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Spatial multiomic mapping of gene function with CRISPRoff

使用 CRISPRoff 进行基因功能的空间多组图谱

基本信息

批准号：
10518318
负责人：
Luke Gilbert
金额：
$ 174.02万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10518318
关键词：
ATAC-seq Alleles Area Biological Assay Biological Models Biology Cell division Cell physiology Cells Chimeric Proteins Chromatin Chromosome Mapping Clustered Regularly Interspaced Short Palindromic Repeats Code Communities DNA Damage DNA Methylation DNA Repair Development Disease Ectoderm Embryonic Development Endoderm Epigenetic Process Financial compensation Gene Expression Gene Silencing Genes Genetic Genetic Transcription Genomic approach Goals Histones Human Human Biology Human Development Human Genome Image Individual Life Maps Measures Mesoderm Messenger RNA Methods Microscopy Modeling Molecular Mutagenesis Neurons Nuclear Organoids Phenotype Population Process Proteins Reproducibility Research Research Personnel Resolution Resources Specificity Technology Testing Time biomedical scientist cell type epigenetic memory functional genomics gene function genetic manipulation human disease human model induced pluripotent stem cell innovation insight interest molecular phenotype multiple omics paralogous gene programs resilience single-cell RNA sequencing stem cell differentiation stem cells transcription factor transcriptome transcriptomics

项目摘要

PROJECT SUMMARY / ABSTRACT A hallmark goal in human biology is to define the relationship between genes and phenotypes. Mapping the function of every gene in human cells will enable us to begin to define how gene expression programs impart specialized and adaptive human cellular functions required for life. We are especially interested in how transcription factors and epigenetic regulators enact cell type specific gene expression programs to dictate cell function during early development. Elucidating how individual genes function to regulate transcription and thus to program cell phenotypes will transform our understanding of human biology, development and disease. A mechanistic understanding of gene function requires scalable approaches for perturbing gene activity, single cell molecular phenotyping assays and robust models of human multicellular biology. We recently developed CRISPRoff— a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that durably and robustly silences gene expression. Unlike CRISPR mutagenesis approaches, CRISPRoff gene silencing effectively programs null alleles at the level of target gene mRNA and protein in polyclonal cell populations without induction of DNA damage or the unpredictability of DNA repair processes. We are proposing to optimize a generalizable multiomic CRISPRoff platform for molecularly phenotyping null alleles at single-cell resolution in multicellular models of human development. We will then use this CRISPRoff platform to create single-cell molecular multiomic maps of nuclear gene function across space and time. Lastly, we will evaluate genetic compensation and paralog functional redundancy in multicellular models. Our proposed research will serve to demonstrate the utility of this multiomics CRISPRoff platform for characterizing null alleles and motivate extending this approach to functionally map null allele phenotypes for all genes encoded by the human genome. The results of the proposed research will serve as a fundamental resource and roadmap for a broad community of biomedical scientists and greatly inform our understanding of gene function in human biology and disease.

项目摘要/摘要