Journal: bioRxiv

Article Title: Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

doi: 10.1101/2022.07.13.499814

Figure Lengend Snippet: a. Schematics of CRISPRi transcription repressor domains and general lentiviral expression construct used for all CRISPRi effectors. b. Experimental design to test effects of stable expression of each CRISPRi effector on growth and transcription in K562 cells. c. Growth defects of effector-expressing cells, measured as the log 2 ratio of mCherry-negative (effector-expressing) to mCherry-positive (not effector-expressing) cells in each well. mCherry levels were measured for 19 days after pooling cells. Data represent mean ± SD from three independent transductions of expression constructs. p -values are from an unpaired two-tailed t-test comparing D19 values for each sample to the D19 value for the “no plasmid” sample. Average percent growth defect per day is the log 2 D19 value divided by the number of days, multiplied by 100 for a percent value. d. Clustered heatmap of correlation of transcript counts from K562 cells expressing indicated CRISPRi effectors or a GFP control. Correlations across samples were calculated using normalized counts (reads per million) for all genes with mean normalized count >1 and then clustered using the Ward variance minimization algorithm implemented in scipy. r 2 is squared Pearson correlation. Data represent three independent transductions of expression constructs. e. Number of differentially expressed genes ( p < 0.05) for cells expressing each effector versus cells expressing GFP only. p -values were calculated using a Wald test and corrected for multiple hypothesis testing as implemented in DeSeq2. See also .

Article Snippet: A modified CROP-seq sgRNA lentiviral expression vector (pJR107) was derived from the parental vector pBA950 ( https://www.addgene.org/122239/ ) by incorporating a GFP fluorescent marker and a UCOE element upstream of the EF1alpha promoter to prevent marker silencing. sgRNA targeting sequences were appended with flanking sequence, BstX1/BlpI overhangs, and PCR adapters.

Techniques: Expressing, Construct, Two Tailed Test, Plasmid Preparation

Journal: iScience

Article Title: Generation and characterization of inducible KRAB-dCas9 iPSCs from primates for cross-species CRISPRi

doi: 10.1016/j.isci.2024.110090

Figure Lengend Snippet:

Article Snippet: Briefly, gRNAs were cloned into the BsmBI-digested (New England BioLabs, R0739S) CROP-seq-opti vector by Gibson Assembly using the NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs, E2621L) and NEB Stable Competent E. coli (New England BioLabs, C3040I) were transformed with the assembled plasmids.

Techniques: Virus, Recombinant, Membrane, Lysis, CRISPR, DNA Extraction, Western Blot, Transfection, Bicinchoninic Acid Protein Assay, SYBR Green Assay, Stripping, Software, Microscopy