Research
Simplifying the Creation of Transgenics
Transgenes are a cornerstone of molecular biology. We have made significant advancements in our ability to design, create, and experimental test transgenes. For Caenorhabditis elegans, there has been several advancements in creating integrated transgenes, such as mosSCI and CRISPR.
However, the process of cloning transgenes into plasmids, and validating the transgene has integrated into the genome properly still poses several technical challenges. To simplify the process, we took advantage of the C. elegans native homology directed repair to ‘clone’ genes within the worm. We found that several fragments can be fused together and integrated into the genome all in a single step.
Additionally, because worms form extrachromosomal arrays from injected DNA, extra steps are needed to ensure the transgene is integrated onto a native chromosome. Generally, this is done by performing several anti-array screening steps, such as inducing a toxic transgene and screening for lack of fluorescent co-markers. We sought to reverse the process. Instead of selecting against arrays, we decided to build a system to select for integrations. Using CRISPR, we engineered synthetic ‘landing pads,’ which contain half of a selectable antibiotic resistance gene. When the transgene is integrated, it brings the second half of the antibiotic resistance gene, restoring functionality. This experimentally provides us with integration-specific selection.
Publication:
Stevenson, Z. C., Moerdyk-Schauwecker, M. J., Jamison, B., & Phillips, P. C. (2020). Rapid Self-Selecting and Clone-Free Integration of Transgenes into Engineered CRISPR Safe Harbor Locations in Caenorhabditis elegans. G3 Genes|Genomes|Genetics, 10(10), 3775–3782. doi.org/10.1534/g3.120.401400
Github website for reagents and updates: https://github.com/phillips-lab/SLP
Bringing Library Transgenesis to Animal Systems
In microbial and cell culture systems, transformation and transduction of large, diverse transgenic libraries have enabled several experimental paradigms. From synthetic biology to optimizing genetic circuits, to functional biology, screening for functional amino acid residues for a protein, all the way to evolutionary biology with large libraries of barcodes, enabling large selection experiments.
However, for all animal systems, there have been significant logistical limitations to testing large synthetic libraries. For Caenorhabditis elegans, we must perform microinjections, generally with the goal of getting one or a few transgenics at a time. Several publications have increased this number allowing a few hundreds of specific edits, however, to screen on the scale of microbial or cell culture systems, there needs to be advancements in methodology.
To overcome this limitation, we developed TARDIS (Transgenic Arrays Resulting In Diversity of Integrated Sequences). TARDIS allows us to screen several thousand variants— more than is possible from a single individual alone. TARDIS works by injecting the DNA library to form a TARDIS array. The array is heritable and passes the genetic library to future generations. This amplification step is critical because it 1) replicates the library and 2) allows it to be used in many individual worms. It is this amplification that allows for several thousand variants to be tested at once from a single injection.
Publication:
Zachary C. Stevenson, Megan J. Moerdyk-Schauwecker, Stephen A. Banse, Dhaval S. Patel, Hang Lu, Patrick C. Phillips, (2023) High-Throughput Library Transgenesis in Caenorhabditis elegans via Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS) eLife12:RP84831 https://doi.org/10.7554/eLife.84831.1
Patent:
Stevenson, Z. C., Banse, S. A., & Phillips, P. C. (2021). Genetic data compression and methods of use (Patent No. US20210332387A1)
Github website for reagents and updates: https://github.com/phillips-lab/TARDIS
Quantitative High throughput Measurement of Selection in an Animal System via Novel Barcode Library Transgenesis
Caenorhabditis elegans is a widely used model organism for studying various biological processes, including neurobiology, aging and experimental evolution. C. elegans have many advantages for experimental evolution, such as a rapid life cycle, large brood size, the capacity to freeze and revive populations, self-fertilization reproduction and easy genetic manipulation via CRISPR.
We are using C. elegans as the first genomically-barcoded experimental evolutionary animal model to compete two different strains under various concentrations of ivermectin as a selective pressure. We introduced a genomic barcode sequence into lineages of each strain using CRISPR genome editing by TARDIS (described above) to create mixed barcoded populations and grown in liquid medium for approximately five generations with various concentrations of ivermectin.
By adopting a liquid protocol, we can grow populations in the millions, making this one of the largest animal experimental evolutions to date. Barcode frequencies are then used to measure the fitness of the individual lineages in the population. We found that at low concentrations of ivermectin, the wild type strain holds an advantage, while higher concentrations tend to favor the resistant strain.
Publication:
In prep
