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

Abstract

Free sialic acid storage disorders (FSASDs) result from pathogenic variations in the SLC17A5 gene, which encodes the lysosomal transmembrane protein sialin. Loss or deficiency of sialin impairs FSA transport out of the lysosome, leading to cellular dysfunction and neurological impairment, with the most severe form of FSASD resulting in death during early childhood. There are currently no therapies for FSASDs. Here, we evaluated the efficacy of CRISPR-Cas9-mediated homology directed repair (HDR) and adenine base editing (ABE) targeting the founder variant, SLC17A5 c.115C>T (p.Arg39Cys) in human dermal fibroblasts. We observed minimal correction of the pathogenic variant in HDR samples with a high frequency of undesired insertions/deletions (indels) and significant levels of correction for ABE-treated samples with no detectable indels, supporting previous work showing that CRISPR-Cas9-mediated ABE outperforms HDR. Furthermore, ABE treatment of either homozygous or compound heterozygous SLC17A5 c.115C>T human dermal fibroblasts demonstrated significant FSA reduction, supporting amelioration of disease pathology. Translation of this ABE strategy to mouse embryonic fibroblasts harboring the Slc17a5 c.115C>T variant in homozygosity recapitulated these results. Our study demonstrates the feasibility of base editing as a therapeutic approach for the FSASD variant SLC17A5 c.115C>T and highlights the usefulness of base editing in monogenic diseases where transmembrane protein function is impaired.

Keywords: CRISPR-Cas9; MT: RNA/DNA editing; SLC17A5; Salla; free sialic acid storage disease.

Graphical abstract

Figure 1

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.

Figure 2

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.

Figure 3

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 S3, Figures S9 and S10.

Figure 4

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.