A CRISPR-Based Assay For The Study of Eukaryotic DNA Repair Onboard The ISS

Astronauts traveling beyond the protective boundaries of Earth’s magnetosphere are at an increased risk of DNA damage caused by ionizing radiation.

Such DNA damage may lead to cancer and other detrimental health effects, raising questions about the safety of long-duration space travel.

Abstract

As we explore beyond Earth, astronauts may be at risk for harmful DNA damage caused by ionizing radiation.

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.

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.

The CRISPR/Cas9 gene editing system offers a model for the safe and targeted generation of double-strand breaks in eukaryotes.

Here we describe a CRISPR-based assay for DNA break induction and assessment of double-strand break repair pathway choice entirely in space.

As necessary steps in this process, we describe the first successful genetic transformation and CRISPR/Cas9 genome editing in space.

Introduction

Astronauts traveling beyond the protective boundaries of Earth’s magnetosphere are at an increased risk of DNA damage caused by ionizing radiation.

Such DNA damage may lead to cancer and other detrimental health effects, raising questions about the safety of long-duration space travel.

In space, a significant portion of the ionizing radiation is Galactic Cosmic Radiation which is mainly composed of high linear energy transfer (LET) particles. These particles can create clustered and complex DNA damage that may be difficult to repair.

Double-strand breaks (DSBs), in which the phosphate backbones of both DNA strands are hydrolyzed, are a particularly harmful type of DNA lesion. On Earth, eukaryotic organisms use at least two mechanisms for repairing DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ).

During HR, a homologous DNA sequence is used as a template for repair so that the DNA sequence remains unchanged. HR repair is typically limited to the S and G2 phases of the cell cycle.

NHEJ, however, can occur at any point in the cell cycle. During NHEJ the cell directly rejoins the two pieces of DNA, often resulting in changes to the original DNA sequence . These alterations may increase the risk of cancer and other detrimental conditions.

Figure-1. (Image Credit: NIH)

Overview of CRISPR genome editing system adapted for use onboard the ISS.

Map of the pVG1 vector [15]. 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.

B. ADE2 mutant colonies are easily distinguished from those bearing the wild type ADE2 sequence. ADE2 is not essential for survival, but S. cerevisiae with mutations in this gene turn red due to the buildup of purine precursors in the vacuole [16].

Wild type S. cerevisiae colonies are white. C. Adaptation of S. cerevisiae transformation and CRISPR/Cas9 genome editing protocols for use onboard the ISS. Prior to launch, cells were grown in liquid culture on Earth, pelleted by centrifugation, and frozen in glycerol at -80°C for transport to the ISS. Step 1, transformation: transformation mixture and pVG1 vector were added to thawed cells.

The miniPCR thermal cycler was used as a heat block to induce transformation. 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.

Step 2, DNA extraction: A pipette tip was used to transfer a small number of cells from four red and four white colonies to the DNA extraction buffer.

Cells were heated in the miniPCR thermal cycler to 95°C to extract the DNA. Step 3, PCR and barcoding: DNA extract was directly added to PCR reagents. PCR was performed to amplify the 5’ end of the ADE2 gene.

Sequencing barcodes were added at this step. Step 4, sample pooling and magnetic bead clean up: PCR product was pooled and purified during a magnetic bead cleanup step.

Step 5, nanopore sequencing: Purified PCR product was sequenced by nanopore sequencing. Data was downlinked to the ground where sequences were assessed.

Figure-2. Astronaut Christina Koch plating S. cerevisiae following transformation. (Image Credit: NASA and NIH)

Successful transformation and CRISPR/Cas9 genome editing onboard the ISS.

1, Astronaut Koch transfers small volumes of liquid culture (~20 μl) onto the Petri dish multiple times so that liquid remains attached to the agar due to surface tension.

2, Cells were spread using a sterile plastic spreader. B. Astronaut Nick Hague examines a Petri dish following six days incubation at room temperature (image credit: NASA).

Both white and red colonies are visible, suggesting successful CRISPR editing of the ADE2 locus. C. Transformed S. cerevisiae colonies from flight and ground.

Four red colonies, labeled R1-R4, and four white colonies, labeled W1-W4, were selected for further assessment by PCR and DNA sequencing. D. Examples of red and white colony phenotypes.

Zoomed in photos of colony R2 and colony W4 from ground control plate A highlight the phenotypic differences between red and white colonies that make it easy to visually identify successfully CRISPR edited colonies. E. Total number of colonies of transformed S. cerevisiae seen in ground and flight experiments after six days of growth at room temperature.

F. Alignment of nanopore sequences from red and white colonies transformed, cultured, extracted, and sequenced in flight or on the ground. Sequences are aligned to either the wild type ADE2 sequence or the ade2 repair template sequence.

Red letters indicate the 12-base-pair insertion in the ade2 repair template sequence. Stop codons are annotated with asterisks and the Cas9 cut site is indicated with an arrow. Yellow letters indicate the six base pairs found in the wild type sequence that are absent from the repair template.

Black shading indicates bases that align to the reference sequence while white shading indicates a mismatch to the reference sequence. Gray shading indicates a match that has <50% coverage relative to other nucleotides.

This method relies on using a CRISPR-based mutagenesis strategy for the targeted generation of DSBs at a defined genomic locus. During CRISPR/Cas9 mediated genome editing, the Cas9 nuclease is directed by an engineered guide RNA to recognize and create a DSB a specific site in the genome.

DNA repair mechanisms then make changes to the DNA sequence at the site of the DSB. NHEJ may introduce random insertions or deletions at the break site, while HR can be harnessed to make specific changes to the DNA sequence through an engineered repair template.

The choice of repair pathway can be determined by analyzing the DNA sequence at the break site to determine whether the sequence includes the expected repair template sequence, indicating repair by HR, or whether the sequence contains random insertions or deletions, indicating NHEJ was used.

This type of DNA sequence analysis can be performed using methods that have been previously established to work in space, specifically amplification by polymerase chain reaction (PCR) followed by  polymerase chain reaction (PCR) followed by nanopore sequencing.

The plates were assessed on the ISS and ground six days after transformation. The ISS crew reported four red colonies and six white colonies. Ground controls contained eight red and twenty-nine white colonies.

The red phenotype is indicative of successful CRISPR/Cas9 mutagenesis resulting in the disruption of the ADE2 locus.

Conclusion

To confirm successful CRISPR/Cas9 genome editing, the ADE2 locus was examined using PCR and DNA sequencing. Four red colonies (labeled R1-R4) and four white colonies (labeled W1-W4) were randomly selected from both ISS and ground transformation plates for DNA extraction.

DNA was extracted from these colonies using a simple protocol where cells were heated to 95°C for 10 minutes in DNA extraction buffer. Purified DNA with DNA extraction buffer was sequenced using the MinION nanopore sequencer. Sequencing data was downlinked to Earth for analysis.

A  significant step towards enabling a better understanding of DNA repair pathway choice in microgravity conditions, this approach has one notable limitation. DSBs generated by Cas9 are much simpler than those generated by high-LET particles found in space.

As the complexity of the DNA damage may influence repair pathway choice, our model may not fully recapitulate the conditions found outside of Earth’s magnetosphere.

Future studies could attempt to better mimic the effects of high-LET radiation by generating more complex breaks. For example, one might imagine creating clustered DNA damage sites using multiple gRNAs simultaneously.

Results demonstrate the first successful use of both transformation and CRISPR/Cas9 genome editing in space and represent a significant expansion of the molecular biology toolkit onboard the ISS.

In addition to establishing a viable platform for furthering our understanding of DNA repair in microgravity, these tools may enable the adaptation of many powerful methods for use in space.

For example, the applications of CRISPR/Cas9 genome editing on Earth are rapidly expanding to include a number of gene editing approaches as well as novel uses of this technology such as viral detection.

One might imagine how CRISPR screens can expand our understanding of biological responses to microgravity or the utility of a simple detection assay in ensuring astronaut safety on long-duration missions.

Similarly, genetic transformation of microbes has many applications, including the production of large amounts of a desired protein on demand.

In the future, transformation in space might allow for on-demand production of critical medicines during deep space missions, or be used for pharmaceutical microgravity research in orbiting laboratories such as those currently in development by several commercial entities .

This study has the potential to impact both our understanding of basic biological processes in microgravity as well as future space exploration and colonization and highlights the importance of basic molecular biology research onboard the ISS National Lab.