Journal: iScience

Article Title: Genome editing is induced in a binary manner in single human cells

doi: 10.1016/j.isci.2022.105619

Figure Lengend Snippet: Measurement of the copy number of RBM20 in HEK293T cells (A) A hypothetical situation that emphasizes the importance of the analysis of genome editing outcomes at the single cell level. Cell populations 1 and 2 consist of cells with totally different genotypes individually. However, the total allelic frequencies of WT, HDR, and NHEJ are exactly the same for both populations. (B) Representative karyotypes of HEK293T cells with three and four chromosome 10s. (C) A CGH analysis of HEK293T cells in comparison to diploid human iPS cells. The relative CGH signal of HEK293T cells normalized to that of diploid iPS cells is shown throughout the genome. (D) CGH copy number peak assignment. In the CGH analysis, there were several peaks of the CGH signal ratio between HEK293T cells and diploid iPS cells based on the chromosomal numbers. The highest peak corresponded to three copies per cell, and was set as the baseline of the CGH log ratio between HEK293T cells and iPS cells. (E) Line of fit between the copy number and the CGH log ratio based on the peak assignment shown in (D). The copy number and the CGH log ratio showed a clear linear correlation. (F) Scattered plot of relative CGH signal of HEK293T cells in comparison to diploid human iPS cells around the RBM20 gene. The relative CGH signals of HEK293T cells normalized by that of iPS cells are represented by +. No microduplications or microdeletions were detected around the RBM20 locus.

Article Snippet: HEK293T cells (RCB2202), HeLa cells (RCB0007) and PC9 cells (RCB4455) are available at RIKEN BioResource Research Center ( https://cell.brc.riken.jp/en/ ) and WTC11 iPS cells (GM25256) are available at Coriell Institute for Medical Research ( https://www.coriell.org/ ).

Techniques: Comparison

Journal: iScience

Article Title: Genome editing is induced in a binary manner in single human cells

doi: 10.1016/j.isci.2022.105619

Figure Lengend Snippet: Schematic illustration of the experiments of this study (A) Design of genome editing in RBM20 as a model case in this study. We introduced the R636S (c.1906C>A) mutation using CRISPR-Cas9 and a single-stranded oligonucleotide donor DNA. The resulting HDR allele has a C to A single nucleotide substitution, whereas the NHEJ alleles have various insertions and deletions. (B) The experimental flow of this study. We expressed Cas9-T2A-EGFP together with the gRNA targeting RBM20 to label HEK293T cells in which Cas9 protein was expressed. Using a microfluidic cell sorter (On-chip Sort), we sorted EGFP+ cells. Then, the sorted cells were plated into four 96-well plates by the SPiS. Cells were cultured for 2 to 4 weeks and then the genome editing outcomes were analyzed by amplicon sequencing. (C) An imaging analysis of single cells dispensed by the SPiS. The SPiS aspirates cell suspension into a specialized microtip and takes two images with a 1-s interval. Only when the SPiS recognized a single cell, the content of the tip was dispensed into a well of a 96-well plate. Otherwise, the SPiS would take another aliquot of the cell suspension to repeat the process.

Article Snippet: HEK293T cells (RCB2202), HeLa cells (RCB0007) and PC9 cells (RCB4455) are available at RIKEN BioResource Research Center ( https://cell.brc.riken.jp/en/ ) and WTC11 iPS cells (GM25256) are available at Coriell Institute for Medical Research ( https://www.coriell.org/ ).

Techniques: Mutagenesis, CRISPR, Cell Culture, Amplification, Sequencing, Imaging, Suspension

Journal: iScience

Article Title: Genome editing is induced in a binary manner in single human cells

doi: 10.1016/j.isci.2022.105619

Figure Lengend Snippet: Genome editing outcomes in RBM20 in individual HEK293T cells edited by Cas9 (A) Genome editing outcomes in isolated clones derived from single HEK293T cells edited by Cas9. We repeated the experiment three times, and isolated more than 90 clones out of 384 cells plated in all three trials. Each bar represents one clone, and the proportions of WT (green), NHEJ (blue), HDR (red), and HDR + NHEJ (purple) in one clone are also shown in each bar. (B) Total allelic frequencies in genome edited HEK293T cells in the three experiments shown in (A). (C) Models of the distributions of HEK293T cell clones with different genome editing outcomes in the Cas9 No.3 experiment, if genome editing randomly occurred at the frequencies shown in (B) in HEK293T cells with three or four copies of RBM20. (D) Comparison of the proportions of WT and full NHEJ clones between the mathematical models with three and four copies of RBM20 and the actually observed cells. Values ±S.E. are shown (n = 3). Student’s t test was used to evaluate differences. ∗p <0.05 and ∗∗p <0.01.

Article Snippet: HEK293T cells (RCB2202), HeLa cells (RCB0007) and PC9 cells (RCB4455) are available at RIKEN BioResource Research Center ( https://cell.brc.riken.jp/en/ ) and WTC11 iPS cells (GM25256) are available at Coriell Institute for Medical Research ( https://www.coriell.org/ ).

Techniques: Isolation, Clone Assay, Derivative Assay, Comparison

Journal: iScience

Article Title: Genome editing is induced in a binary manner in single human cells

doi: 10.1016/j.isci.2022.105619

Figure Lengend Snippet: Genome editing outcomes in RBM20 in individual HEK293T cells edited by HypaCas9 (A) Genome editing outcomes in isolated clones derived from single HEK293T cells edited by HypaCas9 and the single-stranded donor DNA. We repeated the same experiment three times. Each bar represents one clone, and the proportions of WT (green), NHEJ (blue), HDR (red), and HDR + NHEJ (purple) in one clone are also shown in each bar. (B) Total frequencies of WT (green), NHEJ (blue), HDR (red), and HDR + NHEJ (purple) allelic frequencies in genome edited HEK293T cells in the three experiments shown in (A). (C) Comparison of total allelic frequencies of WT, NHEJ, HDR, and HDR + NHEJ between Cas9 and HypaCas9. Values ±S.E. are shown (n = 3). Student’s t test was used to evaluate differences. ∗p <0.05 and NS: not significantly different (p >0.1). (D) Models of the distributions of HEK293T cell clones with different genome editing outcomes in the HypaCas9 No.3 experiment, if genome editing randomly occurred at the frequencies shown in (B) in HEK293T cells with three or four copies of RBM20. (E) Comparison of the proportions of WT and full NHEJ clones between the mathematical models with three and four copies of RBM20 and the actually observed cells. Values ±S.E. are shown (n = 3). Student’s t test was used to evaluate differences. ∗∗p <0.01. (F) Comparison of the proportions of clones with partial editing by HDR and clones with HDR accompanied by NHEJ events between the models with three and four copies of RBM20 and the actually observed cells. Values ±S.E. are shown (n = 3). Student’s t test was used to evaluate differences. ∗p <0.05 and NS: not significantly different (p >0.1).

Article Snippet: HEK293T cells (RCB2202), HeLa cells (RCB0007) and PC9 cells (RCB4455) are available at RIKEN BioResource Research Center ( https://cell.brc.riken.jp/en/ ) and WTC11 iPS cells (GM25256) are available at Coriell Institute for Medical Research ( https://www.coriell.org/ ).

Techniques: Isolation, Clone Assay, Derivative Assay, Comparison

Journal: iScience

Article Title: Genome editing is induced in a binary manner in single human cells

doi: 10.1016/j.isci.2022.105619

Figure Lengend Snippet: Genome editing outcomes in ATP7B and GRN in individual HEK293T cells edited by HypaCas9 (A) Genome editing outcomes in isolated clones derived from single HEK293T cells edited by HypaCas9 and the single-stranded donor DNA targeting ATP7B. We repeated the same experiment three times. Each bar represents one clone, and the proportions of WT (green), NHEJ (blue), HDR (red), and HDR + NHEJ (purple) in one clone are also shown in each bar. (B) Comparison of the proportions of WT and full NHEJ clones between the mathematical models with three copies of ATP7B and the actually observed cells. Values ±S.E. are shown (n = 3). Student’s t test was used to evaluate differences. ∗p <0.05 and ∗∗p <0.01. (C) Comparison of the proportions of clones with partial editing by HDR and clones with HDR accompanied by NHEJ events between the models with three copies of ATP7B and the actually observed cells. Values ±S.E. are shown (n = 3). Student’s t test was used to evaluate differences. NS: not significantly different (p >0.1). (D) Genome editing outcomes in isolated clones derived from single HEK293T cells edited by HypaCas9 and the single-stranded donor DNA targeting GRN. We repeated the same experiment three times. Each bar represents one clone, and proportions of WT (green), NHEJ (blue), HDR (red), and HDR + NHEJ (purple) in one clone are also shown in each bar. (E) Comparison of the proportions of WT and full NHEJ clones between the mathematical models with three and four copies of GRN and the actually observed cells. Values ±S.E. are shown (n = 3). Student’s t test was used to evaluate differences. ∗p <0.05 and ∗∗p <0.01.

Article Snippet: HEK293T cells (RCB2202), HeLa cells (RCB0007) and PC9 cells (RCB4455) are available at RIKEN BioResource Research Center ( https://cell.brc.riken.jp/en/ ) and WTC11 iPS cells (GM25256) are available at Coriell Institute for Medical Research ( https://www.coriell.org/ ).

Techniques: Isolation, Clone Assay, Derivative Assay, Comparison

Journal: iScience

Article Title: Genome editing is induced in a binary manner in single human cells

doi: 10.1016/j.isci.2022.105619

Figure Lengend Snippet:

Article Snippet: HEK293T cells (RCB2202), HeLa cells (RCB0007) and PC9 cells (RCB4455) are available at RIKEN BioResource Research Center ( https://cell.brc.riken.jp/en/ ) and WTC11 iPS cells (GM25256) are available at Coriell Institute for Medical Research ( https://www.coriell.org/ ).

Techniques: Recombinant, Transfection, Library Quantification, Microarray, Control, Amplification, Sequencing, Cloning, Software, FACS, Digital PCR

Journal: Neuroscience Bulletin

Article Title: Distribution and Functional Characteristics of Voltage-Gated Sodium Channels in Immature Cochlear Hair Cells

doi: 10.1007/s12264-019-00415-3

Figure Lengend Snippet: Electrophysiological recordings from CbmNav1.7 variants and related mutants in HEK293T cells. A, B Locations of the substitution and deletion of amino-acids in CbmNav1.7a and 1.7b compared with wtNav1.7 (purple, segment replacement generated by alternative splicing of exon 5; red, segment deletion generated by alternative splicing of exon 11 or exon 20; blue, single amino-acid residue replacement produced by three RNA editing events in exons 15, 16. and 26). C–E Na+ current recordings from wtNav1.7, CbmNav1.7a, and CbmNav1.7b exogenously expressed in HEK293T cells. F–N Na+ current recordings of nine Nav1.7 mutants exogenously expressed in HEK293T cells.

Article Snippet: Whole-Cell Electrophysiological Recordings from HEK293T Cells Whole-cell voltage-clamp recordings were made using an Axon Multiclamp 700B Microelectrode Amplifier (Molecular Devices) at room temperature (21 °C–25 °C).

Techniques: Generated, Produced