lenticrispr sgrna construct  (Addgene inc)

Journal: iScience

Article Title: CASK loss of function differentially regulates neuronal maturation and synaptic function in human induced cortical excitatory neurons

doi: 10.1016/j.isci.2022.105187

Figure Lengend Snippet:

Article Snippet: Lenti-CRISPv2 sgRNA construct , Addgene , RRID: Addgene_52961.

Techniques: Virus, Bacteria, Produced, Recombinant, Electron Microscopy, Protease Inhibitor, RNA Extraction, Plasmid Preparation, Expressing, Next-Generation Sequencing, Generated, CRISPR, Construct, Software, Protein-Protein interactions

Journal: Frontiers in Physiology

Article Title: EDN1-AS , A Novel Long Non-coding RNA Regulating Endothelin-1 in Human Proximal Tubule Cells

doi: 10.3389/fphys.2020.00209

Figure Lengend Snippet: CRISPR/Cas9 Targeted Deletion of GR and E-Box Regulatory Elements within the EDN1-AS Predicted Promoter Region. CRISPR/Cas9 technology was utilized to create biallelic KO of a portion of the predicted Edn1AS promoter region, which contained GR and E-Box regulatory sites. Four guide RNAs (gRNAs) were designed to create deletion of this region (A) . LentiCRISPR was used to deliver the gRNAs to HK2 cells. Puromycin was used to screen cells. Using genotyping primers, GT1F and GT1R (solid arrows), two clones were identified that had a KO of approximately 350 bp generated with gRNAs 2 and 3 (dotted lines) (B) . Sequencing results reviled KO1 has a biallelic KO of 297 bp with 64 bp additional KO 5′ of this region on one allele and 78 bp KO 3′ of this region on the other allele (black solid lines). KO2 has a biallelic KO of 310 bp with 46 bp additional KO 3′ of this region on one allele and 71 bp KO 5′ of this region on the other allele (gray solid lines). Both KOs have a deletion of the 1st E-Box response element and the GR element. In addition, we identified control (CNTL) cells that had undergone the CRISPR process, but retained WT copes of the EDN1-AS regulatory region.

Article Snippet: HEK293 cells were co-transfected with the four LentiCRISPR + gRNA constructs (sgRNA 1, 2, 3, and 4) along with psPAX2 (Addgene #11260) and pMD2.G (Addgene #11259) for generation of pseudotyped lentiviral particles.

Techniques: CRISPR, Clone Assay, Generated, Sequencing, Control