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, 1 (9), 886-898

CRISPR Correction of a Homozygous Low-Density Lipoprotein Receptor Mutation in Familial Hypercholesterolemia Induced Pluripotent Stem Cells


CRISPR Correction of a Homozygous Low-Density Lipoprotein Receptor Mutation in Familial Hypercholesterolemia Induced Pluripotent Stem Cells

Linda Omer et al. Hepatol Commun.


Familial hypercholesterolemia (FH) is a hereditary disease primarily due to mutations in the low-density lipoprotein receptor (LDLR) that lead to elevated cholesterol and premature development of cardiovascular disease. Homozygous FH patients (HoFH) with two dysfunctional LDLR alleles are not as successfully treated with standard hypercholesterol therapies, and more aggressive therapeutic approaches to control cholesterol levels must be considered. Liver transplant can resolve HoFH, and hepatocyte transplantation has shown promising results in animals and humans. However, demand for donated livers and high-quality hepatocytes overwhelm the supply. Human pluripotent stem cells can differentiate to hepatocyte-like cells (HLCs) with the potential for experimental and clinical use. To be of future clinical use as autologous cells, LDLR genetic mutations in derived FH-HLCs need to be corrected. Genome editing technology clustered-regularly-interspaced-short-palindromic-repeats/CRISPR-associated 9 (CRISPR/Cas9) can repair pathologic genetic mutations in human induced pluripotent stem cells.

Conclusion: We used CRISPR/Cas9 genome editing to permanently correct a 3-base pair homozygous deletion in LDLR exon 4 of patient-derived HoFH induced pluripotent stem cells. The genetic correction restored LDLR-mediated endocytosis in FH-HLCs and demonstrates the proof-of-principle that CRISPR-mediated genetic modification can be successfully used to normalize HoFH cholesterol metabolism deficiency at the cellular level.

Conflict of interest statement

Potential conflict of interest: Nothing to report.


Figure 1
Figure 1
Mutation identification in HoFH fibroblasts. (A) HoFHϕ and control fetal IMR90ϕ were treated overnight in 5% lipoprotein‐deficient serum media supplemented with either lovastatin or excess sterols. A significant amount of fluorescent DiI‐LDL was visualized in IMR90ϕ treated with lovastatin that was abrogated with excess sterols. GM03040ϕ showed impaired DiI‐LDL internalization with lovastatin treatment (scale bars, 200 μm). (B) Western blot for LDLR shows IMR90ϕ up‐regulate LDLR in lovastatin and suppress LDLR when exposed to sterols. In contrast, HoFH GM03040ϕ express comparatively little LDLR under the same conditions. (C) Sanger sequencing revealed a homozygous three‐nucleotide deletion in exon 4 of LDLR in GM03040ϕ. Abbreviation: ϕ, Fibroblasts.
Figure 2
Figure 2
CRISPR correction strategy. (A) Schematic of the methods: 1 3040‐iPSCs were transfected with 5 μg Cas9n, 5 μg sgRNA1, 5 μg sgRNA9, and 5 μg ssODN, using 2 a NEPA21 square‐wave electroporator. 3 Double‐positive (GFP+/RFP+) cells were selected for expansion by fluorescence sorting. 4 LDLR + cells were enriched by magnetic sorting and expanded in culture. 5 Clones were isolated on laminin‐521 + E‐cadherin‐Fc substrate then 6 analyzed for correction of the LDLR by RFLP, sequencing, western blot, and LDL internalization. (B) Diagram of the CRISPR/Cas9 design to target the mutation site of the LDLR. Paired single‐guide RNAs (target 1 and target 9) were selected to target the mutation site, and an ssODN template was created to mediate HDR. The ssODN contains a 10‐nucleotide sequence (underlined) as a novel XmnI restriction enzyme site, additional silent mutations (red nucleotides) at the PAM for sgRNA 1 or within 10 bp upstream of the PAM for sgRNA 9 to prevent rebinding of guides and cleavage with Cas9n following repair, and the 3‐bp insertion (purple). (C) 3040‐iPSCs were sorted for double‐positive RFP and GFP cells by FACS after transfection; 13.1% of cells were dual positive and 2.4% of the population was collected and expanded to increase specificity of positively transfected cells. (D) MACS was performed on the FACS‐sorted 3040‐iPSCs to enrich for LDLR+ expressing cells. Pre‐MACS‐sorted cells had a mix of both positive (green dots) and negative (red dots) LDLR+ cells. After MACS sorting, a clear shift to the right showed 19.1% of LDLR+ cells were collected. Abbreviations: FITC, fluorescein isothiocyanate; SSC‐A, side‐scatter.
Figure 3
Figure 3
Analysis of 3040‐iPSC LDLR correction by CRISPR/Cas9. (A) RFLP analysis was used to determine 3040‐iPSC correction status by restriction digest with XmnI. Nontransfected 3040‐iPSCs showed no XmnI cleavage (576 bp) in the presence or absence of enzyme. 3040‐iPSC clone 1 (3040‐C) contained two bands at 384 bp and 192 bp in the presence of XmnI, which remained uncut (576 bp) without enzyme. (B) Sanger sequencing of 3040‐C confirmed the permanent insertion of three nucleotides in exon 4 of the LDLR in both alleles (purple box). Silent mutations (red line), including the novel XmnI site (blue box), were also integrated into the gene. Bold letters beneath red lines indicate original native nucleotide. Two silent mutations (green box) were introduced in only one allele. (C) 3040‐C‐iPSCs and 3040‐iPSCs were incubated overnight in lovastatin or excess sterols. Western blot (representative image, C, i) showed 3040‐iPSCs respond with an up‐regulation of an immature LDLR after treatment with lovastatin that is reduced with excess sterols. 3040‐C‐iPSCs had almost no immature LDLR with lovastatin and increased mature LDLR. (C, ii) Quantification (n = 2 independent experiments) showed a 2‐fold increase of mature LDLR in 3040‐C (2.3e6 ± 1.9e5) treated with lovastatin compared to 3040 (1.1e6 ± 4.5e5). (C, iii) Immature LDLR was highly expressed in 3040 (3.8e6 ± 6.7e5) that was almost lost in 3040‐C (6.5e4 ± 6.3e4). (C, iv) Mature/immature LDLR was over 30 times greater in 3040‐C (35 ± 3.1) than 3040 (0.3 ± 0.6) in lovastatin that was abrogated in excess sterols (1.4 ± 1.5 and 0.3 ± 0.1, respectively). Bars shown as mean ± SEM. (D) LDL internalization showed 3040‐C‐iPSCs respond with an increased DiI‐LDL internalization similar to control H1 cells that is almost nonexistent in noncorrected 3040‐iPSCs. LDL uptake is decreased with excess sterols. scale bars, 50 μm. Abbreviations: Lova, lovastatin; rh, recombinant human; XS, excess sterols.
Figure 4
Figure 4
LDLR‐mediated endocytosis is restored in corrected 3040‐iPSC‐derived HLCs. (A) DiI‐LDL uptake was restored in 3040‐C‐HLCs after treatment with lovastatin that was not obvious in 3040‐HLCs. H1‐HLC was used a control for normal LDLR function. (B) Bar graph (n = 2 independent experiments) shows measured fluorescence intensity of DiI‐LDL that correlates with a 4‐fold increase in DiI‐LDL uptake in 3040‐C‐HLCs (8.2 ± 1.3) relative to 3040‐HLCs (1.8 ± 0.8). Excess sterols were normalized to 1 (H1, 1 ± 0.6; 3040, 1 ± 0.5; 3040‐C, 1 ± 0.7) and lovastatin (H1, 11 ± 2; 3040, 1.8 ± 0.8; 3040‐C, 8.2 ± 1.3) normalized to excess sterols. Bars shown as mean ± SEM; scale bars, 50 μm.

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