Tree Genetics and Molecular Breeding 2024, Vol.14, No.1, 32-42 http://genbreedpublisher.com/index.php/tgmb 38 4.3 Case study on gene activation Gene activation using CRISPR/Cas9 has also been explored in poplar species. In one study, the CRISPR/Cas9 system was employed to target the phytoene desaturase gene (PDS) in Populus tomentosa Carr. Four guide RNAs were designed to target distinct genomic sites of the PtoPDS gene. Following Agrobacterium-mediated transformation, transgenic poplar plants exhibited an albino phenotype, indicating successful gene knockout. Mutation efficiency was estimated at 51.7%, with 30 out of 59 PCR clones being homozygous mutants. This study demonstrated the potential of the CRISPR/Cas9 system for precise genome editing and effective creation of knockout mutations in woody plants (Figure 6) (Fan et al., 2015). Figure 6 Efficient CRISPR/Cas9-mediated targeted mutagenesis inPopulus in the first generation (Adopted from Fan et al., 2015) Image caption: Albinism phenotype of transgenic poplars. (A, B) Albino phenotype of the regenerated poplar shoots generated on the CRISPR/Cas9-PtoPDS transformed leaf discs. (C) Representative CRISPR/Cas9 directed PtoPDS plants (T0). WT, Nontransgenic wild-type poplar; Ho, biallelic homozygous mutant; Hz, biallelic heterozygous mutant. Both Ho and Hz plants show an albino phenotype (Adopted from Fan et al., 2015) In another study, the CRISPR/Cas9 system was used to edit the PDS gene in a hybrid poplar (Populus alba × Populus glandulosa). A single guide RNA was designed, and the construct was delivered via Agrobacterium-mediated transformation. Among 110 transgenic lines, 82 showed either an albino or pale green phenotype, indicating a 74.5% phenotypic mutation efficiency. This confirmed the effectiveness of CRISPR/Cas9-mediated genome editing in hybrid poplar, with no off-target modifications detected (Bae et al., 2021). These case studies collectively highlight the versatility and efficiency of the CRISPR/Cas9 system in modifying lignin biosynthesis pathways in poplar species, paving the way for improved lignocellulosic biomass for biofuel production. 5 Challenges and Limitations 5.1 Technical challenges The CRISPR/Cas9 system, while highly efficient for targeted genome editing, is not without its technical challenges. One of the primary concerns is the occurrence of off-target effects, where the Cas9 nuclease induces double-stranded breaks at unintended genomic locations. This can lead to unintended mutations, which may have deleterious effects on the organism.
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