Journal: Synthetic and Systems Biotechnology
Article Title: Metabolic reprogramming of abscisic acid-producing strain Botrytis cinerea TB-31 toward terpenoid biosynthesis using a CRISPR/Cas9 ribonucleoprotein system
doi: 10.1016/j.synbio.2025.12.002
Figure Lengend Snippet: Gene editing strategy using the CRISPR/Cas9 RNP system and the terpenoid synthesis pathway in B. cinerea strain TB-31. (A)–(B) Schematic illustration of the workflow of CRISPR/Cas9-mediated gene editing in B. cinerea TB-31 using preassembled Cas9-sgRNA RNP complexes. Key steps include the (1) design and synthesis of sgRNAs targeting the gene of interest; (2) in vitro assembly of the Cas9 protein and sgRNAs into RNP complexes; (3) delivery of two RNPs and a vector of telomere pTEL- hyg (A) or a donor DNA containing a G418 box (B) into B. cinerea protoplasts via PEG-mediated transformation; and (4) regeneration and screening of edited strains. (C)∼(F) Genomic diagram of single-gene editing within the ABA BGC. △ bcaba1 (C), △ bcaba2 (D), △ bcaba3 (E), and △ bcaba4 (F); (G)∼(H) Genomic diagram of multigene editing of the ABA BGC. △ bcaba124 (G), △ bcaba1234 (H). Gene structure diagram illustrating the editing events was generated using IBS 2.0 , with the gene length scaled down proportionally. The binding locations of the validation primers are indicated by black arrows.
Article Snippet: The fragment Cas9-4NLSs was amplified with primers PpkiA - Cas9 -F and Cas9 - TglaA -R using pET28a- Cas9 - 4NLSs as template, and cloned into the Nhe I and Pac I sites of vector Anep8-Cas9 (Addgene number: #117169) to generate pAnep8- Cas9 - 4NLSs - AMA1 .
Techniques: CRISPR, In Vitro, Plasmid Preparation, Transformation Assay, Generated, Binding Assay, Biomarker Discovery
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