Results for “Genome editing”

NASA NTRS2015

CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) for Near-Perfect Selective Transformation

The CRISPR (Clustered, Regularly Interspaced, Short Palindromic Repeats)/Cas9 system has revolutionized genome editing by providing unprecedented DNA-targeting specificity. Here we demonstrate that this system can be also applied in vitro to fundamental cloning steps to facilitate efficient plasmid selection for transformation and selective gene insertion into plasmid vectors by cleaving unwanted plasmid byproducts with a single-guide RNA (sgRNA)-Cas9 nuclease complex. Using fluorescent and chromogenic proteins as reporters, we demonstrate that CRISPR/Cas9 cleavage excludes multiple plasmids as well as unwanted ligation byproducts resulting in an unprecedented increase in the transformation success rate from approximately 20% to nearly 100%. Thus, this CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) protocol is a novel, inexpensive, and convenient application to conventional molecular cloning to achieve near-perfect selective transformation.

molecular cloning↗

NASA NTRSDate not supplied

Guanine Oxidation in the Genome, not RNA Editing, Accounts for Single Nucleotide Variation in the Exome of Mice Flown on Board the ISS

We have conducted a further analysis of single nucleotide variation (somatic mutation) in mice flown aboard the ISS. We used data archived in GeneLab from a cohort of 18-week-old mice were flown to the ISS, housed in the Rodent Habitat and therefore subjected to microgravity for 37 days. Mice of similar age, sex and the same strain were used for ground controls housed in identical hardware and simulating, but not matching ISS environmental conditions (temperature, humidity and gas atmosphere). Primary data consists of next generation RNA sequencing for the tissues examined: eye, liver, skeletal muscle and kidney. We used novel software, developed at NASA Ames Research Center and deployed on the NASA Ames Supercomputer, to perform variant calling for single point mutations. Unexpectedly, we discovered a high degree of somatic mutation in ISS-flown mice, compared to controls. We found that the extent of somatic mutation correlated with the degree of gene expression in the four tissue types, with the highest degree of somatic mutation observed in genes with the highest degree of expression. Careful analysis that included measurement of specific nucleotide changes that occurred demonstrated that guanine substitutions were the most frequent, consistent with the hypothesis that reactive oxygen species-mediated guanine oxidation was responsible for the hypermutation events. By contrast, adenine substitutions would be expected if gene editing were responsible for the somatic mutation. These types of substitutions were much less frequent. The implication of these findings for astronaut health in a variety of mission scenarios will be discussed.

International Space Station↗