Journal: Scientific Reports

Article Title: Transiently expressed CRISPR/Cas9 induces wild-type dystrophin in vitro in DMD patient myoblasts carrying duplications

doi: 10.1038/s41598-022-07671-w

Figure Lengend Snippet: Dystrophin correction in immortalized DUPmyo cells electroporated with CRISPR/Cas9-expressing plasmids. ( a ) Schematic of the plasmid where CR0 and CR2 were cloned. ( b ) T7E1 assay performed on HEK293T transfected with the LCR2 plasmid (lentiviral vector expressing sgRNA2), CR0 (negative control plasmid) and CR2 (plasmid expressing sgRNA2). Arrows indicated cleaved bands of expected molecular size in cells expressing the nuclease. LCR2 was used as a reference to evaluate the targeting efficiency of CR2. CR0 did not show cleaved bands as LCR2 and CR2, proving it is a valid negative control for monitoring the targeting effect of CRISPR/Cas9. NT = untreated cells. ( c ) FACS analysis of immortalized DUPmyo-i myoblasts electroporated by NEON. ( d ) T7E1 assay performed on the total pool of electroporated DUPmyo-i cells expressing CR0 and CR2. ( e ) Efficiency of genomic targeting evaluated in T7E1 replicates (n = 3). ( f ) Mutated dystrophin transcript in cells expressing CR0 and CR2 (Kruskall-Wallis test, p = 0.0552) (n = 3 technical replicates/sample). ( g ) Western blot showing dystrophin correction (427 kDa band) in cells expressing CR2. Vinculin (116 KDa band) and meta-vinculin (124 kDa band) were probed as a loading control and a measure of myogenic differentiation, respectively. ( h ) Percentages of mutated dystrophin assessed in electroporated DUPmyo-i (Kruskall-Wallis test, p = 0.0679) (n = 3). i) Comparison of mutated dystrophin observed in patient-myoblasts following LCR2-transduction (n = 4) and CR2 electroporation (n = 3) (Mann–Whitney test, p = 0.4). NT = untreated cells. TotCR0/TotCR2 = total pool of cells expressing CR0/CR2. WT = protein derived from the immortalized murine H2K 2B4 cells, expressing wild-type dystrophin (positive control), LCR2 = transduced cells.

Article Snippet: The best sgRNA (sgRNA2) was also cloned into the integrating pL-CRISPR.EFS.GFP plasmid from Benjamin Ebert’s laboratory (Addgene #57818), following the specified protocol.

Techniques: CRISPR, Expressing, Plasmid Preparation, Clone Assay, Transfection, Negative Control, Western Blot, Control, Comparison, Transduction, Electroporation, MANN-WHITNEY, Derivative Assay, Positive Control

Journal: Autophagy

Article Title: The Epstein-Barr virus deubiquitinase BPLF1 regulates stress-induced ribosome UFMylation and reticulophagy

doi: 10.1080/15548627.2024.2440846

Figure Lengend Snippet: Reagents used in this paper.

Article Snippet: px330-UFSP2 sgRNA2 , Addgene; gift from Yihong Ye , 134637.

Techniques: Recombinant, Diagnostic Assay, Transfection, Mutagenesis, Cloning, cDNA Synthesis, SYBR Green Assay, DC Protein Assay, Protein Purification, Knock-Out, Plasmid Preparation