Viral vector-mediated gene activation to facilitate large-scale genetic analysis in Caenorhabditis elegans.

NIH Pandemic-Era Grants

Pandemic Era Grants

2024

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Principal Investigator: MAUREEN C FERRAN
Organization: UNIVERSITY OF ROCHESTER
Fiscal Year: 2024
Award: $185,620
Funding agency: National Institute of General Medical Sciences

Project Summary
Fulfilling the promise of modern systems biology and grasping the underlying complexity of biological systems
requires a foundation built upon the development of high-throughput functional genomic approaches capable of
generating large datasets. Large scale sequencing efforts reveal correlations, but lacks causal interactions
best provided via genetic approaches. Caenorhabditis elegans has been a workhorse for gene discovery and
pathway analysis, and is the only established system where high-throughput genetic analysis can be
conducted in the context of a living multi-cellular organism (i.e. feeding based RNAi). Despite the power of this
model system, no high-throughput methods to achieve targeted gene overexpression in C. elegans have been
developed. This project will explore how recombinant strains of two different viruses can be adapted as vectors
to enable large-scale genetic analysis of gene overexpression in C. elegans. The objective of Specific Aim 1 is
to achieve promoter-specific gene activation using CRISPRa. This variant form of CRISPR relies on a
cleavage defective isoform of Cas9 (dCas9) fused with a transcriptional activator to drive overexpression of a
gene targeted by the single gene RNA (sgRNA). Specifically, we propose to generate proof-of-principle
evidence that recombinant vesicular stomatitis virus (rVSV) can deliver a sgRNA into transgenic C. elegans
that express the CRISPRa machinery in intestinal cells to induce sgRNA-directed overexpression of a reporter
gene. Ultimately our goal is to develop a comprehensive sgRNA VSV library directed to promoter regions to
allow high-throughput functional genomic screening in C. elegans. The objective of Specific Aim 2 is to develop
Orsay virus (OV) as a vector to deliver functional mRNA exogenously into C. elegans. The use of OV as a
gene delivery system is straightforward as this virus readily enters the animal via the intestinal lumen, and C.
elegans expressing integrated segments of the OV genome have been validated. Briefly, we will use these
existing strains as “packaging lines” to express C. elegans genes of interest capable of being incorporated in
newly generated virion to infect recipient nematodes. These studies represent an initial step towards the use of
OV as an overexpression vector and would accelerate the development large-scale genetic analysis in this
multicellular organism. These viral-based expression tools would integrate easily with existing approaches
widely used by the C. elegans community, which could potentially transform multiple areas of scientific
investigation, and has implications for understanding of many diseases.

Terms: <Acceleration><Active Follow-up><Aging><Animals><Area><Biologic Models><Biologic Phenomena><Biological><Biological Models><Biological Phenomena><Body Tissues><C elegans><C. elegans><C.elegans><COVID crisis><COVID epidemic><COVID pandemic><COVID-19 crisis><COVID-19 epidemic><COVID-19 era><COVID-19 global health crisis><COVID-19 global pandemic><COVID-19 health crisis><COVID-19 pandemic><COVID-19 period><COVID-19 public health crisis><COVID-19 years><CRISPR><CRISPR approach><CRISPR based approach><CRISPR editing screen><CRISPR interference><CRISPR method><CRISPR methodology><CRISPR screen><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based screen><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR-dCas9-mediated repression><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas system><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 screen><CRISPR/Cas9 technology><CRISPR/dCas9 interference><CRISPR/dCas9-mediated transcriptional inhibition><CRISPRi><Caenorhabditis elegans><Cas nuclease technology><Cell Body><Cells><Clustered Regularly Interspaced Short Palindromic Repeats><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats interference><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Communities><Complex><Development><Discipline><Disease><Disorder><Double-Stranded RNA><E coli><E. coli><Escherichia coli><Foundations><Gene Activation><Gene Expression><Gene Targeting><Genes><Genetic><Genetic Screening><Genetic analyses><Genomic approach><Goals><Grips><Guide RNA><Health><High Throughput Assay><Human><Infection><Innate Immune Response><Innate Immunity><Intestinal><Intestines><Investigation><Isoforms><LDL Receptors><Large-Scale Sequencing><Libraries><Light><Lipoprotein LDL Receptors><Low Density Lipoprotein Receptor><Mediating><Messenger RNA><Methods><Model System><Modern Man><Modernization><Native Immunity><Natural Immunity><Nematoda><Nematodes><Network Analysis><Non-Polyadenylated RNA><Non-Specific Immunity><Nonspecific Immunity><ORFs><Open Reading Frames><Organism><Pathogenicity><Pathway Analysis><Phenotype><Photoradiation><Population><Post-Transcriptional Gene Silencing><Posttranscriptional Gene Silencing><Process><Production><Promoter Regions><Promotor Regions><Protein Coding Region><Protein Isoforms><RNA><RNA Gene Products><RNA Interference><RNA Silencing><RNA delivery><RNAi><Reagent><Receptor Protein><Recombinants><Regulation><Reporter Genes><Research><Ribonucleic Acid><SARS-CoV-2 epidemic><SARS-CoV-2 global health crisis><SARS-CoV-2 global pandemic><SARS-CoV-2 pandemic><SARS-coronavirus-2 epidemic><SARS-coronavirus-2 pandemic><Sequence-Specific Posttranscriptional Gene Silencing><Severe Acute Respiratory Syndrome CoV 2 epidemic><Severe Acute Respiratory Syndrome CoV 2 pandemic><Severe acute respiratory syndrome coronavirus 2 epidemic><Severe acute respiratory syndrome coronavirus 2 pandemic><System><Systems Biology><Technology><Testing><Tissues><Transcription Activator><Transcription Coactivator><Transcription Factor Coactivator><Transcriptional Activator><Transcriptional Activator/Coactivator><Transcriptional Coactivator><Transgenic Animals><Transgenic Organisms><VSV><Variant><Variation><Vesicular Stomatitis Virus><Vesicular stomatitis Indiana virus><Viral><Viral Diseases><Viral Genome><Viral Vector><Virion><Virus><Virus Diseases><Virus Particle><Work><active followup><biologic><biological systems><bowel><cell type><clustered regularly interspaced short palindromic repeats screen><coronavirus disease 2019 crisis><coronavirus disease 2019 epidemic><coronavirus disease 2019 global health crisis><coronavirus disease 2019 global pandemic><coronavirus disease 2019 health crisis><coronavirus disease 2019 pandemic><coronavirus disease 2019 public health crisis><coronavirus disease crisis><coronavirus disease epidemic><coronavirus disease pandemic><coronavirus disease-19 global pandemic><coronavirus disease-19 pandemic><developmental><discover genes><dsRNA><expression vector><feeding><flexibility><flexible><follow up><follow-up><followed up><followup><functional genomics><gRNA><gene delivery system><gene discovery><genetic analysis><genetic approach><genetic promoter element><genetic promoter sequence><genetic strategy><genomic effort><genomic strategy><grasp><high throughput screening><improved><innovate><innovation><innovative><insight><interest><knock-down><knockdown><large data sets><large datasets><living system><mRNA><model organism><mutant><overexpress><overexpression><pathogen><promoter><promoter sequence><promotor><receptor><recombinant viral vector><repressing CRISPR-dCas9 system><roundworm><screening><screenings><severe acute respiratory syndrome coronavirus 2 global health crisis><severe acute respiratory syndrome coronavirus 2 global pandemic><success><tool><transcription co-activator><transcriptional co-activator><transgenic><uptake><usability><vector><viral infection><virus genome><virus host interaction><virus infection><virus-induced disease>