benchling crispr design tool  (Benchling Inc)

Journal: International Journal of Molecular Sciences

Article Title: Precise CRISPR-Mediated Editing of the TGFBI R555W Mutation in Patient-Derived Peripheral Blood Mononuclear Cells

doi: 10.3390/ijms27052418

Figure Lengend Snippet: Delivery of CRISPR/Cas9 components and enrichment of PBMCs. ( a ) Pre-electroporation preparation of PBMCs. Representative bright-field microscopy images showing PBMC morphology at 4×, 10×, 20×, and 40× magnification. The schematic also illustrates the CRISPR/Cas9 plasmid construct and ssODN donor used for transfection. ( b ) Electroporation conditions using a single square-wave pulse. ( c ) Fluorescence microscopy images showing GFP expression 24 h post-electroporation, confirming successful plasmid delivery. ( d ) Flow cytometric analysis and sorting of GFP-positive PBMCs, indicating transfection efficiencies for each sgRNA–ssODN combination.

Article Snippet: Therefore, off-target scores of the designed sgRNAs were calculated using the high-confidence Benchling CRISPR Tool, and sgRNAs with the highest on-target and lowest predicted off-target scores were selected for further experiments.

Techniques: CRISPR, Electroporation, Microscopy, Plasmid Preparation, Construct, Transfection, Fluorescence, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Precise CRISPR-Mediated Editing of the TGFBI R555W Mutation in Patient-Derived Peripheral Blood Mononuclear Cells

doi: 10.3390/ijms27052418

Figure Lengend Snippet: High-resolution melting (HRM) screening of CRISPR/Cas9-edited PBMCs. Derivative melting curves (−dF/dT, y -axis, versus temperature, x -axis) are shown for PBMCs transfected with sgRNA1–ssODN1, sgRNA2–ssODN2, and sgRNA3–ssODN3, each compared with mock-electroporated controls. HRM was used as an initial screening step to identify candidate editing events based on differences in melting curve profiles. For semi-quantitative evaluation, peak areas under the melting curves were measured using ImageJ software (version 1.54g; National Institutes of Health, Bethesda, MD, USA). Bar graphs represent descriptive peak area measurements obtained from pooled samples (n = 1 per condition) and are presented for descriptive comparison only. No inferential statistical tests were performed.

Article Snippet: Therefore, off-target scores of the designed sgRNAs were calculated using the high-confidence Benchling CRISPR Tool, and sgRNAs with the highest on-target and lowest predicted off-target scores were selected for further experiments.

Techniques: CRISPR, Transfection, Software, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Precise CRISPR-Mediated Editing of the TGFBI R555W Mutation in Patient-Derived Peripheral Blood Mononuclear Cells

doi: 10.3390/ijms27052418

Figure Lengend Snippet: Next-generation sequencing (NGS) analysis of CRISPR/Cas9-edited TGFBI locus. ( a ) Representative sequencing reads from GFP-positive PBMCs transfected with the sgRNA3–ssODN3 combination showing the targeted nucleotide substitution and ssODN-introduced guide-blocking silent mutations ( b ) Schematic overview of the sgRNA3 target region and detected editing outcomes at the c.1663 site. A total of 811,358 sequencing reads were obtained for this amplicon (see ).

Article Snippet: Therefore, off-target scores of the designed sgRNAs were calculated using the high-confidence Benchling CRISPR Tool, and sgRNAs with the highest on-target and lowest predicted off-target scores were selected for further experiments.

Techniques: Next-Generation Sequencing, CRISPR, Sequencing, Transfection, Blocking Assay, Amplification