A Rapid Hairy Root-Based Platform for CRISPR/Cas Optimization and Guide RNA Validation in Lettuce

Area of Science:

• Plant genomics
• Functional genomics
• Crop improvement

Background:

• Lettuce (Lactuca sativa) is a key crop and model organism in the Asteraceae family.
• CRISPR/Cas technology holds promise for crop improvement, but requires optimized constructs and validated guide RNAs (gRNAs).
• A rapid system for evaluating CRISPR reagents in lettuce is currently unavailable.

Purpose of the Study:

• To develop an efficient hairy root-based system for optimizing CRISPR/Cas genome editing in lettuce.
• To evaluate different CRISPR construct configurations and promoters for enhanced editing efficiency.
• To demonstrate the system's utility by targeting the LsHB2 gene.

Main Methods:

• Developed and optimized a hairy root induction system using four Agrobacterium rhizogenes strains in two lettuce cultivars.
• Compared various CRISPR construct designs, including different promoters for nuclease and gRNA expression.
• Utilized the system to target the LsHB2 gene with SpCas9, SaCas9, and LbCas12a nucleases, followed by genotyping and indel profiling.

Main Results:

• Agrobacterium rhizogenes strain ATCC15834 proved most effective for hairy root induction.
• A plant-derived promoter combined with the At-pU6-26 variant significantly enhanced genome editing efficiency.
• The system facilitated rapid genotyping and quantitative indel profiling for targeted gene editing.

Conclusions:

• The developed hairy root system provides a robust framework for accelerating CRISPR/Cas genome editing optimization in lettuce.
• This platform enables efficient guide RNA selection and construct design for crop improvement.
• The findings contribute to advancing functional genomics and genetic engineering in Lactuca sativa.