*2.16. Mitochondrial Superoxide Stress Quantification*

Mitochondrial superoxide production was quantified using MitoSOX Red mitochondrial superoxide indicator (Thermo Fisher Scientific) according to the manufacturer's protocol. Briefly, the cells were incubated with 5 μM MitoSOX Red in the culture medium in the dark for 60 min at 37 ◦C. Stained cells were counterstained as desired and mounted and analyzed by fluorescent microscopy.

#### *2.17. Annexin V Staining*

Cells were washed once with PBS and analyzed using FITC Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer's protocol.

#### *2.18. GLA Enzyme Activity Assay*

Cells were washed twice with 1X PBS and were lysed in 60 μL lysis buffer (27 mM sodium citrate, 46 mM sodium phosphate dibasic, 0.5% Triton X-100). 10 μL of cell lysate was added to 50 μL assay buffer containing 6 mM 4-methylumbelliferyl-α-d-galactopyranoside (Sigma-Aldrich) and 117 mM N-acetyl-D-galactosamine (Sigma-Aldrich) and incubated at 37 ◦C for 1 hr. The 4-methylumbelliferone (Sigma-Aldrich) dissolved in methanol was used as standard ranging from 0.15 μM to 5000 μM. Thereafter, 70 μL glycine-carbonate solution (pH 10.8) was then added to stop the reaction and fluorescence was detected by microplate reader (em/ex = 365/448 nm). The enzyme activity was normalized by protein concentration of cell lysate.

#### *2.19. Statistical Analysis*

The quantifiable data are presented as the means ± standard deviation (SD) and compared with Student's *t*-test by GraphPad Prism 6 (GraphPad Prism Software). \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.005 and \*\*\*\* *p* < 0.001.

#### **3. Results**

#### *3.1. CRISPR*/*Cas9-Mediated Knockout of Expression of GLA in hESCs*

In order to investigate the functions of *GLA* and its therapeutic potential for FD, we performed CRISPR/Cas9-mediated gene editing to knock out expression of this gene in human embryonic stem cells (hESCs). The procedure was similar to that described in our previous work, where we established the GLA-null HEK293 cell line [8]. Briefly, the *GLA*-specific single-guide RNA (sgRNA) was designed by using Optimized CRISPR Design tool (http://crispr.mit.edu/) to introduce the frameshift mutations in exon 1 of *GLA* gene as shown in Figure 1A. The parental male hESC line H9 (WA09) was transfected with Cas9-encoding plasmid with EGFP reporter, along with the sgRNA. Following the transfection, the proportion of the successfully transfected EGFP-expressing cells was enriched by FACS, and these cells were seeded to 96-well dishes to obtain pure colonies. Out of 36 wells where the single cells were seeded, the clones #20 to #24 and #28 did not grow up. The remaining 30 colonies were screened for GLA protein expression by western blotting (Figure 1B). The GLA protein expression was completely ablated in four clones (#3, #19, #26, and #27) and partially suppressed in two (#25 and #31) (Figure 1B). By performing preliminary differentiation of these clones into cardiomyocytes (CMs), we found that clone #26-derived CMs displayed slight GLA expression, which could be due to contamination with parental non-edited cells, therefore, this clone was excluded from further analysis (Supplementary Figure S1A). *GLA* mRNA levels in these CM clones were not significantly decreased as compared to the control, only in clone #27-derived CMs mRNA levels were downregulated by approximately 50% (Supplementary Figure S1B). On the other hand, GLA enzyme activity was completely absent in clones #3, #19, and #27, which was consistent with the complete absence of GLA protein in these cells (Supplementary Figure S1C). Subsequently, the mutations introduced by CRISPR/Cas9 were identified by Sanger sequencing in the remaining *GLA*-knockout hESC clones #3, #19, and #27. Clone #3 displayed mixed genetic background at the target site, due to putative non-homogeneous population, therefore, it was also excluded from the analysis. (Supplementary Figure S1D). Two of GLA-null hESC clones were selected for the following study because they were hemizygous for 2 bp deletion (clone #19) and 1 bp insertion (clone #27) (Figure 1C). T7 Endonuclease I (T7E1) was used to confirm the presence CRISPR/Cas9-introduced mutations in the *GLA* gene, whereby the cleaved mismatch products were detected in clones #19 and #27 (Figure 1D). On the other hand, T7E1 assay revealed that there were no CRISPR/Cas9-introduced mutations in the potential off-target genes predicted by Optimized CRISPR Design tool (Suppl. 2). Therefore, by conducting the CRISPR/Cas9-mediated genome editing, we successfully established two GLA-null hESC clones.

**Figure 1.** CRISPR/Cas9-mediated knockout of expression of *GLA* in hESCs. (**A**) Schematic depiction of sgRNA-guided Cas9 target site within exon 1 of *GLA* gene. The sgRNA sequence (5 -AGGAACCCAGAACUACAUCU-3 ) is labeled in blue font and PAM recognition sequence highlighted in red. The gRNA targeting site in the *GLA* exon 1 region is highlighted in green and the double-strand breaking site is indicated by the red arrowheads. The start codon is highlighted in yellow. (**B**) Western blot screening of 30 CRISPR/Cas9-corrected clones for expression of GLA protein. H9 cells were used as wild type parental control. GAPDH used as a loading control. (**C**) Sanger sequencing analysis confirming two nucleotides deletion and one nucleotide insertion in CRSIPR/Cas9-edited hESC clones #19 and #27, respectively. (**D**) T7E1 digestion assay of the mutants at the target site in *GLA* gene. The mismatch T7E1 cleavage products are marked with white arrowheads.
