Journal: ACS Nano

Article Title: Robust, Durable Gene Activation In Vivo via mRNA-Encoded Activators

doi: 10.1021/acsnano.1c10631

Figure Lengend Snippet: In vivo demonstration of optimized B4galnt2 gene activation. (a) Representative slide scan images of liver sections showing RNAscope staining for B4galnt2 mRNA (green) and DAPI (blue). Insets depict the relative locations of 4× and 16× views. Scale bars are 800 μm for 1×, 200 μm for 4×, and 50 μm for 16× images. Time courses of B4galnt2 mRNA (b) and activator mRNA (c) copy numbers from liver tissue over 9 days. B4galnt2 mRNA copy numbers (d) and percentage of activated hepatocytes (e) in mice treated with activator mRNA and B4 sgRNA with or without AcrIIA4 co-delivery. The “–AcrIIA4” groups were dosed with activator mRNA and B4 sgRNA on day 0. The “+AcrIIA4” groups were simultaneously dosed with activator mRNA, B4 sgRNA, and AcrIIA4 mRNA on day 0. All VPR and VPH-SS18-treated mice were euthanized at day 1 postinjection, and all p300-treated mice were euthanized at 5 days postinjection. (f) VPH-SS18 time course with redosing. VPH-SS18 mRNA and B4 sgRNAs were delivered to both groups on day 0. AcrIIA4 treatment was given to one group on day 5, followed by euthanasia on day 6. Remaining mice that did not receive AcrIIA4 mRNA were then redosed with VPH-SS18 mRNA and B4 sgRNAs on day 14. Data represent mean ± SEM ( n = 3–4 mice). Statistical significance was assessed using a two-way ANOVA followed by Dunnett’s multiple comparison compared to the NT-treated group (b,c) and a student’s t test (d,e) (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Plasmid DNA constructs coding dCas9-VP64 (Addgene Plasmid #47107), dCas9-VPR (Addgene (Plasmid #63798)), dCas9-p300 (Addgene Plasmid #83889), and VPH-dCas9-SS18 (Charles Gersbach) with cleavable mCherry and AcrIIA4 (Addgene Plasmid #101042) were flanked (3′ and 5′) by UTR regions and synthesized by Genscript Inc. mRNA synthesis was performed as previously described.

Techniques: In Vivo, Activation Assay, RNAscope, Staining, Comparison

Journal: Cell

Article Title: B cell-specific XIST complex enforces X-inactivation and restrains atypical B cells

doi: 10.1016/j.cell.2021.02.015

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To generate the anti-CRISPR virus, we cultured HEK 293T cells at 4 million per 10cm dish and transfected with 4.5 ug pMP.G, 1.5 ug psPAX2 and 6 ug Fuw-AcrIIA4-P2A-GFP vector (Addgene #108247) using OptiMEM and Lipofectamin 3000 at the following day.

Techniques: Recombinant, Sequencing, Mass Spectrometry, Software

Journal: Neuron

Article Title: Mutations in spliceosomal genes PPIL1 and PRP17 cause neurodegenerative pontocerebellar hypoplasia with microcephaly

doi: 10.1016/j.neuron.2020.10.035

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Empty CRISPRi plasmid (PX330-U6–2XBsmBI-gRNA-CBh-dCas9-KRAB-T2a-Puro) was generated on the modified PX330 with 2× BsmBI gRNA cloning sites. dCas9-KRAB-T2a-Puro was amplified from vector pLV-hU6-sgRNA-hUbC-dCas9-KRAB-T2aPuro (Addgene #71236) and cloned inside PX330 to replace original WT Cas9. gRNAs targeting PRP17 or scramble gRNA was further cloned between 2XBsmBI sites.

Techniques: Recombinant, Flow Cytometry, Multiplex Assay, Knock-Out, Knock-In, Mutagenesis, Plasmid Preparation, Software