![]() Following transformation, cells were plated on synthetic defined agar lacking uracil (SDA-URA) and grown at room temperature for six days when the phenotype of the colonies was assessed. The miniPCR thermal cycler was used as a heat block to induce transformation. ![]() Step 1, transformation: transformation mixture and pVG1 vector were added to thawed cells. Prior to launch, cells were grown in liquid culture on Earth, pelleted by centrifugation, and frozen in glycerol at -80☌ for transport to the ISS. cerevisiae transformation and CRISPR/Cas9 genome editing protocols for use onboard the ISS. cerevisiae with mutations in this gene turn red due to the buildup of purine precursors in the vacuole. ADE2 is not essential for survival, but S. ADE2 mutant colonies are easily distinguished from those bearing the wild type ADE2 sequence. This vector contains CRISPR machinery: Cas9, guide RNAs targeting ADE2, a repair template that introduces two stop codons and an EcoRI site into the ADE2 gene, and the URA3 gene for positive selection. These milestones represent a significant expansion of the molecular biology toolkit onboard the International Space Station.Ī. As necessary steps in this process, we describe the first successful genetic transformation and CRISPR/Cas9 genome editing in space. Here we describe a CRISPR-based assay for DNA break induction and assessment of double-strand break repair pathway choice entirely in space. The CRISPR/Cas9 gene editing system offers a model for the safe and targeted generation of double-strand breaks in eukaryotes. However, our understanding of this problem has been limited by technical and safety concerns, which have prevented integral study of the DNA repair process in space. Previous work suggests that space conditions may impact the choice of DNA repair pathway, potentially compounding the risks of increased radiation exposure during space travel. Double-strand breaks are a type of DNA damage that can be repaired by two major cellular pathways: non-homologous end joining, during which insertions or deletions may be added at the break site, and homologous recombination, in which the DNA sequence often remains unchanged. ![]() As we explore beyond Earth, astronauts may be at risk for harmful DNA damage caused by ionizing radiation.
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