Journal: Nature genetics

Article Title: The impact of nonsense-mediated mRNA decay on genetic disease, gene editing and cancer immunotherapy

doi: 10.1038/s41588-019-0517-5

Figure Lengend Snippet: a , a decrease in protein expression due to tiling sgRNAs placed along the length of human and mouse genes (y axis quantifies the sgRNA fold difference between a low-expressing versus high-expressing set of cells) reveals, overall, similar associations with the non-canonical start-proximal NMD rule to the canonical NMD last-exon rule. The CD13 gene demonstrates the effect of the non-canonical long-exon rule. Shaded regions are 95% confidence interval of the loess fit to protein expression. Pearson correlation coefficients and two-sided tests for association were computed by comparing the loess fit to the NMDetective -A NMD efficacy scores. b , evading NMD attenuates the loss of fitness (y axis) due to knockout of essential genes. Data for non-essential genes are in . P values are by Mann-Whitney U test. The knock-out efficiency compares the reduction of sgRNAs in NMD evading regions to the reduction in regions that trigger NMD. c , a ‘saturation genome editing’ CRISPR experiment shows strongly reduced mRNA levels for nonsense mutations in BRCA1, except for those introduced into regions covered by the start-proximal (top) and last-exon NMD evasion rules (bottom panel).

Article Snippet: We therefore used the CRISPR design tools E-CRISP and ‘CRISPRko’ offered by the Genetic Perturbation Platform of the Broad Institute, to design sgRNAs for knock-out experiments of the top 100 most cited genes (from http://doi.org/10.5281/zenodo.1066066 ).

Techniques: Expressing, Knock-Out, MANN-WHITNEY, CRISPR

Journal: Nature genetics

Article Title: The impact of nonsense-mediated mRNA decay on genetic disease, gene editing and cancer immunotherapy

doi: 10.1038/s41588-019-0517-5

Figure Lengend Snippet: a , fitness loss upon targeting a non-essential gene (left) versus an essential gene (right) using a sgRNA directed at gene sections which are covered by various NMD-evasion rules. b - e , distribution of loci targeted by sgRNAs that are NMD-detected or NMD-evading (according to the individual NMD rules) for genome-wide CRISPR libraries ( b , c ) or by sgRNA design tools ( d , e ).

Article Snippet: We therefore used the CRISPR design tools E-CRISP and ‘CRISPRko’ offered by the Genetic Perturbation Platform of the Broad Institute, to design sgRNAs for knock-out experiments of the top 100 most cited genes (from http://doi.org/10.5281/zenodo.1066066 ).

Techniques: Genome Wide, CRISPR

Journal: Scientific Reports

Article Title: Probing the requirement for CD38 in retinoic acid-induced HL-60 cell differentiation with a small molecule dimerizer and genetic knockout

doi: 10.1038/s41598-017-17720-4

Figure Lengend Snippet: CRISPR/Cas9-mediated disruption of CD38 does not affect signalling proteins known to be involved in RA-induced differentiation. Wild-type and CD38 CRISPR cell lines were cultured for 48 h with 1 μM RA as indicated and whole cell lysate was collected. 25 μg of lysate per lane was run. Western blots of PAGE-resolved lysates from wild-type cells and CRISPR 1, 2, and 3 cell lines were probed for the indicated proteins, where GAPDH is a loading control. Membrane images for each protein are cropped to show only the band of interest.

Article Snippet: Lentiviral particles were produced using 2.5 µg pMD2.g (Addgene #12259), 7.5 µg psPAX2 (Addgene #12260), and 10 µg pLentiCRISPR v2 with CRISPR 1, 2, or 3 inserted.

Techniques: CRISPR, Cell Culture, Western Blot