zim3krab dcas9 domain  (Addgene inc)

Journal: Nature Genetics

Article Title: Systematic decoding of cis gene regulation defines context-dependent control of the multi-gene costimulatory receptor locus in human T cells

doi: 10.1038/s41588-024-01743-5

Figure Lengend Snippet: a , Protein expression in T conv (left) and T reg (right) cells for CD28 (sub-left), CTLA4 (sub-middle) and ICOS (sub-right) after 0 hours (black), 6 hours (dark gray), or 24 hours (light gray) restimulation. b , Representative FACS gating strategy for CRISPRi screens in T conv cells. c , Representative FACS gating strategy for CRISPRi screens in T reg cells. d , Examination of high versus low protein bin sgRNA enrichment (log 2 ) matched by gene target in bulk CD4 + T cells from one human donor with technical replicates. Colors indicate significant (adjusted P < 0.05) sgRNA enrichment with the dCas9-ZIM3 (orange), dCas9-KRAB (yellow), or both (purple) CRISPRi systems. e , CRISPRi tiling screen results comparing CRISPRi systems (‘ZIM3’, ‘KRAB’) for each target gene (rows) in CD4 + cells from one human donor (two technical replicates per condition) across the TAD designated in Fig. . CRISPRi tiling results are plotted as in Fig. . f , Correlation of log 2 (fold change) (LFC) sgRNA enrichment between high (adjusted P < 0.05, LFC > 0, gold) and low (adjusted P < 0.05, LFC < 0, blue) protein bins matched by cell type and gene target across two biological replicates plotted in Fig. . Gray sgRNAs were not significantly different between low and high bins (adjusted P > 0.05). Inset includes Pearson statistics for each group. Lines indicate best fit from a general linear model with 95% confidence interval. g , log 2 (fold change) sgRNA enrichment between high versus low protein bins for each gene target and cell type categorized by distance to the target gene transcriptional start site across two biological replicates plotted in Fig. . Significance determined with the one-way ANOVA test for each target per cell type. For all CRISPRi tiling screens, significance was determined using the Wald test with Benjamini-Hochberg correction. Boxplots indicate the sample median (central line), first and third quartiles (box), and 1.5× interquartile range (whiskers).

Article Snippet: The dCas9-ZIM3 plasmid was constructed using NEBuilder HiFi DNA Assembly Master Mix (NEB, E2621), the sequence for the ZIM3 KRAB –dCas9 domain amplified from pLX303-ZIM3-KRAB-dCas9 (Addgene, 154472) by PCR with primers CM_oligo_1 and CM_oligo_2 (Supplementary Table ) and the Lenti-SFFV-mCherry-dCas9-VP64 (ref. ) (Addgene, 180263) lentiviral backbone digested with PmeI (NEB, R0560) and BamHI (NEB, R3136).

Techniques: Expressing

Journal: American Journal of Human Genetics

Article Title: Therapeutic validation of MMR-associated genetic modifiers in a human ex vivo model of Huntington disease

doi: 10.1016/j.ajhg.2024.04.015

Figure Lengend Snippet: Construction and validation of the 125Q HD CRISPRi iPSC lines (A) Schematic showing TALEN-mediated knockin of a dCas9-tagBFP ZIM3 KRAB construct into the CLYBL safe harbor. (B) Single-cell clones were isolated and screened for TagBFP expression and pluripotency-associated marker expression. Scale bars, 50 μm. (C) Accurate knockin was verified by PCR across homology arm junctions (5′ and 3′ junctions) and insert site spanning (5′ to 3′ spanning) PCR alongside an internal dCas9 PCR. (D) Zygosity and off-target integration was assessed by copy-number qPCR. (E) Modal CAG length was determined by fragment analysis. (F) Expression of pluripotency-associated genes was further assessed by qPCR for OCT3/4 and NANOG relative to the parental iPSC line. Clone 1B12 ( ∗ ) used for further work. Mean values ± SEM in triplicate.

Article Snippet: The KOX1 KRAB domain of the dCas9-KRAB fusion in the CLYBL-targeting vector pC13 dCas9-TagBFP-KRAB (addgene #127968 ) was swapped for ZIM3 KRAB domain from pLX303-ZIM3-KRAB-dCas9 (addgene #331490 ).

Techniques: Biomarker Discovery, Knock-In, Construct, Clone Assay, Isolation, Expressing, Marker

Journal: American Journal of Human Genetics

Article Title: Therapeutic validation of MMR-associated genetic modifiers in a human ex vivo model of Huntington disease

doi: 10.1016/j.ajhg.2024.04.015

Figure Lengend Snippet: Homogeneous lowering of MMR factor expression in CRISPRi pools leads to characteristic MMR deficiencies (A) Immunostaining for indicated target proteins in red channel shows knockdown is consistent cell-to-cell. Representative images for a non-targeting control guide and a targeting guide. EGFP from the guide vectors and TagBFP from the dCas9-KRAB knock-in. Scale bars, 50 μm. (B and C) Quantification of fluorescence intensity for each target normalized to NTC1 levels for MutS and LIG1 (B) and MutL (C). Line indicate median values, 1–2,000 cells per plot from four random positions in three independent experiments. (D and E) iPSC colony formation after treatment with the indicated concentration of methylnitronitrosoguanidine (MNNG) for CRISPRi lowered MutS and LIG1 (D) and MutL (E). Mean colony frequency ± SEM in triplicate as a proportion of untreated cells of the same genotype, normalized to non-targeting control iPSCs (NTC) and shown alongside MSH6 knockout (MSH6 KO) iPSC survival. Dotted lines 95% CIs, ∗ p < 0.05.

Article Snippet: The KOX1 KRAB domain of the dCas9-KRAB fusion in the CLYBL-targeting vector pC13 dCas9-TagBFP-KRAB (addgene #127968 ) was swapped for ZIM3 KRAB domain from pLX303-ZIM3-KRAB-dCas9 (addgene #331490 ).

Techniques: Expressing, Immunostaining, Knockdown, Control, Knock-In, Fluorescence, Concentration Assay, Knock-Out