pmscv ires egfp caspase 1 r179d  (Addgene inc)

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

Journal: Nucleic Acids Research

Article Title: Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered, light-switchable anti-CRISPR protein

doi: 10.1093/nar/gkaa1198

Figure Lengend Snippet: Engineering of CASANOVA-C3, a light-switchable anti-CRISPR protein for optogenetic control of Nme Cas9. ( A ) Schematic of CASANOVA-C3 function. ( B ) Structure of AcrIIC3. The nine regions chosen for LOV2 domain insertion (R1–R9) are shown in red (PDB 6J9N). ( C ) Luciferase reporter-based screen of AcrIIC3-LOV2 hybrids. HEK293T cells were co-transfected with vectors encoding (i) a firefly luciferase reporter, (ii) Nme Cas9 and a sgRNA targeting the luciferase reporter and (iii) either wild-type AcrIIC3 (AcrIIC3) or the indicated AcrIIC3-LOV2 hybrid (S11-V100) followed by luciferase assay. The AcrIIC3 residues behind which the LOV2 domain was inserted are indicated. R1–9 correspond to the different regions in B. R, region. Rep, reporter only control. The lead region is labelled in bold. ( D ) Lead panel of AcrIIC3-LOV2 hybrids. Glycine–serine linkers are in green. ( E ) Luciferase assay screen of the AcrIIC3-LOV2 hybrids in D. Cells were transfected as in C and then exposed to blue light or kept in the dark for 48 hours, followed by luciferase assay. Rep, reporter only control. ( F ) HEK293T cells were co-transfected with vectors encoding (i) Nme Cas9 and a sgRNA targeting the endogenous IL2RG locus and (ii) the indicated Acr variant in D. Samples were exposed to blue light or kept in the dark for 72 h. Gene editing was assessed by T7 assay. Representative gel images are shown below the bar charts. The dotted line separates different gels. In, input. T7, T7 cleavage fragments. (C, E, F) Bars represent mean values, error bars the standard deviation and dots individual data points from n = 3 independent experiments.

Article Snippet: AcrIIC3-LOV2 hybrid constructs were created by inserting the LOV2 domain into our published CMV promoter-driven AcrIIC3 expression vector (Addgene plasmid #120301) ( ).

Techniques: CRISPR, Luciferase, Transfection, Variant Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered, light-switchable anti-CRISPR protein

doi: 10.1093/nar/gkaa1198

Figure Lengend Snippet: The LOV2 domain in CN-C3 is located in close proximity to the Nme Cas9 binding surface. ( A ) Analysis of AcrIIC3 residue contacts. Spatially proximate AcrIIC3 residue pairs (distance < 7 Å) are indicated by black squares (triangular plot). α-Helices and β-sheets, indicated by cylinders and arrows according to the published structure (PDB: 6J9N) are shown on the left. Regions into which the LOV2 domain was inserted into AcrIIC3 (see Figure ) are indicated in red and correspond to the labelled regions in the AcrIIC3 structure (lower right). The LOV2 insertion site underlying CN-C3(G) is marked in green. Numbers correspond to AcrIIC3 residues. ( B ) Close-up view on the identified LOV2 insertion site in context of the AcrIIC3:HNH domain complex. The approximate distance between the insertion site on AcrIIC3 and the Nme Cas9 HNH domain is indicated. The angle as well as the distance between the secondary structure elements adjacent to the insertion site are shown. Residues in red mediate direct contact to the HNH domain. ( C ) Computational model of CN-C3 generated by domain assembly simulation. The three most populated conformational clusters of the LOV2 are shown in purple in descending order. ( D ) Cluster 3 does not sterically clash with the HNH-domain. PDB 6J9N, 2V0W.

Article Snippet: AcrIIC3-LOV2 hybrid constructs were created by inserting the LOV2 domain into our published CMV promoter-driven AcrIIC3 expression vector (Addgene plasmid #120301) ( ).

Techniques: Binding Assay, Generated