interference of crispr edits analysis v3 tool  (Synthego Inc)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Cell-targeted gene modification by delivery of CRISPR-Cas9 ribonucleoprotein complexes in pseudotyped lentivirus-derived nanoparticles

doi: 10.1016/j.omtn.2024.102318

Figure Lengend Snippet: Targeted DNA modification in cells treated with VSV-G-pseudotyped CRISPR-Cas9/sgRNA-loaded LVNPs (A) Plasmids used for production of LVNPs. Plasmids are co-transfected into HEK293T producer cells using the calcium phosphate method. Donut diagram shows the amounts of the different plasmids used in a transfection for the production of VSV-G-pseudotyped LVNPs. (B) 8 × 10 4 HEK293T cells were treated with increasing dosages of VSV-G-pseudotyped LVNPs targeting AFF1 or HEK3. Indel rates were determined 3 days after transduction ( n = 3 technical replicates). (C) 8 × 10 4 HEK293T cells were treated with increasing dosages of VSV-G-pseudotyped LVNPs targeting B2M . KO rates were determined by flow cytometry after 7 days using an anti-B2M-PE antibody ( n = 3 technical replicates). (D) Indel rates determined 3 days after treating 1 × 10 5 SupT1 cells with increasing dosages of VSV-G-pseudotyped LVNPs targeting AFF1 or HEK3 ( n = 2 technical replicates). (E) 1 × 10 5 SupT1 cells were treated with increasing dosages of VSV-G-pseudotyped LVNPs targeting B2M . KO rates were determined by flow cytometry after 7 days using an anti-B2M-PE antibody ( n = 2 technical replicates). Data are presented as mean (SD). Indel rates were determined by ICE analysis.

Article Snippet: Indels were analyzed using Synthego’s Interference of CRISPR Edits (ICE) analysis tool.

Techniques: Modification, CRISPR, Transfection, Transduction, Flow Cytometry

Journal: Molecular Therapy. Nucleic Acids

Article Title: Cell-targeted gene modification by delivery of CRISPR-Cas9 ribonucleoprotein complexes in pseudotyped lentivirus-derived nanoparticles

doi: 10.1016/j.omtn.2024.102318

Figure Lengend Snippet: MV-H/F and MV-H/F-SLAM pseudotyping of CRISPR-Cas9/sgRNA-containing LVNPs allows for cell- and gene-targeted DNA cleavage in primary B cells (A) The workflow for transduction of primary B cells. In half of the experiments, B cells were stimulated by IL-4 and IL-21. (B) Representative B cell profiles at day 6 (donor 1). (C) Representative SLAM expression levels measured by flow cytometry after 3 days of stimulation. CD20 staining was used to discriminate between B cells and feeder cells (donor 1). (D) CD40L stimulation resulted in upregulation of SLAM. Black and gray columns show the MFI and the percentage of SLAM + cells, respectively ( n = 3 biological replicates, donors 1 + 2). (E) Further stimulation with IL-4 and IL-21 resulted in higher MFI, translating to higher levels of SLAM on the cell surface ( n = 3 biological replicates, donors 1 + 2). Columns colors as in (D). (F) Pseudotyped LV-mediated EGFP gene transfer in B cells. 5 × 10 4 B cells were seeded on 5 × 10 4 feeder cells. B cells were transduced at an MOI of 5 IU/cell ( n = 3 biological replicates, donors 1–3). (G) Gene transfer in B cells after IL-4 and IL-21 stimulation. B cells were transduced at an MOI of 5 IU/cell ( n = 3 biological replicates, donors 1–3). (H) Indel analysis in B cells after treating 5 × 10 4 B cells with 500 ng p24 pseudotyped LVNPs targeting AFF1 ( n = 3 biological replicates, donors 1 + 2). (I) Stimulation with IL-4 and IL-21 resulted in increased gene modification in B cells treated with MV-H/F- and especially MV-H/F-SLAM-pseudotyped LVNPs ( n = 3 biological replicates, donors 1 + 2). Data are presented as mean (SD). Indel rates were determined by ICE analysis.

Article Snippet: Indels were analyzed using Synthego’s Interference of CRISPR Edits (ICE) analysis tool.

Techniques: CRISPR, Transduction, Expressing, Flow Cytometry, Staining, Modification