Journal: Cell Reports

Article Title: A Disease-Causing Single Amino Acid Deletion in the Coiled-Coil Domain of RAD50 Impairs MRE11 Complex Functions in Yeast and Humans

doi: 10.1016/j.celrep.2020.108559

Figure Lengend Snippet: P1’s Cells Exhibit a Reduced DNA End Resection and HDR (A–C) Immunofluorescence detection of P-KAP1 (A), RPA32 (B), and RAD51 (C) in primary fibroblasts from healthy control and P1 before and 6 h after 10 Gy irradiation. The image is representative of three independent experiments. (D) Quantitative analysis of RAD51 foci before and after IR in SV40-hTERT fibroblasts untransduced or transduced with an empty or WT-RAD50-expressing vector. The number of nuclei scored and statistical significances are noted. (E) Efficiency of HDR assessed by the Cas9-directed knockin of Clover in the Lamin-A (LMNA) coding sequence. Cells were transfected with the mCherry vector alone (used to control transfection efficiency) or in combination with the repair template sequence containing the Clover coding sequence in conjunction, or not, with the pX330-LMNA1 vector encoding Cas9 and the guide RNAs (gRNAs) targeting the LMNA sequence (denoted as CRISPR-Cas9). Relative HDR efficiency was measured by the percentage of Clover + cells triple transfected relative to cells transfected without the CRISPR-Cas9-gRNA-expressing vector. Results represents the mean and SD of triplicates from three independent experiments.

Article Snippet: 1 μg of mCherry vector (PGK-H2BmCherry, Addgene #21217), 4 μg of CRISPR/cas9 vector (pX330-LMNA-gRNA1, Addgene #122507) and 1 μg of donor vector (pCR2.1 Clover-LMNA donor, Addgene #122508) were used.

Techniques: Immunofluorescence, Irradiation, Transduction, Expressing, Plasmid Preparation, Knock-In, Sequencing, Transfection, CRISPR

Journal: Cell Reports

Article Title: A Disease-Causing Single Amino Acid Deletion in the Coiled-Coil Domain of RAD50 Impairs MRE11 Complex Functions in Yeast and Humans

doi: 10.1016/j.celrep.2020.108559

Figure Lengend Snippet:

Article Snippet: 1 μg of mCherry vector (PGK-H2BmCherry, Addgene #21217), 4 μg of CRISPR/cas9 vector (pX330-LMNA-gRNA1, Addgene #122507) and 1 μg of donor vector (pCR2.1 Clover-LMNA donor, Addgene #122508) were used.

Techniques: Recombinant, Fluorsave, Protease Inhibitor, Blocking Assay, Transfection, Mutagenesis, Transduction, 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: Virus, Recombinant, Flow Cytometry, cDNA Synthesis, RNAscope, Multiplex Assay, Control, Knock-Out, Knock-In, Mutagenesis, Plasmid Preparation, Software