protein minipreptm kit  (Zymo Research)

Journal: Journal of Biomedical Science

Article Title: Screening strategy to identify Cas9 variants with higher HDR activity based on diphtheria toxin

doi: 10.1186/s12929-025-01197-9

Figure Lengend Snippet: Scheme of the HDR-inducible Cas9 screening system. A HDR occurred during cell selection by Diphtheria toxin (DT). The human HBEGF (hHBEGF) gene was edited into a E141K mutated hHBEGF (mHBEGF) downstream of the cleavage site through HDR repair . Then, human cells acquire DT resistance. B Off-target effects of EGFP expression on the cell selection system. The PAM sequence of the target site in the EGFP gene is underlined with a blue line. A silent mutation was introduced into the 5′ end of the EGFP target site to insert the SnaBI recognition site. A library of off-target sgRNAs targeting randomly mutated sequences 6 bases from the 5′ end was constructed by removing the target site recognition sgRNA plasmid via SnaBI and exonuclease. C A scheme for the selection of HDR-inducible Cas9 from a mutant library ( C ) was created with BioRender.com

Article Snippet: Purification of His-tagged Cas9 proteins was performed via the His-Spin Protein Miniprep Kit (Zymo Research) according to the manufacturer’s protocol.

Techniques: Selection, Expressing, Sequencing, Mutagenesis, Construct, Plasmid Preparation

Journal: Journal of Biomedical Science

Article Title: Screening strategy to identify Cas9 variants with higher HDR activity based on diphtheria toxin

doi: 10.1186/s12929-025-01197-9

Figure Lengend Snippet: Optimization of screening conditions. A Consideration of the conditions for introducing a random mutation in the nuclease domain of SpCas9 via a Diversify PCR Random Mutagenesis Kit. B Optimization of the amount of template DNA and hHBEGF target sgRNA for screening of hTERT-RPE1 cells via the Neon Transfection System 100 μL kit. The pulse conditions were 1100 V, 20 ms, and two pulses. The left line graph shows the ratio of mCherry-positive cells when the amount of template DNA and sgRNA-encoding plasmid was increased while the molar ratio was maintained at 1:1. The middle line graph shows the ratio of mCherry-positive cells when only template DNA was transfected into hTERT-RPE-1 cells. The right line graph shows the ratio of mCherry-positive cells when the amount of hHBEGF target sgRNA was increased while the amount of template DNA was maintained at 2.5 μg. C Consideration of the amount of the EGFP off-target sgRNA library. The left line graph shows the ratio of EGFP-positive cells when the amount of EGFP target sgRNA was increased. The right date of flow cytometry shows the ratio of EGFP-positive cells when the off-target sgRNA library was introduced into hTERT-RPE1 cells. A wild-type Cas9-encoding lentiviral vector was used, as shown in Fig. B and C

Article Snippet: Purification of His-tagged Cas9 proteins was performed via the His-Spin Protein Miniprep Kit (Zymo Research) according to the manufacturer’s protocol.

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Flow Cytometry

Journal: Journal of Biomedical Science

Article Title: Screening strategy to identify Cas9 variants with higher HDR activity based on diphtheria toxin

doi: 10.1186/s12929-025-01197-9

Figure Lengend Snippet: The activity of the top three obtained mutants (#13, #24, and #27 clones) compared with that of wild-type Cas9. The Cas9 gene was transfected with an episomal vector (pEBMulti. Hyg) via lipofection. Plasmid DNA coding sgRNA and template ssODN, which knock in 12 bases by removing 3 bases from the original sequence, were transfected via electroporation after 3 days of selection with hygromycin B. Editing efficiencies were analyzed via Sanger sequencing and ICE software. The red bar graphs show the results for wild-type Cas9. The green bar graphs show the results for clone #13. The blue bar graphs show the results for clone #24. The purple bar graphs show the results for clone #27. A The ratio of HDR-mediated target knock-in at the AAVS1, EMX1, and VEGFA target sites in HEK293 cells. B The ratio of target mutations at the AAVS1, EMX1, and VEGFA target sites in HEK293 cells. C The ratio of target knock-in per target mutation at the AAVS1, EMX1, and VEGFA target sites in HEK293 cells. All the samples except for the AAVS1 target Cas9 sample were performed in four replicates. One trial of AAVS1 targeting Cas9 could not be performed because of sample loss, but three replicates of AAVS1 targeting Cas9 were performed. The p values in the figures are the p values obtained by performing a Tukey test when the significance level was set at 5%. The raw data sets of each trial are shown in Supplementary Table 3

Article Snippet: Purification of His-tagged Cas9 proteins was performed via the His-Spin Protein Miniprep Kit (Zymo Research) according to the manufacturer’s protocol.

Techniques: Activity Assay, Clone Assay, Transfection, Plasmid Preparation, Knock-In, Sequencing, Electroporation, Selection, Software, Mutagenesis

Journal: Journal of Biomedical Science

Article Title: Screening strategy to identify Cas9 variants with higher HDR activity based on diphtheria toxin

doi: 10.1186/s12929-025-01197-9

Figure Lengend Snippet: Differences in genome editing features between wild-type Cas9 and HSS Cas9 in HEK293 and HeLa cells. pEBMulti. Hyg encoding wild-type Cas9 or HSS Cas9 was transfected into the cells via lipofection. After 3 days of selection with hygromycin B, the sgRNA plasmid and ssODN were introduced via electroporation. Genome editing analysis was performed via Sanger sequencing and ICE software. All red bar graphs show data for wild-type Cas9. All blue bar graphs show HSS Cas9 data. A Comparison of HDR efficiency between wild-type Cas9 and HSS Cas9 when AAVS1, EMX1, and VEGFA target sgRNAs were used in HEK293 cells. B Comparison of on-target mutation efficiency between wild-type Cas9 and HSS Cas9 when AAVS1, EMX1, and VEGFA target sgRNAs were used in HEK293 cells. C Comparison of off-target mutation efficiency between wild-type Cas9 and HSS Cas9 when AAVS1, EMX1, and VEGFA target sgRNAs were used in HEK293 cells. D Comparison of HDR efficiency between wild-type Cas9 and HSS Cas9 when AAVS1, EMX1, and VEGFA target sgRNAs were used in HeLa cells. E Comparison of on-target mutation efficiency between wild-type Cas9 and HSS Cas9 when AAVS1, EMX1, and VEGFA target sgRNAs were used in HeLa cells. F Comparison of off-target mutation efficiency between wild-type Cas9 and HSS Cas9 when AAVS1, EMX1, and VEGFA target sgRNAs were used in HeLa cells. All the samples were analyzed in triplicate. The p values in the figures are the p values obtained via Student’s t test. The raw data sets of each trial are shown in Supplementary Table 4

Article Snippet: Purification of His-tagged Cas9 proteins was performed via the His-Spin Protein Miniprep Kit (Zymo Research) according to the manufacturer’s protocol.

Techniques: Transfection, Selection, Plasmid Preparation, Electroporation, Sequencing, Software, Comparison, Mutagenesis

Journal: Journal of Biomedical Science

Article Title: Screening strategy to identify Cas9 variants with higher HDR activity based on diphtheria toxin

doi: 10.1186/s12929-025-01197-9

Figure Lengend Snippet: Droplet digital PCR analysis of the edited genome using wild-type Cas9 or HSS Cas9. Plasmid DNA encoding wild-type Cas9 or HSS Cas9 and each target sgRNA was transfected with ssODN into HEK293 cells via lipofection. The transfected cells were selected with puromycin for 3 days. Two types of ssODNs were used in this experiment. One is the normal phosphodiester bond oligo DNA shown as PO in the figure, and the other is the phosphorothioate bond oligo DNA at 2 bases from two ends shown as PS in the figure. WT shows wild-type Cas9. HSS shows HSS Cas9. A HDR efficiency of each sample when ATP7B, GRN, or RBM20 target sgRNA was used. B indels efficiency of each sample when ATP7B, GRN, or RBM20 target sgRNA was used. C HDR/indels ratio of each sample when ATP7B, GRN, or RBM20 target sgRNA was used. All the samples were analyzed in triplicate. The p values in the figures are the p values obtained by performing a Tukey test when the significance level was set at 5%. The raw data sets of each trial, including the data in Fig. , are shown in Supplementary Table 5. The calculated data sets of each trial, including the data in Fig. , are shown in Supplementary Table 6. The data in Figs. 5 and were taken from the same experiments

Article Snippet: Purification of His-tagged Cas9 proteins was performed via the His-Spin Protein Miniprep Kit (Zymo Research) according to the manufacturer’s protocol.

Techniques: Digital PCR, Plasmid Preparation, Transfection

Journal: Journal of Biomedical Science

Article Title: Screening strategy to identify Cas9 variants with higher HDR activity based on diphtheria toxin

doi: 10.1186/s12929-025-01197-9

Figure Lengend Snippet: Droplet digital PCR analysis of the edited genome via cell cycle-dependent activation of wild-type Cas9 or HSS Cas9. Plasmid DNA encoding AcrIIA4-Cdt1 and wild-type Cas9 or HSS Cas9 and each target sgRNA was transfected with ssODN into HEK293 cells via lipofection. The transfected cells were selected with puromycin for 3 days. Two types of ssODNs were used in this experiment. One is the normal phosphodiester bond oligo DNA shown as PO in the figure, and the other is the phosphorothioate bond oligo DNA at 2 bases from two ends shown as PS in the figure. WT shows wild-type Cas9. HSS shows HSS Cas9. A4CT2A shows AcrIIA4-Cdt1-T2A-Cas9 (WT or HSS). IRESA4C shows Cas9 (WT or HSS)-IRES2-AcrIIA4-Cdt1. All the data in Figs. and 6 were taken from the same experiments, and the WT-PO data are the same as those in Fig. . A Detailed gene cassettes of each sample. B HDR efficiency of each sample when ATP7B, GRN, or RBM20 target sgRNA was used with A4CT2AWT/HSS gene cassettes and PO-ssODN. C Indels efficiency of each sample when ATP7B, GRN, or RBM20 target sgRNA was used with A4CT2AWT/HSS gene cassettes and PO-ssODN. D HDR/indels ratio of each sample when ATP7B, GRN, or RBM20 target sgRNA was used with A4CT2AWT/HSS gene cassettes and PO-ssODN. E HDR efficiency of each sample when ATP7B, GRN, or RBM20 target sgRNA was used with WT/HSSIRESA4C gene cassettes and PS-ssODN. F Indels efficiency of each sample when ATP7B, GRN, or RBM20 target sgRNA was used with WT/HSSIRESA4C gene cassettes and PS-ssODN. G HDR/indels ratio of each sample when ATP7B, GRN, or RBM20 target sgRNA was used with WT/HSSIRESA4C gene cassettes and PS-ssODN. All the samples were analyzed in triplicate. The p values in the figures are the p values obtained by performing a Tukey test when the significance level was set at 5%. The raw data sets of each trial are shown in Supplementary Table 5. The calculated data sets of each trial, including the data in Fig. , are shown in Supplementary Table 6. The data in Figs. and 6 were taken from the same experiments. All p-values by all two samples combinational comparison from ( B – G ) were shown in Supplementary Table 8 and 9. All p-values by all two samples combinational comparison in Figs. and 6 were shown in Supplementary Table 10 and 11

Article Snippet: Purification of His-tagged Cas9 proteins was performed via the His-Spin Protein Miniprep Kit (Zymo Research) according to the manufacturer’s protocol.

Techniques: Digital PCR, Activation Assay, Plasmid Preparation, Transfection, Comparison

Journal: Journal of Biomedical Science

Article Title: Screening strategy to identify Cas9 variants with higher HDR activity based on diphtheria toxin

doi: 10.1186/s12929-025-01197-9

Figure Lengend Snippet: Optimization of the gene cassette for cell cycle-dependent activation in the co-delivery of Cas9, sgRNA, and ssODN. Plasmid DNA encoding AcrIIA4-Cdt1 and either wild-type Cas9 or HSS Cas9 along with each target sgRNA was transfected into HEK293 cells together with ssODN via lipofection. Transfected cells were selected with puromycin for 3 days. Two types of ssODNs were used: a standard phosphodiester oligo (denoted as PO in the figure), and an oligo with phosphorothioate bonds at two terminal bases (PS). WT refers to wild-type Cas9. HSS refers to HSS Cas9. A4CT2A indicates AcrIIA4-Cdt1-T2A-Cas9 (WT or HSS). IRESA4C indicates Cas9 (WT or HSS)-IRES2-AcrIIA4-Cdt1. All data shown in ( B – G ) were obtained from the same experimental set, as were those in ( H – J ). A Schematic of gene cassettes used in each sample. B – D HDR efficiency, indel frequency, and HDR/indel ratio, respectively, for each sample when ATP7B, GRN, or RBM20 sgRNAs were used with WT Cas9 and PO-ssODN across five types of gene cassettes. E – G Same as B–D, but with PS-ssODN. All data in ( B – G ) were analyzed in triplicate. H – J HDR efficiency, indel frequency, and HDR/indel ratio, respectively, for each sample using WT or HSS A4CT2A gene cassettes and PS-ssODN. n = 6. p-values shown in the figures were calculated using Tukey’s test with a significance level of 5%. Raw data for each trial are provided in Supplementary Table 12, and the corresponding processed data in Supplementary Table 13. All pairwise comparisons and their p-values from Fig. ( B – G ) and ( H – J ) are summarized in Supplementary Tables 14, 15, and 16

Article Snippet: Purification of His-tagged Cas9 proteins was performed via the His-Spin Protein Miniprep Kit (Zymo Research) according to the manufacturer’s protocol.

Techniques: Activation Assay, Plasmid Preparation, Transfection