Journal: Nature Communications

Article Title: Accurate determination of CRISPR-mediated gene fitness in transplantable tumours

doi: 10.1038/s41467-022-31830-2

Figure Lengend Snippet: a Numbers of unique sgRNA-UMI clones per million reads in transduced tumours grown in 21 PDX lines (bars show mean + /− SEM for each PDX line, individual tumour datapoints overlaid). b Diversity measures in transduced PDX tumours. Panels compare sgRNA-UMI clone number with two measures of diversity; the Shannon diversity index and the area under a Lorenz curve plot of cumulative sequence reads against cumulative clone numbers. All measures are evaluated per million sequence reads. Lower clone numbers per tumour are strongly associated with a greater heterogeneity in clone sizes. c Lorenz curves of cumulative sequence reads against cumulative clone numbers, illustrating clone size heterogeneity in sample tumours from six PDX lines, and in the initiating sgRNA-UMI library plasmid (black). Solid and dotted lines show data from control and gene-targeting guide subsets. A greater deviation from the x = y diagonal indicates a greater unevenness in clone sizes. d Reduced diversity with passaging time. Panels show clone numbers (left) and Shannon diversity index (right) for replicate transplants from two PDX lines harvested at variable timepoints after transplantation. e Transplant clone diversity is not associated with genomic clonal diversity. Panels show clone numbers (left) and Shannon diversity index (right) against the number of genomic clonal clusters inferred from bulk WGS by population structure model PyClone-VI in 21 PDX lines (sequence from one PDX tumour analyzed per line). Boxplot lines at 25th, 50th and 75th centiles, vertical lines extend to furthest datapoint within 1.5 interquartile range distance of box limits. f GSEA pathway enrichment graph derived from RNAseq transcriptomes from the six highest compared with six lowest sgRNA-UMI diversity PDX lines. Node size indicates the number of genes in curated pathways, from Reactome Hallmark pathway set. Edge line width indicates numbers of genes shared between pathways. High sgRNA-UMI diversity PDX lines show differential activity in cell-cycle pathways, low sgRNA-UMI diversity lines in immune pathways. Source data are provided as Source Data files.

Article Snippet: Pooled sgRNA oligonucleotides were incorporated into BsmBI/Esp3I (Thermo Fisher Scientific) digested lentiCRISPRv2 plasmids at 3:1 insert-to-vector molar ratio with Quick Ligase (New England Bio labs).

Techniques: Clone Assay, Sequencing, Plasmid Preparation, Passaging, Transplantation Assay, Derivative Assay, Activity Assay

Journal: iScience

Article Title: Genome-wide CRISPR-Cas9 screen analyzed by SLIDER identifies network of repressor complexes that regulate TRIM24

doi: 10.1016/j.isci.2023.107126

Figure Lengend Snippet:

Article Snippet: Oligonucleotides were resuspended to 100uM, annealed and treated with T4 PNK (NEB) at 10uM in 10uL reactions, diluted 1:200, and ligated into Esp3I (Thermo) digested lentiCRISPRv2 (Addgene 52961) for viral CRISPR-knockout by Cas9 or Esp3I digested pXPR_502 (Addgene 96923) for viral CRISPR-activation by dCas9-VP64 using 1uL diluted oligo and 100-200ng digested vector in 20uL T4 ligase (NEB) reactions.

Techniques: Virus, Recombinant, Mutagenesis, Protease Inhibitor, Membrane, SYBR Green Assay, Plasmid Preparation, cDNA Synthesis, Cell Culture, Purification, TA Cloning, Bicinchoninic Acid Protein Assay, Software