter using a single crRNA.

Main Results:

• Successfully modified the CsPDS gene in Duncan grapefruit.
• Achieved editing of the EBE_PthA4-CsLOB1 promoter in 35S-LbCas12a transformed plants.
• Transgenic line #D₃₅ s4 exhibited high mutation rates and alleviated XccΔpthA4:dCsLOB1.4 infection.
• No LbCas12a-mediated indels were observed in Yao-LbCas12a transformed plants.
• No potential off-target mutations were detected.

Conclusions:

• CRISPR-LbCas12a is a potent and effective tool for citrus genome editing.
• This technology can be applied to modify citrus genes, including those related to disease susceptibility.
• CRISPR-LbCas12a holds significant potential for advancing citrus breeding and disease management strategies.