puc grna  (Addgene inc)

Journal: Journal of Fungi

Article Title: Establishment of a CRISPR/Cas9-Mediated Efficient Knockout System of Trichoderma hamatum T21 and Pigment Synthesis PKS Gene Knockout

doi: 10.3390/jof9050595

Figure Lengend Snippet: Schematic representation of knockout genes by a CRISPR/Cas9-mediated homologous recombination strategy. CRISPR/Cas9 system releases generate functional sgRNAs and direct Cas9 cleavage to complementary sites in the genome. Primary sgRNA transcripts contain HDV and HH ribozymes, which are self-cleaved to release functional sgRNAs that direct Cas9 cleavage to complementary sites in the genome. The black scissors indicate the cleavage sites. The target gene cut site is cut by a gRNA/Cas9 complex, and a double-strand break will be formed, activating cellular repair mechanisms. Cas9 cutting sites are indicated with blue scissors. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are two major pathways of DNA double-strand break repair, leading to loss of gene capacity and achieving gene knockout.

Article Snippet: The PUC-Cas9-neo-gRNA plasmid was digested with EcoRV and BglII (New England Biolabs, Ipswich, MA, USA) to recover the linear fragment.

Techniques: Knock-Out, CRISPR, Homologous Recombination, Functional Assay, Gene Knockout

Journal: Journal of Fungi

Article Title: Establishment of a CRISPR/Cas9-Mediated Efficient Knockout System of Trichoderma hamatum T21 and Pigment Synthesis PKS Gene Knockout

doi: 10.3390/jof9050595

Figure Lengend Snippet: Phenotypes and molecular identification of Δ Thpyr4 mutants and analysis of Cas9 protein expression. ( A , B ) Fluorescent images and bright field of the Thpyr4 knockout strain. ( C ) Growth phenotypes of mutants on medium containing uridine, uridine + 5-fluoroorotic acid. ( D ) Molecular identification of the Δ Thpyr4 mutant. M, Marker. Lanes 1 to 5, amplification of five knockout strains. Lane 6, positive control, amplification of the T21 wild-type strain. Lane 7, negative control, amplification of water. Lanes 8 to 14, template validation, templates same with 1–7 amplified by Thpks1-f and Thpks1-r. ( E ) RT-PCR analysis confirming Cas9 expression in the Δ Thpyr4 mutants. M, Marker. Lane 1, T21 wild type. Lanes 2 to 4, Δ Thpyr4 mutants. Lane 5, positive control, amplification of the CRISPR/Cas9 plasmid. Lane 6, negative control.

Article Snippet: The PUC-Cas9-neo-gRNA plasmid was digested with EcoRV and BglII (New England Biolabs, Ipswich, MA, USA) to recover the linear fragment.

Techniques: Expressing, Knock-Out, Mutagenesis, Marker, Amplification, Positive Control, Negative Control, Reverse Transcription Polymerase Chain Reaction, CRISPR, Plasmid Preparation