NEW YORK – Researchers at JES Tech, NASA, MiniPCR Bio, MIT, and Boeing Defense, alongside high school students participating in the Genes in Space program have developed a CRISPR-Cas9 assay to be used entirely in space for the study of eukaryotic DNA repair.
In a paper published on Wednesday in PLOS One, the researchers noted that astronauts who are exposed to ionizing radiation are at risk for DNA damage, and that previous studies have shown that conditions in space can often affect the body's choice of repair pathway: non-homologous end joining, or NHEJ, during which insertions or deletions may be added at the break site, versus homologous recombination, or HR, in which the DNA sequence often remains unchanged. The choice of repair pathway can compound the risks of increased radiation exposure during space travel, increasing an astronaut's risk of developing cancer or other health problems, they added.
Until now, it's been challenging to study this problem directly in space. However, the use of CRISPR-Cas9 gene editing allowed the researchers to develop a model for the safe and targeted generation of double-strand breaks, or DSBs, in eukaryotes, and the subsequent assessment of DSB repair pathway choice, entirely in space.
Previous studies have often relied on the generation of DSBs on Earth, after which the biological material was frozen and sent to space to assess DNA repair choice under microgravity conditions. But the initial recognition of the DNA break and the assembly of DNA repair factors at the break site are thought to be important determinants of repair pathway choice and may occur soon after the DSB, making it important for the DSB to occur in space if accurate conditions are to be replicated in a model.
Therefore, the researchers sought to develop a method to study DSB break induction and repair entirely in the microgravity environment onboard the International Space Station National Laboratory. They used a CRISPR-based mutagenesis strategy for the targeted generation of DSBs at a defined genomic locus in Saccharomyces cerevisiae. DNA repair mechanisms then made changes to the DNA sequence at the site of the DSB, either through NHEJ to introduce random insertions or deletions at the break site, or through HR to make specific changes to the DNA sequence through an engineered repair template.
The researchers worked with S. cerevisiae that carried ADE2 mutations, making them turn red, allowing for visual identification of mutant colonies. The CRISPR plasmid they engineered contained a repair template that introduced early stop codons into the ADE2 gene, introducing a DSB in the ADE2 sequence and leading to the induction of DNA repair. As S. cerevisiae relies heavily on homology-directed repair, the researchers hypothesized that transformed cells would utilize the repair template on the CRISPR plasmid to repair the DSB instead of NHEJ. Given that NHEJ repair would also introduce mutations at the break site, the ADE2 gene would be mutated following CRISPR editing regardless of the repair mechanism, resulting in a red phenotype and enabling visual identification of the edited cells.
To confirm successful editing, the researchers examined the ADE2 locus using PCR and DNA sequencing. Four red colonies and four white colonies were randomly selected from experiments done on both the ISS and labs on the ground for DNA extraction, and the researchers found that seven of the eight white colonies aligned to the wild type ADE2 sequence while all eight of the red colonies aligned to the repair template.
Importantly, they said, the sequencing data confirmed the first transformation of live cells and the first successful CRISPR-Cas9 genome editing event in space. The protocol in space deviated from standard methods for the study of DNA repair on Earth, but it was generally sufficient to determine the mechanism of repair.
Overall, the researchers obtained a lower number of reads from each red colony sequenced than from the white colonies, likely due to the smaller colony size. Regardless, the sequencing data was of sufficient quality to deduce the repair mechanism used by the yeast cells, confirming that all red colonies sampled aligned to the repair template sequence, indicating that they were repaired using homologous recombination.
The authors did note that the challenges of adapting Earth-based protocols to the space environment were significant, including the difficulties posed by microgravity to liquid handling and safety concerns. Therefore, the protocol required reduced total sample volumes and used reagents that were premixed and frozen rather than prepared fresh as is usually done. This likely resulted in lower transformation efficiencies for both the flight and ground controls compared to what is typically seen in traditional S. cerevisiae transformation experiments.