Proof-of-Concept Gene Editing for the Murine Model of Inducible Arginase-1 Deficiency

Abstract

Arginase-1 deficiency in humans is a rare genetic disorder of metabolism resulting from a loss of arginase-1, leading to impaired ureagenesis, hyperargininemia and neurological deficits. Previously, we generated a tamoxifen-inducible arginase-1 deficient mouse model harboring a deletion of Arg1 exons 7 and 8 that leads to similar biochemical defects, along with a wasting phenotype and death within two weeks. Here, we report a strategy utilizing the Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system in conjunction with piggyBac technology to target and reincorporate exons 7 and 8 at the specific Arg1 locus in attempts to restore the function of arginase-1 in induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHLCs) and macrophages in vitro. While successful gene targeted repair was achieved, minimal urea cycle function was observed in the targeted iHLCs compared to adult hepatocytes likely due to inadequate maturation of the cells. On the other hand, iPSC-derived macrophages expressed substantial amounts of "repaired" arginase. Our studies provide proof-of-concept for gene-editing at the Arg1 locus and highlight the challenges that lie ahead to restore sufficient liver-based urea cycle function in patients with urea cycle disorders.

Figure 1

Generation of Arg1-deficient iPSCs from PMEF cultures. (a) Schematic diagram depicting the genotype of cell source. The arrows indicate the positions of the primers used for genotyping the resulting cells. (b) PCR genotyping to confirm the deletion of exons 7 and 8. Arg1-Cre PMEFs showed bands at 1.2 kb and 252 bp (indicative of intact exons 7 and 8), while both Arg1 ^∆ PMEFs and derived iPSCs produced only one band at 195 bp. Skin fibroblasts isolated from a tamoxifen-induced Arg1-deficient mouse were used as a positive control, while untreated Arg1-Cre PMEFs served as negative control. (c) Alkaline phosphatase as a pluripotent marker. (d) Formation of embryoid bodies and (e) teratoma assay to demonstrate trilineage differentiation in vitro and in vivo, respectively. Shown are gutlike epithelial tissues, cartilage and neural tissues, depicting the formation of endoderm, mesoderm and ectoderm, respectively.

Figure 2

CRISPR/Cas9-mediated gene targeting in mouse iPSCs. (a) Schematic diagram showing the site of Arg1 gene modification using CRISPR/Cas9. The gRNA was designed to target a 20 nt region in intron 6 of Arg1. (b) Cas9:gRNA off-target analysis. A table showing the output from CRISPR design tool. The on-site target is given at the top, followed by off-target sequences organized in decreasing Cas9-cleavage probability. (c) Detection of Cas9:gRNA-mediated on-target cleavage of Arg1 by the Surveyor nuclease assay. The cleavage products were shown as extra bands (149 bp + 197 bp) indicated by the arrows. Mutation frequency (indel) was calculated by measuring the band intensities. (d) Sequencing data from TOPO-cloned PCR amplicons of CRISPR/Cas9-modified genomic DNA showing a few examples of NHEJ-mediated indel mutations at the desired location. gRNA target site is in bold. Deletions are shown in dashes. Nucleotide substitution is shown in lowercase. The net change in length caused by each indel mutation is to the right of each sequence.

Figure 3

Reincorporation of Arg1 exons 7 and 8 via homology-directed repair. (a) The experimental strategy for precise genome modification using CRISPR/Cas9 and piggyBac transposon methodology. Arrows indicate primers for PCR-based screening to confirm the selected clones. The remnant LoxP left from the initial Cre-excision of exons 7 and 8 would be removed upon targeting vector integration. PBx, piggyBac transposase; T2A, viral sequence for ribosomal skipping; Puro, puromycin; ITR, inverted terminal repeat; TK, thymidine kinase. (b) Representative gel images of puromycin-resistant iPSC colonies screened for correctly targeted clones. Each homology arm was amplified independently by PCR. Amplicon sizes of the left and right arm were 1530 and 1660 bp, respectively, while random integration should yield a product of ≈300 bp. (c) PCR-based genotyping of alleles after piggyBac excision. Upper panel shows primer combination to uniquely identify different alleles. Lower panel shows a representative gel of different banding patterns and corresponding genotypes. Amplicon sizes of the repaired and targeted alleles were 668 bp and 530 bp, respectively. (d) Footprint analysis by sequencing. Sequences in transposon-free repair clones indicating the exact repair after transposon excision. TTAA target sites are boxed.

Figure 4

Generation of hepatocyte-like cells from repaired mouse iPSCs. (a) Schematic diagram showing the stepwise hepatic differentiation protocol. (b) Representative phase contrast images showing sequential morphological changes in iPSCs at different stages of differentiation (10x magnification). The cells are positive for PAS staining at day 25 after the start of the differentiation procedure. (c) RT-PCR gene expression of iHLCs for the hepatocyte markers alpha-fetoprotein (Afp), albumin and Arg1. Expression of genes was normalized to β-actin. (d) Representative immunostaining images from two independent batches of hepatic differentiation showing the expression profile. (e) Functional characterization of iHLCs. After 24 h of culture, supernatants were collected for urea production assay. Values were normalized by the number of cells seeded. Values are mean ± SD for n = 3 − 4. (f) qPCR expression analysis of the five urea cycle enzymes. The C_t values of all genes were normalized to the C_t values of 18S rRNA. The y-axis represents the fold-change of gene expression compared with undifferentiated iPSCs. Values are mean ± SD for n = 3. Statistical significance between groups was determined by Student’s t-test (*P < 0.05).

Figure 5

Characterization of iPSC-derived macrophages by assessing mRNA expression of macrophage phenotypic markers. (a) Phase contrast (upper panel) and cytocentrifuge preparations of macrophages for Wright–Giemsa staining (lower panels). The cells appear purplish and granular. 20x magnification. (b) RT-PCR analysis of macrophage marker genes in macrophages. Differentiated macrophages demonstrated M2-like characteristics. Expression of genes was normalized to β-actin. (c) RFP signal observed in the repaired iPSC-derived macrophages. (d) mRNA expression levels of Arg1-RFP in macrophages after treatment with IL-4, IL-10, sodium butyrate (NaB) as assessed by RT-PCR.