Current in vitro approaches for laboratory- and cell-based toxicology studies do not capture the interindividual variability in responses within the human population. Single nucleotide gene polymorphisms, gene heterozygosity, variations in gene expression and in some cases gene loss can yield highly variable responses to genotoxic compounds, ranging from hypersensitivity to complete resistance. Further, toxicological analysis based on model organisms such as bacteria, rats or mice do not adequately provide such response variability. A defined panel of human cells with appropriate genetic diversity, especially in genes and gene families that alter the response outcome to genotoxins, may begin to offer such toxicodynamic variability. Here, we propose to employ our Barcoded Exon Tagging And Gene (BETA-Gene) disruption platform to create barcoded control, heterozygous gene knockout (KO) and homozygous gene KO panels of diploid human cells for high-throughput, multiplexed genotoxin screens. The availability of panels of such cells would provide a level of genetic diversity currently unavailable for cyto-toxicological analysis. To address this significant need and in response to RFA-ES-20-008, we have outlined three specific aims. In Aim 1, we propose to develop a 99-cell panel of barcoded, human diploid RPE-1 cells engineered with a single or double allele gene disruption in genotoxin-response gene families: DNA damage response/repair, cell death and stress response. This approach, BETA-Gene disruption, utilizes the CRISPR/cas9 gene editing system for simultaneous exon deletion/disruption and gene-specific barcode tagging with preference for a single allele in diploid cells. This will then yield the development of a barcoded 48-cell line heterozygous gene KO panel, a barcoded 48-cell line homozygous gene KO panel and three barcoded, unmodified control cells amenable for multiplexed, cytotoxicity analysis. Goals of Aim 2 will include genetic validation and functional genotoxin-response testing of the RPE-1 BETA-Gene disrupted cell lines and in Aim 3, we will validate the barcoded, multiplex genotoxin screening platform to demonstrate the variability in response of the RPE-1 BETA-Gene disrupted heterozygous gene-KO and homozygous gene-KO cell line pools upon exposure to genotoxic and non-genotoxic compounds. This system will provide a rapid and high-throughput, barcode-based multiplex analysis of toxicodynamic variability coupled with mechanistic insight that contributes to the variability in genotoxin response.
Public Health Relevance Statement: Project Narrative This Phase I/II fast track proposal will yield the development of a defined panel of barcoded, human cells with genetic diversity in genotoxin-response gene families: DNA damage response/repair, cell death and stress response. This system will provide a rapid and high-throughput, barcode-based analysis of toxicodynamic variability coupled with mechanistic insight that contributes to the variability in genotoxin response.
Project Terms: Alleles; Allelomorphs; Bacteria; Bar Codes; barcode; Cell Cycle; Cell Division Cycle; Cell Death; necrocytosis; Cell Line; CellLine; Strains Cell Lines; cultured cell line; Cell Survival; Cell Viability; Cells; Cell Body; Diploidy; Diploid; DNA Damage; DNA Injury; Eligibility Determination; Eligibility; Protocol Screening; Exons; Flow Cytometry; Flow Cytofluorometries; Flow Cytofluorometry; Flow Microfluorimetry; Flow Microfluorometry; flow cytophotometry; Gene Expression; Genes; Goals; Grant; Heterozygote; heterozygosity; Human; Modern Man; Hypersensitivity; Allergy; In Vitro; Laboratories; Leadership; Mus; Mice; Mice Mammals; Murine; Mutagens; Genotoxins; genotoxic agent; Nucleotides; Genetic Polymorphism; polymorphism; Quality Control; Rattus; Common Rat Strains; Rat; Rats Mammals; Research Personnel; Investigators; Researchers; Testing; Toxicology; Genetic Variation; Genetic Diversity; Work; biological adaptation to stress; reaction; crisis; stress response; stress; reaction; Sequence Analysis; SEQ-AN; Sequence Analyses; base; repaired; repair; Phase; Variant; Variation; insight; Toxicity Tests; Toxicity Testing; Funding; Genetic; Exposure to; System; preference; Lytotoxicity; cytotoxicity; genotoxicity; knockout gene; Animal Models and Related Studies; model of animal; model organism; Animal Model; response; cell engineering; cellular engineering; Drops; Diploid Cells; Address; Cellular Stress; cell stress; Gene Family; Small Business Innovation Research Grant; SBIR; Small Business Innovation Research; Validation; Knock-out; Knockout; Development; developmental; novel strategies; new approaches; novel approaches; novel strategy; Outcome; Population; Coupled; Resistance; resistant; screening; Genomic DNA; gDNA; CRISPR/Cas technology; CRISPR method; CRISPR methodology; CRISPR technique; CRISPR technology; CRISPR-CAS-9; CRISPR-based method; CRISPR-based technique; CRISPR-based technology; CRISPR-based tool; CRISPR/Cas method; CRISPR/Cas9; CRISPR/Cas9 technology; Cas nuclease technology; 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; inter-individual variation; inter-individual variability; interindividual variability; interindividual variation
