Rapid and Efficient Identification of Caenorhabditis elegans Legacy Mutations Using Hawaiian SNP-Based Mapping and Whole-Genome Sequencing.
The production of viable embryos requires the coordination of many cellular processes, including protein synthesis, cytoskeletal reorganization, establishment of polarity, cell migration, cell division, and in Caenorhabditis elegans, eggshell formation. Defects in any of these processes can lead to embryonic lethality. We examined six temperature-sensitive mutants as well as one nonconditional mutant that were previously identified in genetic screens as either embryonic lethal (maternal-effect or zygotic lethal) or eggshell defective. The responsible molecular lesion for each had never been determined. After confirmation of temperature sensitivity and lethality, we performed whole-genome sequencing using a single-nucleotide polymorphism mapping strategy to pinpoint the molecular lesions. Gene candidates were confirmed by RNA interference phenocopy and/or complementation tests and one mutant was further validated by CRISPR (Clustered Regularly Interspaced Short Palidromic Repeats)/Cas9 gene editing. This approach identified new alleles of several genes that had only been previously studied by RNA interference depletion. Our identification of temperature-sensitive alleles for all of these essential genes provides an extremely useful tool for further investigation for the C. elegans community, such as the ability to address mutant phenotypes at various developmental stages and the ability to carry out suppressor/enhancer screens to identify other genes that function in a specific cellular process.
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Aimee
A
Jaramillo-Lambert
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Abigail S
AS
Fuchsman
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Amy S
AS
Fabritius
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Harold E
HE
Smith
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 andyg@mail.nih.gov.
Andy
A
Golden
D000483:Alleles; D000818:Animals; D064113:CRISPR-Cas Systems; D017173:Caenorhabditis elegans / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics*; D002874:Chromosome Mapping; D005260:Female; D020043:Genes, Essential; D005816:Genetic Complementation Test; D049751:Genome, Helminth; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing; D008297:Male; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D012641:Selection, Genetic
D029742:Caenorhabditis elegans Proteins
CRISPR/Cas9; Hawaiian SNP mapping; cdc-25; essential genes; whole-genome sequencing