Journal: STAR protocols

Article Title: Protocol for iterative enrichment of integrated sgRNAs via derivative CRISPR-Cas9 libraries from genomic DNA of sorted fixed cells.

doi: 10.1016/j.xpro.2024.103493

Figure Lengend Snippet: Figure 1. Flow diagrams depicting the two CRISPR-Cas9 enrichment techniques mentioned in the main text Method A illustrates the conventional enrichment of single guide RNAs (sgRNA) from live cells, which involves Fluorescence-Activated Cell Sorting (FACS) and the expansion of phenotypically-selected live cells. Method B presents our proposed enrichment method, involving FACS of fixed cells and extraction of genomic DNA (gDNA) from fixed sorted cells to generate derivative enriched libraries. In Method B, the green sections indicate the introduction of convenient pause points.

Article Snippet: Design primers that amplify the sgRNA region while keep restriction sites that facilitate subsequent cloning into the CRISPR backbone plasmid (pKLV-U6gRNA(BbsI)PGKpuro2ABFP; Addgene#50946).18 The PCR product will thus represent a pool of sgRNAs targeting the desired genes.

Techniques: CRISPR, Fluorescence, FACS, Extraction

Journal: STAR protocols

Article Title: Protocol for iterative enrichment of integrated sgRNAs via derivative CRISPR-Cas9 libraries from genomic DNA of sorted fixed cells.

doi: 10.1016/j.xpro.2024.103493

Figure Lengend Snippet: Figure 2. Dot-histogram plot confirming Cas9 stable integration in the cell line used in the CRISPR-Cas9 screen CHO-a1ATH334D/Cas9 cells harboring an endogenous fluorescent CHOP::GFP reporter were analyzed by flow cytometry under basal conditions (UT, red) or following treatment with Tunicamycin (Tm) alone (blue) or transfection with an individual lentiGuide-Puro plasmid (without expression of Cas9) targeting GFP (UK1717, orange). ER stress treatments with 2.5 mg/mL Tm lasted 24 h. The GFP high expression population that remained after transfection with a GFP-targeting sgRNA (orange) and treatment with Tm represents cells that either have not incorporated the GFP- targeting sgRNA or have not caused frameshift mutation.

Article Snippet: Design primers that amplify the sgRNA region while keep restriction sites that facilitate subsequent cloning into the CRISPR backbone plasmid (pKLV-U6gRNA(BbsI)PGKpuro2ABFP; Addgene#50946).18 The PCR product will thus represent a pool of sgRNAs targeting the desired genes.

Techniques: CRISPR, Cytometry, Transfection, Plasmid Preparation, Expressing, Mutagenesis

Journal: STAR protocols

Article Title: Protocol for iterative enrichment of integrated sgRNAs via derivative CRISPR-Cas9 libraries from genomic DNA of sorted fixed cells.

doi: 10.1016/j.xpro.2024.103493

Figure Lengend Snippet: Figure 6. Schematic diagram of the cloning PCR amplification strategy to generate derivative enriched libraries from fixed cells Genomic DNA (gDNA) is extracted from pools of sorted cells exhibiting the phenotype of interest (in our case brightest Mab2C1 polymer signal). This gDNA is subjected to PCR amplification targeting the integrated sgRNA region using specific primers (Pr2182 and Pr1758). The resulting 535 bp PCR product is then digested with MluI and BamHI to generate a 382 bp sgRNA insert, which is ligated into the parental lentiviral backbone (pKLV- U6gRNA(BbsI)-PGKpuro2ABFP) at a ratio 3:1, generating new lentiviral sgRNA enriched libraries denoted as Lib1 and Lib2.

Article Snippet: Design primers that amplify the sgRNA region while keep restriction sites that facilitate subsequent cloning into the CRISPR backbone plasmid (pKLV-U6gRNA(BbsI)PGKpuro2ABFP; Addgene#50946).18 The PCR product will thus represent a pool of sgRNAs targeting the desired genes.

Techniques: Cloning, Polymer