U6 grna cmv cas9 gfp

The CRISPR plasmid U6-gRNA/CMV-Cas9-GFP containing a guide RNA targeting mouse DKK3 was purchased from Sigma (MM0000346166). CAF1 cells (CAF-WT) were transfected with the plasmid using Lipofectamine (Life Technologies) following manufacturer’s instructions and GFP positive cells were single sorted into 96 well plates after 24 h. GFP-negative clones were also sorted as controls (e.g. WT.9). Individual cell clones were expanded and the DKK3 locus targeted by CRISPR was sequenced for knockout validation. In addition, loss of DKK3 protein expression was also confirmed by immunoblotting to generate DKK3-null CAFs (CAF-KO). DKK3-null clones were infected with DKK3-expressing lentivirus to generate KO-CAFs re-expressing DKK3 (CAF-KO-REC).

5 × 10⁵DUPmyo (or DUPmyo-i) myoblasts were electroporated with 1 μg of the highly pure CRISPR/Cas9-GFP plasmids by using the NEON device (Thermo Fisher Scientific) provided by Julie Dumonceaux and Virginie Mariot, as specified by the manufacturer instructions. Number of pulses, duration and voltage intensity were suggested by Dumonceaux and Mariot, as follows: 1 pulse, 20 ms, 1400 V. Electroporated plasmids were U6gRNA-CMVCas9-GFP (Sigma-Aldrich) expressing sgRNA0 and sgRNA2 (named CR0 and CR2).

GES1 and HFE145 cells were transiently transfected with CRISPR Cas single format vector with a backbone of U6-gRNA/CMV-Cas9-GFP (Sigma-Aldrich) and containing guide RNAs targeting exon 3 of CBS gene (HS0000002142 [clone1], HS0000002144 [clone 2], Sigma-Aldrich) or control vector (CRISPR universal negative control 1, Sigma-Aldrich) using Lipofectamine 2000. After 48 h, ~ 960 GFP positive single cells from each transfection were sorted by the Duke-NUS Flow core facility. After ~ 3 weeks, at least ten clones from each vector were cultured and screened and knockouts verified by loss of CBS protein using Western blotting. For each cell line model, 2 CBS-deficient clones, 1 CRISPR control, and parental cells were utilized for all downstream experiments.