Journal: eLife

Article Title: Novel mechanism for tubular injury in nephropathic cystinosis

doi: 10.7554/eLife.94169

Figure Lengend Snippet: ( A ) Outline of RNP CRISPR editing. The CTNS gene is located on chromosome 17p13.3 and consists of 12 exons, of which the first 2 are non-coding. Therefore, the guide RNA was targeted towards exon 3 to completely knockout the functional CTNS gene. CRISPR-Cas9 ribonucleoproteins (crRNPs) were synthesized in vitro to knockout CTNS gene and delivered to immortalize RPTECs by nucleofection. These cells were expanded for molecular validation of gene editing and downstream functional assays. ( B ) The guide RNA target sequence and associated PAM are highlighted on the right. We performed Sanger sequencing and the TIDE output calculating percent indels from chromatograms is depicted in the bar-graph. ( C ) Validation of CTNS knockout (-/-) in immortalized RPTEC by using two primers targeting different exons – CTNS #1 targets between exon 2–3 and CTNS #2 targets between exon 9–10. We have shown that in the CRISPR/Cas9 CTNS -/- RPTEC there is a significant reduced Cystinosin RNA levels with both the primers. ( D ) Validation of CTNS -/- in immortalized RPTEC. Increased intracellular accumulation of cystine is shown by HPLC-MS/MS method in control and CTNS -/- RPTECs. Student’s t-test was used. Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Transfected Construct (CRISPR/cas9) ( Homo-sapiens ) , guide RNA, tracrRNA, and Cas9 , Benchling/Dharmacon , , .

Techniques: CRISPR, Knock-Out, Functional Assay, Synthesized, In Vitro, Biomarker Discovery, Sequencing, Tandem Mass Spectroscopy, Control

Journal: eLife

Article Title: Novel mechanism for tubular injury in nephropathic cystinosis

doi: 10.7554/eLife.94169

Figure Lengend Snippet:

Article Snippet: Transfected Construct (CRISPR/cas9) ( Homo-sapiens ) , guide RNA, tracrRNA, and Cas9 , Benchling/Dharmacon , , .

Techniques: Transfection, Construct, CRISPR, Sequencing, Microarray, Isolation, Recombinant, Plasmid Preparation, Membrane

Journal: eLife

Article Title: Novel Mechanism for Tubular Injury in Nephropathic Cystinosis

doi: 10.7554/elife.94169

Figure Lengend Snippet: Figure 3. Experimental overview for the development of immortalized CTNS-/- RPTECs using CRISPR-Cas9 RNPs. (A) Outline of RNP CRISPR editing. The CTNS gene is located on chromosome 17p13.3 and consists of 12 exons, of which the first 2 are non-coding. Therefore, the guide RNA was targeted towards exon 3 to completely knockout the functional CTNS gene. CRISPR-Cas9 ribonucleoproteins (crRNPs) were synthesized in vitro to knockout CTNS gene and delivered to immortalize RPTECs by nucleofection. These cells were expanded for molecular validation of gene editing and

Article Snippet: resource Designation Source or reference Identifiers Additional information Cell line (Homo- sapiens) RPTECs; Skin Fibroblasts Gift from Drs. Gahl and Racusen Primary Cell line (Homo- sapiens) RPTEC Cambrex Biosciences Primary Cell line (Homo- sapiens) Skin Fibroblasts Coriell Cell Repositories Primary Cell line (Homo- sapiens) Immortalized RPTEC Kerafast Cat No. ECH001 Immortalized cell line, Male Drug Astaxanthin (ATX) Millipore Sigma Cat. No SML0982 Drug Cysteamine Millipore Sigma Cat. No M9768 Drug Vitamin E Selleckchem Cat. No S4686 Transfected Construct (CRISPR/cas9) (Homosapiens) guide RNA, tracrRNA, and Cas9 Benchling/Dharmacon Sur et al. eLife 2024;13:RP94169.

Techniques: CRISPR, Knock-Out, Functional Assay, Synthesized, In Vitro, Biomarker Discovery