Abstract
Genome editing using programmable nucleases has revolutionized biomedical research. CRISPR-Cas9 mediated zygote genome editing enables high efficient production of knockout animals suitable for studying development and relevant human diseases. Here we report efficient disabling pancreatogenesis in pig embryos via zygotic co-delivery of Cas9 mRNA and dual sgRNAs targeting the PDX1 gene, which when combined with chimeric-competent human pluriopotent stem cells may serve as a suitable platform for the xeno-generation of human tissues and organs in pigs.
Highlights
Interspecies blastocyst complementation combines the chimera-forming capability of donor pluripotent stem cells (PSCs) and organogenesis-disabled hosts, which allows for the enrichment of donor cells in a target organ[1, 2]
In contrast to single single-guide RNA (sgRNA), bi-allelic and mono-allelic mutants generated by dual sgRNAs could be identified by simple PCR amplification of the targeted region and DNA gel electrophresis
Dual sgRNA-guided Cas[9] nuclease yielded 59% of mutant blastocysts (13/22) among which 77% (10/13) were mono-allelic and 23% (3/13) bi-allelic, a major improvement over single sgRNA for pig embryos (Supplementary information, Figure S2B). These results demonstrate that the combination of Cas[9] and dual-sgRNAs is a powerful platfrom for gene knockout in pig embryos and deletion of a large DNA fragment faciliates mutant screening
Summary
To prepare sgRNA mRNA, the purified PCR product was in vitro transcribed by MEGAshortscript T7 Transcription Kit (Invitrogen) following the manufacturer’s instruction. To determine genotypes of gene modified E28 pig embryos, tail tips were used for genomic DNA extraction using DNeasy Blood & Tissue kit (QIAGEN). Presumptive zygotes were mechanically denuded by vortexing for 3–5 minutes in a 1.5 mL tube and 100 uL of SOF-HEPES medium[14] and cultured in groups of 15–20 in 50 μL drops of potassium simplex optimized medium supplemented with amino acids and 4 mg/mL of BSA (KSOMaa, pH = 7.4, 275 mOsm) (Evolve ZEBV-100, Zenith Biotech, Guilford, CT, USA) for 7 days. After 4 days of culture, the culture medium was supplemented with 10% FBS (Gemini Bio-Product, CA, USA) at 38.5 °C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2. The primary antibodies used were rabbit anti-PDX1 (1:1000, Abcam, ab47267) and rabbit anti-SOX9 (1:100, Abcam, ab185230)
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