Biology subjects

Li-Leger, E.

Publications and source records attributed to Li-Leger, E..

2 recordsLinked to original sources

bioRxiv2021

Identification of essential genes in Caenorhabditis elegans through whole genome sequencing of legacy mutant collections

It has been estimated that 15-30% of the [~]20,000 genes in C. elegans are essential, yet many of these genes remain to be identified or characterized. With the goal of identifying unknown essential genes, we performed whole genome sequencing on complementation pairs from legacy collections of maternal-effect lethal and sterile mutants. This approach uncovered maternal genes required for embryonic development and genes with putative sperm-specific functions. In total, 58 essential genes were identified on chromosomes III, IV, and V, of which 49 genes are represented by novel alleles in this collection. Of these 49 genes, 19 (40 alleles) were selected for further functional characterization. The terminal phenotypes of embryos were examined, revealing defects in cell division, morphogenesis, and osmotic integrity of the eggshell. Mating assays with wild-type males revealed previously unknown male-expressed genes required for fertilization and embryonic development. The result of this study is a catalogue of mutant alleles in essential genes that will serve as a resource to guide further study toward a more complete understanding of this important model organism. As many genes and developmental pathways in C. elegans are conserved and essential genes are often linked to human disease, uncovering the function of these genes may also provide insight to further our understanding of human biology.

genetics↗

bioRxiv2018

Optimizing guide RNA selection and CRISPR/Cas9 methodology for efficient generation of deletions in C. elegans.

The Caenorhabditis elegans Gene Knockout (KO) Consortium is tasked with obtaining null mutations in each of the more than 20,000 open reading frames (ORFs) of this organism. To date, approximately15,000 ORFs have associated putative null alleles. A directed approach using CRISPR/Cas9 methodology is the most promising technique to complete the task. While there has been substantial success in using CRISPR/Cas9 in C. elegans, there has been little emphasis on optimizing the method for generating large insertions/deletions in this organism. To enhance the efficiency of using CRISPR/Cas9 to generate gene knockouts in C. elegans we have developed an online species-specific guide RNA selection tool (http://genome.sfu.ca/crispr). When coupled with previously developed selection vectors, optimization for homology arm length, and the use of purified Cas9 protein, we demonstrate a robust, efficient and effective protocol for generating deletions. Debate and speculation in the larger scientific community about off- target effects due to non-specific Cas9 cutting has prompted us to investigate through whole genome sequencing the occurrence of single nucleotide variants and indels accompanying targeted deletions. We did not detect any off-site variants above the natural spontaneous mutation rate and therefore conclude this modified protocol does not generate off-target events to any significant degree in C. elegans.

genetics↗