Journal: Frontiers in Genome Editing

Article Title: T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds

doi: 10.3389/fgeed.2023.1130736

Figure Lengend Snippet: Obligate-heterodimerization mitigates off-target activity of CCR5 -TALEN (A) Schematic of possible TALEN pairing combinations. Combinations are displayed for TALENs with wild-type (WT) and obligate-heterodimeric (OH) FokI domains. (B) Genotyping. Results of T7E1 assay of WT-TALEN and OH-TALEN (KKR-ELD and KVR-EAD configuration) are shown. 32°C indicates that T cells were subjected to transient cold-shock after electroporation, while control cells were constantly cultured at 37°C. * and ** specify p -values of <0.05 or <0.01 (Student’s t-test, n = 3–5). (C) TALEN expression. Percentage of TALEN-expressing T cells upon mRNA transfer as determined by flow cytometry (n = 1). (D,E) Structural variations. Circos plots illustrate T-CAST results with enlargement of the chromosome three region encompassing CCR5 and CCR2 loci. Lines represent chromosomal rearrangements with the CCR5 target site: OMTs with >20 hits in red, ambiguous classification (OMT/HMT) in yellow. (F,G) Genotyping. NGS at denoted sites was performed on untreated (UT) T cells, and T cells edited with WT-TALENs or OH-TALENs as indicated. The group in which the samples were originally identified is indicated on the bottom. *** specifies p -value<0.001 (Fisher’s exact test, Bonferroni corrected p -values).

Article Snippet: Cells were resuspended in 50 μl CliniMACS ® Electroporation Buffer (Miltenyi Biotec).

Techniques: Activity Assay, TALENs, Electroporation, Control, Cell Culture, Expressing, Flow Cytometry

Journal: Frontiers in Genome Editing

Article Title: T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds

doi: 10.3389/fgeed.2023.1130736

Figure Lengend Snippet: T-CAST analysis for TRAC -targeting TALENs (A) Genotyping. Results of T7E1 assay of WT-TALEN and OH-TALEN (KKR-ELD and KVR-EAD configuration) are shown. 32°C indicates that T cells were subjected to transient cold-shock after electroporation, while control cells were constantly cultured at 37°C. * specifies p -value<0.05 (Student’s t-test, n = 3–6). (B) TCR expression. Displayed is the fraction of TCRα/β-negative T cells upon transfer of TALEN-encoding mRNA as determined by flow cytometry. Where indicated (32°C) T cells were subjected to a transient cold-shock. *** specifies p -value<0.001 (Student’s t-test; n = 3–7). (C–E) Structural variations. Circos plots illustrate T-CAST results with enlargement of the chromosome 14 region encompassing TRAC . Red lines represent chromosomal rearrangements (OMTs with >20 hits) with the TRAC target site. (F) Genotyping. NGS at denoted sites was performed on untreated (UT) T cells, and T cells edited with WT-TALENs or OH-TALENs as indicated. The group in which the samples were originally identified is indicated on the bottom. *** specifies p -value<0.001 (Fisher’s exact test, Bonferroni corrected p -values).

Article Snippet: Cells were resuspended in 50 μl CliniMACS ® Electroporation Buffer (Miltenyi Biotec).

Techniques: TALENs, Electroporation, Control, Cell Culture, Expressing, Flow Cytometry

Journal: Journal of Cell Science

Article Title: A CRISPR-del-based pipeline for complete gene knockout in human diploid cells

doi: 10.1242/jcs.260000

Figure Lengend Snippet: Quantitative analyses of length-dependent DNA deletion efficiency for the optimized CRISPR-del method using flow cytometry. (A) Schematic representation of the chromosome region around the HNRNPA1 gene locus. The mNG tag was inserted into the chromosomal site at the C-terminus of HNRNPA1 gene. The target positions of sgRNAs and the expected lengths of large deletions are shown. (B) Fluorescence imaging of HNRNPA1–mNG in RPE1 HNRNPA1-mNG cells electroporated with Cas9 protein and the indicated sgRNA pairs. Images are representative of three experimental repeats. Arrowheads indicate cells without the expression of HNRNPA1–mNG. Scale bars: 10 µm. (C) FACS analyses for the mNG expression in RPE1 HNRNPA1-mNG cells electroporated with the indicated sgRNAs as in B. Cells at 8 days after Cas9/sgRNAs electroporation were analyzed. (D) Quantification of mNG negative cells for each sgRNA pair from C. N =three biologically independent samples. ≥10,000 cells were analyzed for each sample. The percentage of mNG-negative cells for each sample is indicated on top of the histogram. (E) FACS analyses with the indicated conditions, as in C. (F) Quantification of E, as in D. Data are represented as mean±s.d. * P <0.05; ** P <0.01; *** P <0.001; N.S., not significant (Tukey–Kramer test).

Article Snippet: A.s.Cpf1 Ultra (1 μM, IDT) and crRNA (1 μM) were pre-incubated in buffer R and mixed with RPE1 cells (0.125×10 5 cells/μl), Cpf1 electroporation enhancer (1.8 μM, IDT) and the repair template (33 nM). mNG-positive cells were sorted using FACS Aria III (BD Biosciences), equipped with 355, 405, 488, 561 and 633 nm lasers to generate a mono-allelic HNRNPA1-mNG clone.

Techniques: CRISPR, Flow Cytometry, Fluorescence, Imaging, Expressing, Electroporation