חיפוש מתקדם
Plant Cell Reports
  • Manoj Kumar
  • Dana Ayzenshtat
    Adar Marko
  • Samuel Bocobza

While gene-editing methodologies, such as CRISPR–Cas9, have been developed and successfully used in many plant species, their use remains challenging, because they most often rely on stable or transient transgene expression. Regrettably, in all plant species, transformation causes epigenetic effects such as gene silencing and variable transgene expression. Here, UBIQUITIN10 promoters from several plant species were characterized and showed their capacity to direct high levels of transgene expression in transient and stable transformation assays, which in turn was used to improve the selection process of regenerated transformants. Furthermore, we compared various sgRNAs delivery systems and showed that the combination of UBIQUITIN10 promoters and tRNA–sgRNA complexes produced 80% mutant phenotype with a complete knockout of the four allelic copies, while the remaining 20% exhibited weaker phenotype, which suggested partial allelic knockout, in the T0 generation of the allotetraploid Nicotiana tabacum. These data provide valuable information to optimize future designs of gene editing constructs for plant research and crop improvement and open the way for valuable gene editing projects in non-model Solanaceae species.

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הספר "אוצר וולקני"
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תנאי שימוש
Optimization of T-DNA configuration with UBIQUITIN10 promoters and tRNA–sgRNA complexes promotes highly efficient genome editing in allotetraploid tobacco
41
  • Manoj Kumar
  • Dana Ayzenshtat
    Adar Marko
  • Samuel Bocobza
Optimization of T-DNA configuration with UBIQUITIN10 promoters and tRNA–sgRNA complexes promotes highly efficient genome editing in allotetraploid tobacco

While gene-editing methodologies, such as CRISPR–Cas9, have been developed and successfully used in many plant species, their use remains challenging, because they most often rely on stable or transient transgene expression. Regrettably, in all plant species, transformation causes epigenetic effects such as gene silencing and variable transgene expression. Here, UBIQUITIN10 promoters from several plant species were characterized and showed their capacity to direct high levels of transgene expression in transient and stable transformation assays, which in turn was used to improve the selection process of regenerated transformants. Furthermore, we compared various sgRNAs delivery systems and showed that the combination of UBIQUITIN10 promoters and tRNA–sgRNA complexes produced 80% mutant phenotype with a complete knockout of the four allelic copies, while the remaining 20% exhibited weaker phenotype, which suggested partial allelic knockout, in the T0 generation of the allotetraploid Nicotiana tabacum. These data provide valuable information to optimize future designs of gene editing constructs for plant research and crop improvement and open the way for valuable gene editing projects in non-model Solanaceae species.

Scientific Publication
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