Journal: Molecular Therapy. Methods & Clinical Development

Article Title: In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9

doi: 10.1016/j.omtm.2020.10.007

Figure Lengend Snippet: Design and Use of CRISPR gRNAs in HEK293T Cell Line (A) Exon 9 of the human PKLR gene is represented. Vertical lines and arrows are reported mutations, corresponding to one or more than 1 nt affected, respectively. Horizontal dashed lines represent the two gRNAs used. Vertical dashed lines represent predicted cut sites of gRNAs selected. (B) Sorting of green fluorescence based on the ZsGreen present in the electroporated plasmids. A 530/40- versus 580/30-nm dot plot was used to distinguish ZsGreen fluorescence from autofluorescence. The figure represents the ZsGreen fluorescence of the cell electroporated with the plasmid DNA containing only the Cas9 and the ZsGreen cDNAs (C), or containing the Cas9, the ZsGreen cDNAs and the sequences of the guide I (I) or the guide II (II). (C) Surveyor assay of the DNA obtained from the cells electroporated with the plasmids containing no guide, guide I, guide II, or both guides together (I+II). The gene editing generates a characteristic band pattern for guide I (160 bp + 135 bp), for guide II (191 bp + 104 bp), and a mixed pattern when both are used at the same time (160 bp + 104 bp). (D) Scheme of the band sizes that appear in (C) showing the 31-bp deletion resulting after the simultaneous electroporation of guides I and II.

Article Snippet: The HEK293T cell line (human embryonic kidney cell line competent to replicate vectors carrying the SV40 T antigen; ATCC, CRL-3219) was cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco), HyClone fetal bovine serum (10%; GE Healthcare), and penicillin/streptomycin (1%; Gibco).

Techniques: CRISPR, Fluorescence, Plasmid Preparation, Electroporation

Journal: Neuron

Article Title: A Selectivity Filter Gate Controls Voltage-Gated Calcium Channel Calcium-Dependent Inactivation

doi: 10.1016/j.neuron.2019.01.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Virus Strains Dh5α competent E. coli MCLAB Cat#DA-196 Chemicals, Peptides, and Recombinant Proteins ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) Sigma-Aldrich Cat#E4378 1,2-Bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetrakis (BAPTA) Santa Cruz Biotechnology Cat#sc-202076 Dulbecco’s modified Eagle’s medium (DMEM) GIBCO Cat#11965-092 fetal bovine serum (FBS) GIBCO Cat#16140-071 L-glutamine GIBCO Cat#25030-081 penicillin/streptomycin UCSF Cell Culture Facility Cat#CCFGK004-153K01 Lipofectamine 2000 Invitrogen Cat#11668-019 Matrigel BD Biosciences Cat#354234 Critical Commercial Assays T7 mMessenger kit Thermo Fisher Scientific Cat# AM1344 QuikChange Site-Directed Mutagenesis Kit Agilent Cat#200515 Experimental Models: Cell Lines HEK293 ATCC Cat#CRL-1573 Xenopus oocytes This study N/A Experimental Models: Organisms/Strains Xenopus Laevis Nasco Cat# LM00531 Recombinant DNA human Ca V 1.2/pcDNA3.1 Findeisen and Minor, 2009 N/A human Ca V 1.2 D707A/pcDNA3.1 Shaya et al., 2014 N/A human Ca V 1.2 D707N/pcDNA3.1 Shaya et al., 2014 N/A human Ca V 1.2 D707G/pcDNA3.1 Shaya et al., 2014 N/A human Ca V 1.2 D707E/pcDNA3.1 This study N/A human Ca V 1.2 E1115A/pcDNA3.1 This study N/A human Ca V 1.2 D367A/pcDNA3.1 This study N/A human Ca V 1.2 E1119A/pcDNA3.1 This study N/A human Ca V 1.2 D1420A/pcDNA3.1 This study N/A human Ca V 1.2 G364D/D707G/pcDNA3.1 This study N/A human Ca V 1.2 D707G/G1116D/pcDNA3.1 This study N/A human Ca V 1.2 D707A/A1417D/pcDNA3.1 This study N/A rat Ca V 1.3 42a /pcDNA6/V5-His ABC Xu and Lipscombe, 2001 Addgene#26577 rat Ca V 1.3 D726A/pcDNA6/V5-His ABC This study N/A rat Ca V 1.3 E1121A/pcDNA6/V5-His ABC This study N/A human Ca V 2.1/pcDNA3.1 Kim et al., 2008 N/A human Ca V 2.1 D667A/pcDNA3.1 This study N/A human Ca V 2.1 E1461A/pcDNA3.1 This study N/A rat Ca V β 2a /pTracer-CMV2-GFP Findeisen and Minor, 2009 N/A rabbit Ca V β 3 /pSport This study N/A rabbit Ca V α 2 δ-1/pcDNA3.1 Findeisen and Minor, 2009 N/A sv40 T-antigen/Bluescribe addgene Cat#21826 Software and Algorithms pCLAMP 9 Molecular Devices N/A Clampfit 10 Molecular Devices N/A SigmaStat 3.1 Systat N/A Open in a separate window KEY RESOURCES TABLE Ca V selectivity filter forms the calcium-dependent inactivation (CDI) endpoint Conserved Ca V domain II selectivity filter (+1) aspartate plays an active role in CDI Ca V selectivity filter asymmetry is important for CDI Ca V s gating relies on an SF-based gating framework shared among the VGIC superfamily

Techniques: Recombinant, Modification, Cell Culture, Mutagenesis, Software