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<p>Sugarcane (<i>Saccharum officinarum</i>), a high-biomass C<sub>4</sub> crop, possesses substantial potential for biofuel production. While first-generation biofuels are primarily derived from sucrose, the utilization of complex polysaccharides, such as cellulose and hemicellulose, remains limited due to lignin-induced recalcitrance. Lignin, a complex polymer composed of syringyl, guaiacyl, and hydroxyphenyl units, reinforces the plant cell wall and impedes enzymatic saccharification. Genetic engineering, particularly the CRISPR/Cas9 system, offers a powerful approach to modify lignin biosynthesis pathways. In this study, we aimed to knock out the <i>cinnamyl alcohol dehydrogenase</i> (CAD) gene, a key enzyme involved in monolignol biosynthesis, in the sugarcane variety Co 86,032. A guide RNA (gRNA) targeting the second class of the <i>CAD</i> gene was designed, cloned into the CRISPR/Cas9 vector pRGEB31, and introduced into <i>Agrobacterium tumefaciens</i> LBA4404 for sugarcane transformation. During shoot regeneration, approximately 90% of the transformed plants developed white, albino shoots. Sequence analysis revealed unintended partial homology between the designed CAD gRNA, upstream of the PAM sequence and the coding sequence of the <i>phytoene desaturase</i> (<i>PDS</i>) gene (at 609–616&#xa0;bp), a key gene involved in carotenoid biosynthesis. Real-time PCR analysis of albino plants showed markedly reduced <i>PDS</i> expression, further confirmed by PCR amplification. Chlorophyll a &amp; b, total carotenoid estimation also revealed significantly lower levels in albino plants, validating the off-target disruption of the <i>PDS</i> gene by the CAD-specific gRNA. These findings highlight the importance of precise gRNA design to minimize off-target effects and emphasize the need to redesign a highly specific gRNA for targeted editing of the <i>CAD</i> gene in sugarcane.</p>

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CRISPR/Cas9 Off-Target Effect Induces Albinism Via Phytoene Desaturase (PDS) Gene Disruption in Sugarcane

  • 期刊论文

  • 发表日期：

    2025年9月8日

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  • Lakshmi Kasirajan,
  • Rachel Lissy Vargheese,
  • Indusha Yazhini Sankararaj,
  • Akila Dharshini Venkatachalam

摘要

Sugarcane (Saccharum officinarum), a high-biomass C4 crop, possesses substantial potential for biofuel production. While first-generation biofuels are primarily derived from sucrose, the utilization of complex polysaccharides, such as cellulose and hemicellulose, remains limited due to lignin-induced recalcitrance. Lignin, a complex polymer composed of syringyl, guaiacyl, and hydroxyphenyl units, reinforces the plant cell wall and impedes enzymatic saccharification. Genetic engineering, particularly the CRISPR/Cas9 system, offers a powerful approach to modify lignin biosynthesis pathways. In this study, we aimed to knock out the cinnamyl alcohol dehydrogenase (CAD) gene, a key enzyme involved in monolignol biosynthesis, in the sugarcane variety Co 86,032. A guide RNA (gRNA) targeting the second class of the CAD gene was designed, cloned into the CRISPR/Cas9 vector pRGEB31, and introduced into Agrobacterium tumefaciens LBA4404 for sugarcane transformation. During shoot regeneration, approximately 90% of the transformed plants developed white, albino shoots. Sequence analysis revealed unintended partial homology between the designed CAD gRNA, upstream of the PAM sequence and the coding sequence of the phytoene desaturase (PDS) gene (at 609–616 bp), a key gene involved in carotenoid biosynthesis. Real-time PCR analysis of albino plants showed markedly reduced PDS expression, further confirmed by PCR amplification. Chlorophyll a & b, total carotenoid estimation also revealed significantly lower levels in albino plants, validating the off-target disruption of the PDS gene by the CAD-specific gRNA. These findings highlight the importance of precise gRNA design to minimize off-target effects and emphasize the need to redesign a highly specific gRNA for targeted editing of the CAD gene in sugarcane.