CRISPR/CAS9-MEDIATED GENE EDITING IN THE ARGINASE-1 DEFICIENT MOUSE GENOME
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Arginase-1 deficiency is a rare, autosomal recessive disorder of the urea cycle that normally converts ammonia to less toxic urea. Symptoms include neurodegeneration, spastic diplegia and mental retardation. We designed a Clustered Regularly Interspaced Palindromic Repeat (CRISPR)/Cas9 system to correct an induced Arg1 exon 7 and 8 deletion (Arg1∆) that produces arginase-1 deficiency in mice. Three cell lines were generated to study this disease. The first was primary mouse embryonic fibroblasts isolated from Arg1∆-mice (Arg1∆-PMEF). The second was mouse induced-pluripotent stem cells converted from the aforementioned PMEFs (Arg1∆-miPSC). The third was human embryonic 293T kidney cells with introduced Arg1∆-transgenic DNA (Arg1∆-HEK). These three cell lines were transfected with a targeted Cas9:guide-RNA (gRNA) plasmid. PCR amplicons were subjected to a heteroduplex cleavage assay and sequenced to confirm Cas9 cleavage just upstream of the exon 7 and 8 deletion site. DNA cleavage by Cas9:gRNA can promote homologous recombination with a single-stranded oligodeoxynucleotide (ssODN) to produce small edits in the genome of miPSCs. This mimicked an editing strategy that could be used to correct single nucleotide mutations in Arg1. Homologous recombination was also used to introduce a targeting vector containing both wild-type exon 7 and 8 cDNA and a removable positive-negative selection cassette in miPSCs. PCR analysis and sequencing of “targeted” (positively selected) cells has shown the presence of the repair vector in the correct location in Arg1. Attempts were made to subsequently remove the selection cassette by PiggyBac transposase. The “repaired” (negatively selected) miPSCs were probed by PCR to test for the correct incorporation of exons 7 and 8, showing unclear results. These cells were converted to hepatocyte-like cells showing the correct morphology and staining positive for glycogen. Future tests will be conducted to introduce these hepatocyte-like cells into Arg1-deficient mice. These experiments will serve as a functional model to test gene editing in arginase-1 deficiency. Because the CRISPR/Cas9 system can be designed to target any gene precisely, it has far-reaching applications, particularly for genetic disorders.