codon optimized cas9  (Addgene inc)

Journal: bioRxiv

Article Title: Unbiased CRISPR Synthetic Lethal Screening for Genetic Vulnerabilities in Succinate Dehydrogenase (SDH)-loss Model of Paraganglioma

doi: 10.1101/2025.08.26.672429

Figure Lengend Snippet: (A) Schematic workflow of the pooled CRISPR/Cas9 gene knockout screen. Cas9-expressing chromaffin cells were generated via lentiviral transduction and blasticidin selection. Cells were then transduced with a genome-wide sgRNA library, selected with puromycin, and cultured for 10 population doublings before genomic DNA extraction. sgRNA abundance was quantified at initial and final times by deep sequencing, and data were analyzed using the MAGeCK pipeline. (B) Ranked Robust Rank Aggregation (RRA) scores of the most significantly depleted genes from the screen. (C) Ranked RRA scores of the most significantly enriched genes from the screen. (D) Volcano plot of CRISPR screen results showing significantly depleted genes (blue) versus non-significant genes (gray). The x-axis represents log₂(fold change) (Log₂FC) in sgRNA abundance, and the y-axis shows –log₁₀ P values from MAGeCK analysis. (E) Volcano plot showing significantly enriched genes (red) versus non-significant genes (gray).

Article Snippet: Parental imCC lines were made to express Cas9 by transduction with 3 rd generation lentiviral particles carrying codon-optimized S. pyogenes Cas9 protein and blasticidin resistance driven from the EFS promoter (Addgene # 52962-LV).

Techniques: CRISPR, Gene Knockout, Expressing, Generated, Transduction, Selection, Genome Wide, Cell Culture, DNA Extraction, Sequencing