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Screening of T. harzianum mutants resistant to 5′-FOA. ( A ) Schematic representation of vectors used to transform fungus spores. ( B ) Genomic DNA of T. harzianum wild-type (WT) and FOA-resistant mutants ( ΔP3 , ΔP4 , ΔP7 , and ΔP13 ) was isolated and screened by PCR for the presence of the Cas9 gene which yielded a specific amplicon of 1242-bp. ( C ) Phenotype and growth of T. harzianum wild-type (WT) and FOA-resistant mutants ( ΔP3 , ΔP4 , ΔP7 , and ΔP13 ). *Bars marked with asterisk differ significantly ( P < 0.05). ( D ) A fragment from pyr4 , which was used for sequencing, confirmed mutants’ indels at target region. The sgRNA guiding sequence is highlighted in bold.

Journal: Scientific Reports

Article Title: Generation of Trichoderma harzianum with pyr4 auxotrophic marker by using the CRISPR/Cas9 system

doi: 10.1038/s41598-020-80186-4

Figure Lengend Snippet: Screening of T. harzianum mutants resistant to 5′-FOA. ( A ) Schematic representation of vectors used to transform fungus spores. ( B ) Genomic DNA of T. harzianum wild-type (WT) and FOA-resistant mutants ( ΔP3 , ΔP4 , ΔP7 , and ΔP13 ) was isolated and screened by PCR for the presence of the Cas9 gene which yielded a specific amplicon of 1242-bp. ( C ) Phenotype and growth of T. harzianum wild-type (WT) and FOA-resistant mutants ( ΔP3 , ΔP4 , ΔP7 , and ΔP13 ). *Bars marked with asterisk differ significantly ( P < 0.05). ( D ) A fragment from pyr4 , which was used for sequencing, confirmed mutants’ indels at target region. The sgRNA guiding sequence is highlighted in bold.

Article Snippet: For the construction of the CRISPR/Cas9 editing system, the Cas9 gene coding sequence from S. pyogenes was codon-optimized for expression in Trichoderma harzianum and synthesized by Epoch Life Science, Inc. (Sugar Land, TX, USA) ( ).

Techniques: Isolation, Amplification, Sequencing