Journal: The CRISPR journal

Article Title: CRISPR-AsCas12a Efficiently Corrects a GPR143 Intronic Mutation in Induced Pluripotent Stem Cells from an Ocular Albinism Patient.

doi: 10.1089/crispr.2021.0110

Figure Lengend Snippet: FIG. 1. CRISPR-SpCas9 targets and sgRNA screening in OA1 patient-derived iPSCs. (A) Sequence of the double- stranded GPR143 intron 7 surrounding the patient’s G > A mutation (highlighted in red). The sequence of the two tested sgRNAs is also shown, with the 3¢ PAM sequence underlined. (B) In vitro digestion assay for the positive control (C+) sgRNA targeting the CDC42BPB gene. The ND PCR product size is 478 bp, whereas the estimated sizes of the CRISPR-SpCas9-digested bands are 407 and 71 bp, with only the higher band visible in the agarose gel. (C) In vitro digestion assay for the sgRNAs targeting the GPR143 intronic mutation. The ND PCR product size is 576 bp, whereas the estimated sizes of the CRISPR-SpCas9-digested bands are *315 and *261 bp. (D) Surveyor assay of OA1 patient-derived iPSCs treated with the C+ sgRNA targeting the CDC42BPB gene, and (E) with the sgRNAs targeting the GPR143 intronic mutation. NT cells were used as control. S- and S+, samples incubated in the absence or presence of the Surveyor nuclease, respectively. (F) Percentage of indels in the CRISPR-SpCas9-treated patient-derived iPSCs estimated by the TIDE and the ICE analyses. iPSCs, induced pluripotent stem cells; MW, molecular-weight ladder; ND, non-digested; NT, non-treated; OA1, ocular albinism type 1; PAM, protospacer adjacent motif; PCR, polymerase chain reaction; sgRNA, single-guide gRNA; TIDE, Tracking of Indels by DEcomposition.

Article Snippet: Cells were observed using the FluoView FV1000 confocal laserscanning microscope (Olympus, JSEI Core facility). iPSC in vitro differentiation assay The corrected patient-derived iPSC clones were differentiated using the Human Pluripotent Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, MN) following the manufacturer’s protocol with minor changes.

Techniques: CRISPR, Derivative Assay, Sequencing, Mutagenesis, In Vitro, Positive Control, Agarose Gel Electrophoresis, Control, Incubation, Molecular Weight, Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: Zebularine regulates early stages of mESC differentiation: effect on cardiac commitment

doi: 10.1038/cddis.2013.88

Figure Lengend Snippet: Characterization of HS-181 cell line after treatment with zebularine. ( a ) RT-PCR of cardiac markers after treatment with zebularine. Myh7, Myh6, Actc, cTnI and Serca2 present more expression after treatment. ( b ) Immunostaining of treated cells to detect cardiac-specific proteins. Scale bars; 50 μ m. ( c ) Blots of Proteome Profiler Array and the resulting quantification histograms demonstrating inhibition of pluripotency marker expression and ( d ) increased levels of mesodermic proteins after zebularine treatment (black arrows)

Article Snippet: Protein expression profiles were assayed using the specific human pluripotent Stem Cell array kit ‘Proteome Profiler Array' (R&D Systems Europe, Abingdon, UK; ARY010) following the manufacturer's instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Immunostaining, Inhibition, Marker

Journal: Cell reports

Article Title: NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency

doi: 10.1016/j.celrep.2023.112372

Figure Lengend Snippet: Key resources table

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Acetylated lysine Cell Signaling Technology Cat# 9441 Actin Sigma-Aldrich Cat# MABT1333 ATG5 Nano Tools Cat# 0262-100/ATG5-7C6 53BP1 Abcam Cat# ab36823 LC3B Novus Biologicals Cat# NB100-2220 LC3B NanoTools Cat# 0231-100/LC3-5F10 Cleaved caspase-3 (Asp175) Cell Signaling Technology Cat# 9661 GAPDH Sigma-Aldrich Cat# G8795 GFP Clontech Cat# 632375 Goat anti-mouse IgG (H+L), Alexa Fluor 488 Invitrogen Cat# A-11001 Goat anti-mouse IgG (H+L), Alexa Fluor 594 Invitrogen Cat# A-11005 Goat anti-rabbit IgG (H+L), Alexa Fluor 488 Invitrogen Cat# A-11008 Goat anti-rabbit IgG (H+L), Alexa Fluor 594 Invitrogen Cat# A-11012 Donkey anti-goat IgG (H+L), Alexa Fluor 488 Invitrogen Cat# A-11055 Goat anti-mouse IgG, H&L chain specific peroxidase conjugate Calbiochem Cat# 401253 Goat anti-rabbit IgG, H&L chain specific peroxidase conjugate Calbiochem Cat# 401393 Ki-67 Cell Signaling Technology Cat# 9449 MAP2 Invitrogen Cat# PA5-17646 NANOG R&D Systems Cat# SC009 NESTIN BioLegend Cat# 656802 OCT-3/4 R&D Systems Cat# SC009 p62 BD Biosciences Cat# 610832 p70 S6 kinase Cell Signaling Technology Cat# 9202 PARP1 Santa Cruz Cat# sc-8007 PARP2 Santa Cruz Cat# sc-393310 PAX6 BioLegend Cat# 901301 Phospho-Histone H2A.X (Ser139) Millipore Cat# 05-636 Phospho-p70 S6 kinase (Thr389) Cell Signaling Technology Cat# 9206 Phospho-S6 ribosomal protein (Ser235/236) Cell Signaling Technology Cat# 2211 Poly(ADP-ribose) Enzo Life Sciences Cat# ALX-804-220-R100 Rabbit anti-goat IgG, H&L chain specific peroxidase conjugate Calbiochem Cat# 401515 S6 ribosomal protein Cell Signaling Technology Cat# 2217 SIRT1 Abcam Cat# ab7343 SIRT2 Proteintech Cat# 19655-1-AP SOX2 R&D Systems Cat# AF2018 SOX2 R&D Systems Cat# SC009 SSEA4 R&D Systems Cat# SC009 Tom20 Santa Cruz Biotech.

Techniques: Recombinant, Cell Culture, Knock-Out, Cytotoxicity Assay, CyQUANT Assay, Proliferation Assay, TUNEL Assay, Isolation, Quantitation Assay, Activity Assay, TALENs, Software