Targeted multiplex gene knockouts in Lemna minor using CRISPR/Cas9

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genomics

Background:

• Lemna minor (duckweed) is a valuable aquatic plant for recombinant protein production due to its rapid growth and high yield.
• Developing advanced genetic tools, including CRISPR/Cas9, is crucial for maximizing duckweed's biotechnological potential.
• The polycistronic tRNA-sgRNA (PTG)/Cas9 system offers a promising approach for multiplex gene editing in plants.

Purpose of the Study:

• To investigate the efficacy of the PTG/Cas9 system for multiplex genome editing in Lemna minor.
• To simultaneously target and modify two plant-specific glycosyltransferase genes (FucT and XylT) in L. minor.

Main Methods:

• Engineered a PTG construct with four sgRNAs targeting α-1,3-fucosyltransferase (FucT) and β-1,2-xylosyltransferase (XylT) genes.
• Introduced the PTG/Cas9 system into L. minor via callus transformation.
• Analyzed gene editing outcomes using PCR, RT-PCR, sequencing, and Western blot analysis.

Main Results:

• Successfully induced insertions and deletions (indels) at the target FucT and XylT loci in regenerated L. minor plants.
• Confirmed successful multiplex editing through sequencing and RT-PCR analysis.
• Western blot analysis revealed truncated XylT proteins and differential FucT expression in homozygous edited lines.

Conclusions:

• This study represents the first successful application of the PTG/Cas9 system for multiplex genome editing in Lemna minor.
• The findings validate the PTG/Cas9 system's capability for efficient multiplex editing in duckweed.
• This advancement opens new avenues for sophisticated genetic engineering of L. minor for biotechnological applications.