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. Author manuscript; available in PMC: 2024 Feb 1.
Published in final edited form as: Mol Genet Metab. 2023 Jan 13;138(2):107508. doi: 10.1016/j.ymgme.2023.107508

Figure 1. Generation of CRISPR/Cas9 Mouse Model.

Figure 1.

a. Mus musculus Glb1 gene diagram to develop β-gal deficiency mouse model of Type II GM1 gangliosidosis diseases applying CRISPR/Cas9 genome editing by utilizing two sgRNAs in exons 2 and 6, common sites of mutations in Type II GM1 gangliosidosis patients; The left white bar represents the first 5’ UTR, and the right white bar represents the 3’ UTR. b. and c. Sanger sequencing verification of CRISPR editing revealing a 17bp deletion in exon 2 and a 28bp deletion in exon 6 of Glb1−/− mice. PCR amplification of exons 2 and 6, and Sanger sequencing were performed using genomic DNA collected from homozygous Glb1−/− mice. d. Schematic of frameshift and predicted premature termination. e. Percentage of relative β-gal activity in Glb1−/− tissues as compared to Glb1+/+ control from 4 independent experiments, each with n=4 (mixed gender and ages) Glb1−/− tissues compared to Glb1+/+ control. f. Body weight (g) of female (n=4–5) and males (n=3–10) at 8-, 20- and 32-weeks old (two-tailed unpaired t-test with Welch’s correction). g. Glb1−/− mice demonstrated decreased lifespan with disease progressing more rapidly in female mice progressing than in male mice. Mice were euthanized when they became moribund and unable to move around the cage and/or lost more than 15% of their body weight. Kaplan-Meier survival curve shows that Glb1−/− female mice (42.7 weeks old, ±1.1, n=8) die approximately six weeks sooner than Glb1−/− male mice (49.1 weeks old, ±1.1, n=11).