kcnj13  (Proteintech)

Journal: The Journal of Clinical Investigation

Article Title: Nonviral base editing of KCNJ13 mutation preserves vision in a model of inherited retinal channelopathy

doi: 10.1172/JCI171356

Figure Lengend Snippet: ( A ) Construct design to generate HEK293 FRT stable cells harboring the KCNJ13 W53X allele. ( B ) Chromatogram generated from HEK293 FRT stable cells showing the W53X codon marked in the red box and the downward black arrow indicating the specific nucleotide change (G>A).( C ) Schematic of the hKCNJ13 locus highlighting the mutation c.158G>A (blue box marked with asterisk) and position of the W53X targeting sgRNA (black line) with TGG PAM (red line). ( D ) Base-editing efficiencies are shown as the percentages of sequencing reads with the corrected WT allele (and no other silent changes, bystander edits, or indels) in HEK293 W53X cells following electroporation of ABE8e protein+sgRNA (RNP) or ABE8e mRNA+sgRNA ( n = 3). Markers (diamonds) represent the individual biological replicates ( n = 3), and error bars represent SEM by 2-tailed Student’s t test. ( E ) Percentages of sequencing reads with indels in ABE8e RNP– and ABE8e mRNA–treated stable cells ( n = 3). Markers (diamonds) represent the individual biological replicates ( n = 3), and error bars represent SEM by 2-tailed Student’s t test. ( F ) Kir7.1 expression in ABE8e mRNA–treated cells assessed by immunocytochemistry. GFP primary antibody was used to enhance the endogenous signal. DAPI was used to stain the nucleus. Scale bars: 50 μm. White arrows mark membrane localization in cells.

Article Snippet: The Kcnj13 W53X mouse model was generated by Cyagen Biosciences using CRISPR/Cas9-mediated genome engineering.

Techniques: Construct, Generated, Mutagenesis, Sequencing, Electroporation, Expressing, Immunocytochemistry, Staining, Membrane

Journal: The Journal of Clinical Investigation

Article Title: Nonviral base editing of KCNJ13 mutation preserves vision in a model of inherited retinal channelopathy

doi: 10.1172/JCI171356

Figure Lengend Snippet: ( A ) Left: snapshots of Kir7.1 current profile in WT stable cells. Center: current-sweep plot represents the experimental timeline and is shown for 1 representative cell. Right: Rb + - and Cs + -sensitive current in HEK WT stable cells ( B ) Left: snapshots of Kir7.1 current profile in HEK W53X stable cells. Center: current-sweep plot is shown for 1 representative cell. Right: Rb + - and Cs + -sensitive current in HEK W53X stable cells. ( C ) Left: snapshots of Kir7.1 current profile in HEK W53X base-edited cells using ABE8e mRNA. Cells marked with asterisks showed recovery of K + channel functions after base editing. Center: current-sweep plot is shown for 1 representative cell. Right: Rb + - and Cs + -sensitive current in HEK W53 base-edited cells.

Article Snippet: The Kcnj13 W53X mouse model was generated by Cyagen Biosciences using CRISPR/Cas9-mediated genome engineering.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Nonviral base editing of KCNJ13 mutation preserves vision in a model of inherited retinal channelopathy

doi: 10.1172/JCI171356

Figure Lengend Snippet: ( A ) Representative bright-field images of base-editor treated and untreated iPSC-RPE W53X/W53X . Scale bars: 100 μm. ( B ) Base-editing efficiencies following treatment (BE) with ABE8e mRNA and sgRNA encapsulated in SNC-PEG in iPSC-RPE W53X/W53X as compared with untreated cells. Reads from the untreated and treated cells ( n = 3) were categorized into 4 subtypes based on their sequences, unedited, W53*>WT, indels, and substitutions. ( C ) Reads generated by ABE8e mRNA treatment showing the nucleotide distribution around the cleavage site for sgRNA. Substitutions are highlighted in bold. The scatterplot shows the frequency of alleles observed in treated cells ( n = 3). Data are represented as means ± SEM. ( D ) Manual single-cell patch-clamp assays on iPSC-RPE W53X/W53X cells after treatment with ABE8e. Of the 13 cells assessed for Kir7.1 activity, each could be binned into 1 of 3 classes: low-responding single cells, which appeared to be unedited mutant cells; medium-responding single cells, which showed a low level of Rb + response; and high-responding single cells, which showed Rb + response like WT iPSC-RPE cells. The number ( n ) of cells binned into each class is shown at the top of each graph. ( E ) Current-sweep plot from a representative cell of each bin across a time course of being exposed to physiological HR solution (gray), Rb + stimulation (red), and subsequent wash with HR solution (green).

Article Snippet: The Kcnj13 W53X mouse model was generated by Cyagen Biosciences using CRISPR/Cas9-mediated genome engineering.

Techniques: Generated, Patch Clamp, Activity Assay, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: Nonviral base editing of KCNJ13 mutation preserves vision in a model of inherited retinal channelopathy

doi: 10.1172/JCI171356

Figure Lengend Snippet: ( A and B ) Two different sgRNAs targeting the Kcnj13 gene at exon 2 and a ssODN sequence with the desired nucleotide change to generate the Kcnj13 W53X/W53X mouse model by CRISPR/Cas9 and HDR genome-editing technique by microinjecting them into the pronuclei of the zygote. Double asterisks indicate postnatal day 1 lethal. ( C ) RFLP analysis of the Kcnj13 gene from the generated mice digested with Nhe1 enzyme on 2% agarose gel. ( D ) Chromatograph confirming the mouse genotype. ( E ) OCT images showing comparison between Kcnj13 +/+ , Kcnj13 W53X/+ , and WT allele–disrupted Kcnj13 W53X/+ΔR mice. ( F ) Averaged c wave response confirming WT allele disruption in the RPE of Kcnj13 W53X/+ΔR using the targeted guide (T). One-way ANOVA with post hoc Tukey’s HSD test was used for comparisons between the groups. NT, nontargeting sgRNA.

Article Snippet: The Kcnj13 W53X mouse model was generated by Cyagen Biosciences using CRISPR/Cas9-mediated genome engineering.

Techniques: Sequencing, CRISPR, Generated, Agarose Gel Electrophoresis, Comparison, Disruption

Journal: The Journal of Clinical Investigation

Article Title: Nonviral base editing of KCNJ13 mutation preserves vision in a model of inherited retinal channelopathy

doi: 10.1172/JCI171356

Figure Lengend Snippet: ( A ) Kcnj13 W53X allele–specific sgRNA. Black arrow represents the sgRNA spacer sequence, the desired base editing site is indicated by an asterisk, and the PAM is shown in yellow. ( B ) Workflow of in vivo base-editing strategy. ( C ) RPE florets after SNC-PEG-ATRA packaged ABE8e mRNA, W53X sgRNA, and GFP mRNA or empty SNC-PEG-ATRA/PBS as a mock treatment subretinal delivery. ( D ) W53X>WT corrected cell percentages observed in Kcnj13 W53X/– mice treated with 2 μg or 3 μg of ABE8e. ( E ) Indel percentages observed in Kcnj13 W53X/– mice treated with 2 μg or 3 μg of ABE8e. ( F ) In vivo experiment time line. Baseline ERG prior to the disruption of the WT allele and after 6 weeks follow-up. ERG prior to injection of the base editor. Recovery monitored for 10 weeks. ( G ) Representation of the c wave amplitude in Kcnj13 W53X/+ mice with retina OCT image. ( H ) Reduced c wave amplitude in the Kcnj13 W53X/+ mice at 6 weeks after disrupting the WT allele with Cas9 protein and WT-specific sgRNA. ( I ) The c wave and mfERG traces following the injection of base editor with a nontargeting guide (red) and base editor with a targeting guide (green). The faded traces represent comparisons before the disruption of the WT allele (gray) and injection of the base editor (orange). ( J ) Average c wave amplitude 6 weeks after the disruption of the WT allele (blue) or after the injection of base editor with a nontargeting guide (red) and targeting guide (green). ( K ) Normalized c wave amplitude in the eyes injected with nontargeting and targeting guides at weeks 2, 6, and 10. One-way ANOVA with post hoc Tukey’s HSD test was used for comparisons between the groups.

Article Snippet: The Kcnj13 W53X mouse model was generated by Cyagen Biosciences using CRISPR/Cas9-mediated genome engineering.

Techniques: Sequencing, In Vivo, Disruption, Injection

Journal: Acta biochimica et biophysica Sinica

Article Title: Identification of lipid metabolism-related genes as prognostic indicators in papillary thyroid cancer.

doi: 10.1093/abbs/gmab145

Figure Lengend Snippet: Figure 3. Performance of the four-gene signature in the entire TCGA dataset and the GSE33630 dataset (A) Distribution of patients’ risk score, TTR time and recrudescence state, and four-gene expression in the entire TCGA set. (B) ROC curves for TTR at different time points in the entire TCGA set. P-values show the AUROC of the four-gene signature. (C) Kaplan–Meier method and log-rank test of the prognosis difference between patients in the indicated groups. (D) The expressions of PDZK1IP1, LRP2, and KCNJ13 in the GSE33630 dataset.

Article Snippet: After being blocked with 3% BSA (A8020; Solarbio) at room temperature for 30min, the TMA was incubated with the primary antibodies at 4◦C overnight, including PDZK1IP1 (dilution of 1:150, Ab156014; Abcam, Cambridge, UK), LRP2 (dilution of 1:500, Ab76969; Abcam), KCNJ13 (dilution of 1:100, 12657-1-AP; Proteintech, Rosemont, USA), and TMC3 (dilution of 1:4000, Ab243537; Abcam), followed by incubation with a horseradish peroxidase-conjugated goat antirabbit secondary antibody (GB23303; Servicebio).

Techniques: Gene Expression