Journal: Molecular Therapy. Nucleic Acids

Article Title: CRISPR-Cas9-mediated reactivation of the uricase pseudogene in human cells prevents acute hyperuricemia

doi: 10.1016/j.omtn.2021.08.002

Figure Lengend Snippet: Schematic for CRISPR-Cas9-mediated knockin of exogenous DNA fragment into human AAVS1 locus in HEK293 cells Top: the human AAVS1 allele. gRNA sequences are designated (g1 and g2) and located upstream of exon 2. Center: donor plasmid is represented schematically. Dashed lines show homology arms (∼800 bp on each side of donor) between genomic locus and plasmid DNA. A splice acceptor sequence is inserted upstream of RFP gene to allow proper RNA splicing using the endogenous transcriptional promoter. T2A sequences are inserted both upstream and between RFP/AncUOX to allow the release of the individual proteins after translation. SV40 poly(A) signal sequence is designated downstream of the donor DNA. Bottom: engineered allele. Post Cas9 cuts at the site corresponding to the gRNA sequences, donor DNA is integrated into the breakpoint through homology directed repair (HDR).

Article Snippet: To construct the pU6-(Bbs1)_CBh-Cas9-T2A-GFP-p2A-Ad4E4orf6 plasmid (CRISPR plasmid), we digested px458 (Addgene, #48138) and pU6-(BbsI)_CBh-Cas9-T2A-mCherry-P2A-Ad4E4orf6 (Addgene, #64222) with FseI/BsrGI (New England Biolabs).

Techniques: CRISPR, Knock-In, Plasmid Preparation, Sequencing

Journal: bioRxiv

Article Title: Post-Synaptic Density Proteins in Oligodendrocytes are Required for Activity-Dependent Myelin Sheath Growth

doi: 10.1101/2025.02.10.637467

Figure Lengend Snippet: A. Representation of the plasmid micro-injected into one-cell stage of Tg(s1020t:Gal4) (B, C), Tg(Olig1:KalTA4) (D, E) or Tg(Mbp:Gal4) (F-I) to induce cell-specific dlg4a loss-of-function with the Cas9 endonuclease. B-C. Quantitative RT-PCR, on 1020+ mCherry+ whole larvae, of (B) dlg4a and (C) dlg4b gene expression at 5dpf. Student’s t test; N=4 versus 7 groups of animals, each group containing RNA from 20 dissociated fish. Data are represented as mean ± SD. D. Olig1+ cells in control and dlg4 KO spinal cords at 5 dpf exhibit distinct morphologies. In dlg4a KO, Olig1+ cells are more ramified than in the control. Mature myelinating OLs were mainly observed in control animals. E. Quantification of the oligodendroglial cell surface in control and dlg4a KO animals with IMARIS. Mann-Whitney test; N=117 vs 162 cells, respectively in control and KO fish. F. MBP+ cells with myelin segments in control and KO spinal cords at 5 dpf. G. Quantification of the number of myelin segments / cell. Mann-Whitney test; N=20 vs 30 cells, respectively. H. Quantification the internode length. Mann-Whitney test; N=107 vs 165 internodes. I. Quantification of the internode length per cell. Mann-Whitney test; N=36 vs 41 cells.

Article Snippet: To generate UAS:Cas9-P2A-mCherry; U6-dlg4; U6-dlg4 tol2-flanked cell-specific CRISPR-Cas9 plasmid, we started from the commercially available plasmid pUAS:Cas9-T2A-GFP ; U6-sgRNA1; U6-sgRNA2 (Cat #74009, Addgene) generated by Dr F. Del Bene .

Techniques: Plasmid Preparation, Injection, Quantitative RT-PCR, Expressing, Control, MANN-WHITNEY

Journal: Nature genetics

Article Title: Genetic screening for single-cell variability modulators driving therapy resistance

doi: 10.1038/s41588-020-00749-z

Figure Lengend Snippet: a, To compare the frequency of drug resistance between WM989-A6-G3 and its daughter cell line WM989-A6-G3-Cas9–5a3, we cultured an equal number of cells from each cell line in 1 μM vemurafenib for 3 weeks. Then, we counted the number of colonies that resulted from each cell line. Each dot represents the number of resistant colonies normalized to 10,000 cells alive in each sample before the addition of vemurafenib. The bars represent the mean number of colonies over triplicates, normalized to 10,000 cells alive in each sample before the addition of vemurafenib. Error bars represent the standard error of the mean. b, To compare the frequency of NGFR HIGH /EGFR HIGH cells between WM989-A6-G3 and its daughter cell line WM989-A6-G3-Cas9–5a3, we looked at the distribution of expression of NGFR and EGFR using immunofluorescence. c, WM989-A6-G3-Cas9–5a3 cells expressing NGFR, EGFR, and both NGFR and EGFR are more likely to survive and proliferate in the presence of vemurafenib . Here we show the number of colonies that grew upon vemurafenib exposure in a mixed population of WM989-A6-G3-Cas9–5a3 or in the same population but enriched for EGFR HIGH cells, NGFR HIGH cells, or NGFR HIGH /EGFR HIGH cells. d, In this plot, we show the single guide RNA representation (as percent GFP-positive cells) of controls over time in WM989-A6-G3 cells with or without Cas9 expression. Negative controls (black) are single guide RNAs aimed at ROSA26 , a non-expressing gene in human melanoma. Positive controls (red) target proteins necessary for cell viability. Only cells expressing both Cas9 and a positive control single guide RNA should disappear from the population over time.

Article Snippet: All the Cas9-positive melanoma cell lines in this study were derived by lentiviral transduction with a Cas9 expression vector (EFS-Cas9-P2A-Puro, Addgene: 108100).

Techniques: Cell Culture, Expressing, Immunofluorescence, Positive Control

Journal: Nature genetics

Article Title: Genetic screening for single-cell variability modulators driving therapy resistance

doi: 10.1038/s41588-020-00749-z

Figure Lengend Snippet: We assessed the robustness and generality of the effect of hits identified in the priming and resistance screens (WM989-A6-G3-Cas9–5a3, black bars) by carrying out secondary priming (left) and resistance screens (right) targeting 86 different proteins in WM989-A6-G3-Cas9 (orange bars) as well as in another BRAF V600E melanoma cell line (451Lu-Cas9, blue). On the left, we plot the log 2 fold change in frequency of NGFR HIGH /EGFR HIGH cells (normalized by non-targeting controls) for each sgRNA (dots) targeting 34 of the high confidence hits (Tiers 1 and 2) we identified in the priming screen. We found that 25 of the 34 high confidence hits showed at least a two fold change (as a median across sgRNA triplicates; see ) in the frequency of NGFR HIGH /EGFR HIGH cells concordant with the effects detected in the original screening clonal cell line (WM989-A6-G3-Cas9–5a3). In 451Lu-Cas9 cells, 20 of the 34 targets also showed a change in the frequency of NGFR HIGH /EGFR HIGH cells, with 11 of those exhibiting at least a two-fold change (as a median across sgRNA triplicates; see ). On the right, we plot the log 2 fold change in frequency of cells resistant to vemurafenib (normalized by non-targeting controls) for each sgRNA (dots) targeting 9 of the high confidence hits (Tiers 1 and 2) identified through the resistance screen. In WM989-A6-G3-Cas9, we found that 7 of the 9 targets replicated the effect we observed originally. For 451Lu-Cas9, the same 7 factors showed similar effects. Within each plot, the color of the target label indicates the effect observed in the primary screens. See for the results of Tier 3 and Tier 4 targets.

Article Snippet: All the Cas9-positive melanoma cell lines in this study were derived by lentiviral transduction with a Cas9 expression vector (EFS-Cas9-P2A-Puro, Addgene: 108100).

Techniques:

Journal: Nature genetics

Article Title: Genetic screening for single-cell variability modulators driving therapy resistance

doi: 10.1038/s41588-020-00749-z

Figure Lengend Snippet: a, Frequency of NGFR HIGH cells following the knockout of select targets. Dots represent the change in number of NGFR HIGH cells (as the log 2 fold change over non-targeting sgRNA controls). A star indicates targets where, after excluding samples with low cell numbers (< 500 cells), n = 1. Tier refers to the degree of confidence we have in each particular hit (see ). We performed this analysis for hits from both the priming screen (top) and the resistance screen (bottom). 21 of 34 high confidence showed at least a 50% increase or decrease in the frequency of NGFR HIGH cells (see ). 21 of 49 targets from Tiers 3 and 4 increased or decreased the frequency of NGFR HIGH cells by ≥ 50%. b, Resistance phenotype of melanoma cells following the knockout of hits. Bars represent the log 2 fold change over non-targeting control in the number colonies able to grow in vemurafenib. The number of colonies is normalized to the number of cells present before BRAF V600E inhibition (see ). In the left panel, we labeled in green and gray the effect a given target has on the frequency of NGFR HIGH /EGFR HIGH cells (based on the initial priming screen). In the right panel, we labeled in red and gray the effect a given target has on the number of cells that resist BRAF V600E inhibition (based on the initial resistance screen). Each bar represents one experimental replicate (see for replicates). c, These images show the effect of CSK knockout on a cell’s ability to develop resistance to BRAF V600E inhibition. We exposed CSK-knockout WM989-A6-G3-Cas9–5a3 cells to 1 μM vemurafenib for 3 weeks and counted the number of resulting colonies. The number of resistant cells is too large to accurately identify individual colonies; thus, the number of colonies reported is an underestimate.

Article Snippet: All the Cas9-positive melanoma cell lines in this study were derived by lentiviral transduction with a Cas9 expression vector (EFS-Cas9-P2A-Puro, Addgene: 108100).

Techniques: Knock-Out, Control, Inhibition, Labeling

Journal: Nature genetics

Article Title: Genetic screening for single-cell variability modulators driving therapy resistance

doi: 10.1038/s41588-020-00749-z

Figure Lengend Snippet: Tumor volume as a function of time in xenografts (NOD/SCID mice) treated with a BRAF V600E inhibitor (top) or vehicle control (bottom). Here, we inject each mouse with DOT1L -, LATS2 -, or BRD2 -knockout WM989-A6-G3-Cas9 cells (orange) or with the same cell line without a gene knockout (gray). The values plotted represent the mean tumor volume across mice carrying the same knockout. Error bars represent the standard error of the mean. All p values were obtained with a one-sided t -test. *** are timepoints at which the difference in tumor volume between knockout and control groups reached P ≤ 0.05. Similarly, * represents P = 1 ≤≥ 0.05 (see ). Each group started with n = 6 mice, and we plotted the mean tumor volume up until both knockout and negative control groups have at least n = 3 each. The n at each timepoint and the respective P value at each timepoint are presented in and .

Article Snippet: All the Cas9-positive melanoma cell lines in this study were derived by lentiviral transduction with a Cas9 expression vector (EFS-Cas9-P2A-Puro, Addgene: 108100).

Techniques: Control, Knock-Out, Gene Knockout, Negative Control

Journal: iScience

Article Title: High-throughput screening for myelination promoting compounds using human stem cell-derived oligodendrocyte progenitor cells.

doi: 10.1016/j.isci.2023.106156

Figure Lengend Snippet: Figure 1. Generation and validation of an advanced hES reporter system (A) Schematic of the endogenous PDGFRA, PLP1 and MBP locus of the Ptt-P1-MsnL reporter line after CRISPR-Cas9- based genetic knock-in of reporter sequences. (B) Expression of the tdTomato and sfGFP fluorescent reporters in the differentiating oligodendrocyte cultures Scale bar: 400 mm (C) secNluc activity in the cell culture medium of differentiating PtT-P1-MsNL reporter line measured with Nano-Glo assay. Culture media from different days of differentiation (xaxis) were removed for the assay. Increase in Nano-Luc activity (yaxis), which corresponds to MBP expression increases as cells mature. (D) A higher magnification image of the reporter expression on d100 culture shown in (B). GFP+ processes and branches resembling oligodendrocytes morphology are further magnified in the inset. Scale bar: 200 mm (E) MACS-based enrichment of the GFP+ cells using O4 antigen microbeads. 60% GFP+ cells were achieved from d105 cells. (F) qPCR shows enrichment of OL markers in the O4+ cells compared to the O4- cells. Data presented as mean +/- SEM

Article Snippet: Guide sequence with minimal off target and very high activity score was chosen and cloned into the BbsI restriction site of the Cas9 plasmid (Cas9-P2A-Puro modified from Addgene #6298854). pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t. net/addgene:62988).

Techniques: Biomarker Discovery, CRISPR, Knock-In, Expressing, Activity Assay, Cell Culture, Glo Assay