Biology subjects

Sternberg, P. W.

Publications and source records attributed to Sternberg, P. W..

3 recordsLinked to original sources

bioRxiv2018

An efficient genome editing strategy to generate putative null mutants in Caenorhabditis elegans using CRISPR/Cas9

Null mutants are essential for analyzing gene function. Here, we describe a simple and efficient method to generate Caenorhabditis elegans null mutants using CRISPR/Cas9 and short single stranded DNA oligo repair templates to insert a universal 43-nucleotide-long stop knock-in (STOP-IN) cassette into the early exons of target genes. This cassette has stop codons in all three reading frames and leads to frameshifts, which will generate putative null mutations regardless of the reading frame of the insertion position in exons. The STOP-IN cassette also contains an exogenous Cas9 target site that allows further genome editing and provides a unique sequence that simplifies the identification of successful insertion events via PCR. As a proof of concept, we inserted the STOP-IN cassette right at a Cas9 target site in aex-2 to generate new putative null alleles by injecting preassembled Cas9 ribonucleoprotein and a short synthetic single stranded DNA repair template containing the STOP-IN cassette and two 35-nucleotide-long homology arms identical to the sequences flanking the Cas9 cut site. We showed that these new aex-2 alleles phenocopied an existing loss-of-function allele of aex-2. We further showed that the new aex-2 null alleles could be reverted back to the wild-type sequence by targeting exogenous Cas9 cut site included in the STOP-IN cassette and providing a single stranded wild-type DNA repair oligo. We applied our STOP-IN method to generate new putative null mutants for additional 20 genes, including three pharyngeal muscle-specific genes (clik-1, clik-2, and clik-3), and reported a high insertion rate (46%) based on the animals we screened. We showed that null mutations of clik-2 cause recessive lethality with a severe pumping defect and clik-3 null mutants have a mild pumping defect, while clik-1 is dispensable for pumping. We expect that the knock-in method using the STOP-IN cassette will facilitate the generation of new null mutants to understand gene function in C. elegans and other genetic model organisms.\n\nSummaryWe report a simple and efficient CRISPR/Cas9 genome editing strategy to generate putative null C. elegans mutants by inserting a small universal stop knock-in (STOP-IN) cassette with stop codons in three frames and frameshifts. The strategy is cloning-free, with the mixture consisting of preassembled Cas9 ribonucleoprotein and single stranded repair DNA oligos directly injected into gonads of adult C. elegans. The universal STOP-IN cassette also contains a unique sequence that simplifies detection of successful knock-in events via PCR and an exogenous Cas9 target sequence that allows further genome editing.

genetics↗

bioRxiv2017

Genetic Analysis of a Metazoan Pathway using Transcriptomic Phenotypes

RNA-seq is commonly used to identify genetic modules that respond to perturbations. In single cells, transcriptomes have been used as phenotypes, but this concept has not been applied to whole-organism RNA-seq. Linear models can quantify expression effects of individual mutants and identify epistatic effects in double mutants. To make interpretation of these high-dimensional measurements intuitive, we developed a single coefficient to quantify transcriptome-wide epistasis that accurately reflects the underlying interactions. To demonstrate our approach, we sequenced four single and two double mutants of Caenorhabditis elegans. From these mutants, we reconstructed the known hypoxia pathway. In addition, we uncovered a class of 56 genes that have opposing changes in expression in egl-9(lf) compared to vhl-1(lf) but the egl-9(lf); vhl-1(lf) mutant has the same phenotype as egl-9(lf). This class violates the classical model of HIF-1 regulation, but can be explained by postulating a role of hydroxylated HIF-1 in transcriptional control.\n\nSignificance StatementTranscriptome profiling is a way to quickly and quantitatively measure gene expression level. Because of their quantitative nature, there is widespread interest in using transcriptomic profiles as a phenotype for genetic analysis. However, a source of major concern is that whole-animal transcriptomic profiles mix the expression signatures of multiple cellular states, making it hard to accurately reconstruct genetic interactions. Additionally, it has been difficult to quantify epistasis, the signature of genetic interaction between two genes, in these molecular phenotypes. Here, we show that it is possible to accurately reconstruct genetic interactions between genes using whole-animal RNA sequencing, and we demonstrate a powerful new way to measure and understand epistasis arising from these measurements. This suggests that whole-organism RNA-seq can be a powerful tool with which to understand genetic interactions in entire organisms and not only in isolated cells. With the advent of genome engineering tools, generating mutants has become easier and faster for many organisms. As mutants become easier to create, phenotyping them has become a major bottleneck in understanding the biological functions of the genes in question. Our work presents a possible solution to this problem, because transcriptome profiling is fast and sensitive to genetic perturbations regardless of the context they operate in.

genetics↗

bioRxiv2017

Phenotype and gene ontology enrichment as guides for disease modeling in C. elegans

Genome-wide experiments have the capacity to generate massive amounts of unbiased data about an organism. In order to interpret this data, dimensionality reduction techniques are required. One approach is to annotate genes using controlled languages and to test experimental datasets for term enrichment using probabilistic methods. Although gene, phenotype and anatomy ontologies exist for C. elegans, no unified software offers enrichment analyses of all the ontologies using the same methodology. Here, we present the WormBase Enrichment Suite, which offers users the ability to test all nematode ontologies simultaneously. We show that the WormBase Enrichment Suite provides valuable insight into different biological problems. Briefly, we show that phenotype enrichment analysis (PEA) can help researchers identify disease phenologs, phenotypes that are homologous across species, which can inform disease modeling in C. elegans. The WormBase Enrichment Suite analysis can also shed light on RNA-seq datasets by showing what molecular functions are enriched, which phenotypes these functions are implicated in and what tissues are overrepresented in the dataset. Finally, we explore the phenotype-anatomy relationship, showing that a small subset of highly specific tissues are disproportionately likely to cause an Egl phenotype, but inferring tissue expression from an Egl phenotype is limited to the largest tissues.

bioinformatics↗