unaffected control pcs 201 010  (ATCC)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Base editing corrects the common Salla disease SLC17A5 c.115C>T variant

doi: 10.1016/j.omtn.2023.08.024

Figure Lengend Snippet: ABE restores SLC17A5 c.115C sequence without creating indels in HDFs (A) DNA and respective protein sequences of SLC17A5 around locus c.115. (Top left) c.115C site (in green) and CGT coding for arginine at codon 39 of the SLC17A5 protein. (Top right) pathogenic variant SLC17A5 c.115C>T (in red), coding for cysteine at codon 39 of the SLC17A5 protein. (Middle) sgRNA design for HDR and ABE. The SLC17A5 c.115 site for the targeted cytosine-to-thymine transition is highlighted in red. Horizontal arrows indicate sequences of sgRNAs targeting the SLC17A5 c.115 site. Protospacer adjacent motifs (PAMs) are highlighted in color corresponding with the respective sgRNA: for HDR, in blue, and base editing, in orange. (Bottom) Sequence of single-stranded oligonucleotide (ssODN) used for HDR around SLC17A5 c .115C>T. PAM motifs are indicated in blue. A synonymous substitution to inactivate the PAM site ( SLC17A5 c.135G>A) is underlined. (B) Comparison of editing efficiency (green) and indel (gray) (%) between HDR and ABE in HDFs harboring the SLC17A5 c.115C>T variant (affected). (C) Temporal assessment of editing efficiency in HDFs following base editing from day zero to day 14 post-nucleofection. (D) Electropherograms of SLC17A5 sequence flanking c.115 in unaffected control, affected, HDR-treated, and ABE-treated HDFs, respectively. Shaded blue regions in the background of chromatograms represent Phred base calling quality scores. SLC17A5 c.115 site distinguished by arrows. (Top left) SLC17A5 c.115 unaffected control HDFs. Note the canonical ‘C’ nucleotide at position c.115. (Top right) Homozygous SLC17A5 c.115C>T variant HDFs. Note the mutated ‘T’ nucleotide at position c.115. (Bottom left) HDR-treated SLC17A5 c.115C>T HDFs. Multiple electropherogram peaks indicative of indels. (Bottom right) ABE-treated SLC17A5 c.115C>T affected HDFs. Note the dual peak for ‘T’ and ‘C’, indicating partial correction of the pathogenic ‘T’ to the canonical ‘C’ nucleotide at c.115. Bystander edit at c.114 is marked with an asterisk. Data generated from at least three independent experiments are shown as mean ± standard deviation. Editing efficiency (%) and indel (%) were analyzed using unpaired t tests. Temporal assessment was analyzed using one-way ANOVA with the Tukey post hoc test. ∗∗∗∗p < 0.0001.

Article Snippet: For determination of LAMP1 and CD63 signal by fluorescence microscopy, unaffected control (HDFs, PCS-201-010; ATCC), affected SLC17A5 c.115C>T (GM08497), and ABE-treated SLC17A5 c.115C>T HDFs were grown on eight-chamber cell culture slides (CellTreat Scientific Products).

Techniques: Sequencing, Variant Assay, Comparison, Control, Generated, Standard Deviation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Base editing corrects the common Salla disease SLC17A5 c.115C>T variant

doi: 10.1016/j.omtn.2023.08.024

Figure Lengend Snippet: ABE reduces markers of endo-lysosomal pathology and normalizes FSA levels in HDFs (A and C) LAMP1(A) and CD63 (C) staining in unaffected control (+/+), affected homozygous SLC17A5 c.115C>T (−/−), and ABE-treated SLC17A5 c.115C>T HDFs (ABE). Scale bar, 200 μm. (B and D) Quantification of LAMP1 (B) and CD63 (D) signal intensity in HDFs, measured in mius. The editing efficiency of the ABE-treated group used for staining corresponds with 53%. (E) Quantification of FSA in HDFs. Increased FSA levels in untreated affected HDFs (−/−) compared to unaffected control HDFs (+/+). Data generated from at least (n = 3) independent experiments are shown as mean ± standard deviation. All comparisons were analyzed using a one-way ANOVA with the Tukey post-hoc test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001. See also Figures S1– S8 .

Article Snippet: For determination of LAMP1 and CD63 signal by fluorescence microscopy, unaffected control (HDFs, PCS-201-010; ATCC), affected SLC17A5 c.115C>T (GM08497), and ABE-treated SLC17A5 c.115C>T HDFs were grown on eight-chamber cell culture slides (CellTreat Scientific Products).

Techniques: Staining, Control, Generated, Standard Deviation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Base editing corrects the common Salla disease SLC17A5 c.115C>T variant

doi: 10.1016/j.omtn.2023.08.024

Figure Lengend Snippet: Targeted amplicon deep sequencing of putative off-target sites nominated in silico show no significant adenine deamination Putative off-target (OT) sites were determined by Cas-OFFinder using the c.115T 20-nucleotide query sequence. (A) OT site frequencies predicted by the Cas-OFFinder algorithm using a mismatch parameter of five or fewer nucleotides (5MM) compared with three or fewer nucleotides (3MM). (B) Predicted OT sites separated by genomic location type, with a majority of predicted OTs found to be in intergenic or intronic regions. (C) Heatmap (left) representing percentage of non-adenine base identities where adenine is expected, or indels (right), across in silico -nominated off-target sites after targeted amplicon deep sequencing and CRISPResso2 analysis. No significant differences are observed between ABE-treated SLC17A5 c.115C>T HDFs (ABE; bottom) and mock (top) samples. Associated OT sequences are listed at right, and adenines with the potential for deamination are in red (n = 3, unpaired Student t tests). See also Tables S2 and , Figures S9 and .

Article Snippet: For determination of LAMP1 and CD63 signal by fluorescence microscopy, unaffected control (HDFs, PCS-201-010; ATCC), affected SLC17A5 c.115C>T (GM08497), and ABE-treated SLC17A5 c.115C>T HDFs were grown on eight-chamber cell culture slides (CellTreat Scientific Products).

Techniques: Amplification, Sequencing, In Silico

Journal: Molecular Therapy. Nucleic Acids

Article Title: Base editing corrects the common Salla disease SLC17A5 c.115C>T variant

doi: 10.1016/j.omtn.2023.08.024

Figure Lengend Snippet: ABE restores Slc17a5 c.115C in a murine cell line (A) Human and murine sgRNA comparison. Murine and human sequences are identical, except a single nucleotide difference, highlighted in yellow. SLC17A5/Slc17a5 c.115C>T is in red. (B) Base-editing efficiency (green) and indel (gray) (%) MEFs harboring the SLC17A5 c.115C>T variant. (C) FSA quantification in MEFs. Increased FSA levels in untreated Slc17a5 c.115C>T MEFs (−/−) compared to unaffected control MEFs (+/+). ABE-treated Slc17a5 c.115C>T MEFs (ABE) demonstrate normalization of FSA. Data generated from at least (n = 3) independent experiments are shown as mean ± standard deviation. All comparisons were analyzed using a one-way ANOVA with the Tukey post hoc test. ∗∗∗∗p < 0.0001.

Article Snippet: For determination of LAMP1 and CD63 signal by fluorescence microscopy, unaffected control (HDFs, PCS-201-010; ATCC), affected SLC17A5 c.115C>T (GM08497), and ABE-treated SLC17A5 c.115C>T HDFs were grown on eight-chamber cell culture slides (CellTreat Scientific Products).

Techniques: Comparison, Variant Assay, Control, Generated, Standard Deviation