Biology subjects

Moerdyk-Schauwecker, M. J.

Publications and source records attributed to Moerdyk-Schauwecker, M. J..

4 recordsLinked to original sources

bioRxiv2022

High-Throughput Library Transgenesis in Caenorhabditis elegans via Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS)

High-throughput transgenesis using synthetic DNA libraries is a powerful method for systematically exploring genetic function. Diverse synthesized libraries have been used for protein engineering, identification of protein-protein interactions, characterization of promoter libraries, developmental and evolutionary lineage tracking, and various other exploratory assays. However, the need for library transgenesis has effectively restricted these approaches to single-cell models. Here we present Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS), a simple yet powerful approach to large-scale transgenesis that overcomes typical limitations encountered in multicellular systems. TARDIS splits the transgenesis process into a two-step process: creation of individuals carrying experimentally introduced sequence libraries, followed by inducible extraction and integration of individual sequences/library components from the larger library cassette into engineered genomic sites. Thus, transformation of a single individual, followed by lineage expansion and functional transgenesis, gives rise to thousands of genetically unique transgenic individuals. We demonstrate the power of this system using engineered, split selectable TARDIS sites in Caenorhabditis elegans to generate (1) a large set of individually barcoded lineages and (2) transcriptional reporter lines from pre-defined promoter libraries. We find that this approach increases transformation yields up to approximately 1000-fold over current single-step methods. While we demonstrate the utility of TARDIS using C. elegans, in principle the process is adaptable to any system where experimentally generated genomic loci landing pads and diverse, heritable DNA elements can be generated.

genetics↗

bioRxiv2021

Post-Insemination Selection Dominates Pre-Insemination Selection in Driving Rapid Evolution of Male Competitive Ability

Sexual reproduction is a complex process that contributes to differences between the sexes and divergence between species. From a males perspective, sexual selection can optimize reproductive success by acting on the variance in mating success (pre-insemination selection) as well as the variance in fertilization success (post-insemination selection). The balance between pre- and post-insemination selection has not yet been investigated using a strong hypothesis-testing framework that directly quantifies the effects of post-insemination selection on the evolution of reproductive success. Here we use experimental evolution of a uniquely engineered genetic system that allows sperm production to be turned off and on in obligate male-female populations of Caenorhabditis elegans. We show that enhanced post-insemination competition increases the efficacy of selection and surpasses pre-insemination sexual selection in driving a polygenic response in male reproductive success. We find that after 30 generations post-insemination selection increased male reproductive success by an average of 5- to 7-fold. Contrary to expectation, enhanced pre-insemination competition hindered selection and slowed the rate of evolution. Furthermore, we found that post-insemination selection resulted in a strong polygenic response at the whole-genome level. Our results demonstrate that post-insemination sexual selection plays a critical role in the rapid optimization of male reproductive fitness. Therefore, explicit consideration should be given to post-insemination dynamics when considering the population effects of sexual selection.

evolutionary biology↗

bioRxiv2020

Rapid self-selecting and clone free integration of transgenes into engineered CRISPR safe harbor locations in Caenorhabditis elegans

Precision genome editing for model organisms has revolutionized functional analysis and validation of a wide variety of molecular systems. To date, the capacity to insert transgenes into the model nematode Caenorhabditis elegans has focused on utilizing either transposable elements or CRISPR-based safe harbor strategies. These methods require laborious screening processes that often result in false positives from heritable extrachromosomal arrays or rely on co-CRISPR markers to identify likely edited individuals. As a result, verification of transgene insertion requires anti-array selection screening methods or extensive PCR genotyping respectively. These approaches also rely on cloning plasmids for the addition of transgenes. Here, we present a novel safe harbor CRISPR-based integration strategy that utilizes engineered insertion locations containing a synthetic guide RNA target and a split-selection system to eliminate false positives from array formation, thereby providing integration-specific selection. This approach allows the experimenter to confirm an integration event has taken place without molecular validation or anti-array screening methods, and is capable of producing integrated transgenic lines in as little as five days post-injection. To further increase the speed of generating transgenic lines, we also utilized the C. elegans native homology-based formation of extra-chromosomal arrays to assemble transgenes in-situ, removing the cloning step. We show that complete transgenes can be made and inserted into our split-selection safe harbor locations starting from PCR products, providing a clone-free and molecular-validation-free strategy for single-copy transgene integration. Overall, this combination of approaches provides an economical and rapid system for generating highly reproducible complex transgenics in C. elegans.

genetics↗

bioRxiv2018

Proteomic and Evolutionary Analyses of Sperm Activation Identify Uncharacterized Genes in Caenorhabditis Nematodes

BackgroundNematode sperm have unique and highly diverged morphology and molecular biology. In particular, nematode sperm contain subcellular vesicles known as membranous organelles that are necessary for male fertility, yet play a still unknown role in overall sperm function. Here we take a novel proteomic approach to characterize the functional protein complement of membranous organelles in two Caenorhabditis species: C. elegans and C. remanei.\n\nResultsWe identify distinct protein compositions between membranous organelles and the activated sperm body. Two particularly interesting and undescribed gene families--the Nematode-Specific Peptide family, group D and the here designated Nematode-Specific Peptide family, group F--localize to the membranous organelle. Both multigene families are nematode-specific and exhibit patterns of conserved evolution specific to the Caenorhabditis clade. These data suggest gene family dynamics may be a more prevalent mode of evolution than sequence divergence within sperm. Using a CRISPR-based knock-out of the NSPF gene family, we find no evidence of a male fertility effect of these genes, despite their high protein abundance within the membranous organelles.\n\nConclusionsOur study identifies key components of this unique subcellular sperm component and establishes a path toward revealing their underlying role in reproduction.

evolutionary biology↗