Using CRISPR/Cas9 to Obtain Sugarcane with Low Lignin Content

Sugarcane is the most important source of raw material for bioethanol production. Its biomass can be used for bioethanol production. In bioethanol production, pretreatment is a costly step, and sugarcane feedstock with low lignin content can reduce the need for pretreatment and reduce production costs. Sugarcane lignin content can be reduced through molecular breeding methods such as RNAi, TALENs and CRISPR/Cas9. CRISPR/Cas9 is considered an effective tool for gene editing in plants with complex genomes such as sugarcane due to its flexibility and high efficiency. The transcription factor LIM can regulate the expression of lignin biosynthetic pathway genes. Lignin content can be reduced by inhibiting the expression of LIM in tobacco, eucalyptus and cotton.

On January 13, 2024, Sontichai Chanprame's team at the Kasetsart University published an article titled "Lignin reduction in sugarcane by performing CRISPR/Cas9 site-direct mutation of SoLIM transcription factor" in Plant Science. This study used CRISPR/Cas9 technology to successfully down-regulate the expression of SoLIM, a key transcription factor for lignin synthesis in sugarcane, thereby obtaining a sugarcane strain with lower lignin content. This is of great significance for improving the ethanol conversion efficiency of sugarcane biomass.

The authors first cloned and sequenced the transcription factor gene SoLIM from sugarcane variety KK3. The results showed that the SoLIM gene contains 5 exons and 4 introns. SoLIM cDNA is 603 bp in length, encoding a peptide chain containing 200 amino acids, and has high similarity with the LIM genes of sorghum, millet, wheat and other plants.

The author designed the sgRNA sequence based on the SoLIM gene, transformed the expression vector into sugarcane callus through Agrobacterium-mediated method, and obtained transgenic sugarcane plants containing different sgRNA. Analysis of the edited sugarcane plants showed that the expression level of SoLIM in the edited sugarcane plants was lower than that of the wild type. In plants with insertion or deletion mutations, the expressions of SoPAL, SoC4H and SoCAD were also significantly reduced. This indicates that SoLIM is a transcription factor that regulates the lignin biosynthetic pathway, and its expression inhibition can reduce the expression of subsequent genes.

Next, the authors measured the lignin, cellulose and hemicellulose contents. The results showed that compared with the wild type, the lignin content of the edited positive plants decreased by 9.74-51.46%, with the lignin in lines 2-5 decreasing the most. And while the lignin content decreased, the cellulose content increased. The results of hemicellulose content are not uniform, with lines 2-20 having the highest hemicellulose content. The results showed that the decrease in lignin content was associated with the increase in cellulose and hemicellulose content.

Through LC-MS analysis, it was found that the content of S-type monomeric lignin in the edited positive plants increased and the S/G ratio increased. Histochemical analysis also showed an increase in S-type lignin in the edited samples. SEM results showed that the cell wall thickness of the edited sample was reduced compared with the wild type. The above results confirm that editing SoLIM can effectively reduce sugarcane lignin content.

Fig. 1. The cell wall thickness of the edited sample was reduced compared with the wild type. (Laksana, C., et al. 2024)

This study successfully used CRISPR/Cas9 technology to reduce the lignin content in sugarcane for the first time. Three gRNAs targeting SoLIM were designed, a CRISPR/Cas9 expression vector was constructed, and the edited cell line was obtained through Agrobacterium transformation. Downregulation of SoLIM can reduce the expression of SoPAL, SoC4H and SoCAD, leading to a decrease in lignin content. The lignin content in the edited strain obtained was reduced by up to 51.46%, and the S/G ratio was increased by 2 times. Low-lignin sugarcane will facilitate the production of second-generation bioethanol, reducing pretreatment requirements and production costs. CRISPR/Cas9 technology demonstrates the potential for precise gene editing in crops with complex genomes such as sugarcane. This study lays the foundation for using gene editing to obtain low-lignin sugarcane suitable for biofuel production.

Reference

1. Laksana, C., et al. Lignin reduction in sugarcane by performing CRISPR/Cas9 site-direct mutation of SoLIM transcription factor. Plant Sci. 2024, 340: 111987.

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