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Principal Investigator: Steven Michael Romanelli
Organization: UNIVERSITY OF MICHIGAN AT ANN ARBOR
Fiscal Year: 2020
Award: $36,743
Funding agency: National Institute of Diabetes and Digestive and Kidney Diseases
PROJECT SUMMARY
The prevalence of obesity has reached pandemic levels with nearly 2 billion people worldwide considered
overweight and one-third of that obese. Obesity is associated with an increased risk for type II diabetes, heart
disease, and certain types of cancer which severely debilitates quality of life and adds considerably to national
healthcare budgets. While diet and lifestyle choices are the main contributors, dysregulation of adipogenic or
metabolic pathways also predispose individuals towards obesity. Thus, it is essential we understand the
underlying mechanisms of adipocyte development and metabolism to help address this global health crisis.
Traditionally, we have relied upon knockout or knock-in approaches to study the function of adipogenic genes
in a physiological context. While this method has been useful to uncover mechanistic pathways, it remains
inefficient as generating transgenic mice is time consuming, challenging, and expensive. Moreover, it limits the
scope of gene targets as embryonically lethal genes cannot be explored. To circumvent these issues, we
developed a robust, adipose-specific CRISPR/Cas9 model to improve our ability to study adipocyte physiology.
CRISPR/Cas9 enables an unprecedented ability to target the genome and introduce permanent, site specific
mutations in DNA. Recently, several strategies to model disease in vivo have utilized CRISPR/Cas9 via
delivery or transgenic expression of its components in a tissue specific manner. The use of CRISPR/Cas9 to
explore gene function in adipose tissue, however, has remained limited. Our approach focuses on an adipose-
specific inducible Cas9-mouse model to which single-guide RNAs (sgRNAs) are delivered to target genes of
interest. This method is innovative in that it: a.) reduces the time required to generate a transgenic mouse, b.)
affords the ability to probe multiple gene targets simultaneously, c.) allows targeting of embryonically lethal
genes in adult mice, and d.) controls Cas9 activity to limit off-target effects. We have created a cloning pipeline
strategy and efficiency assay to validate sgRNAs in cultured Cas9-preadipocytes and are optimizing sgRNA
delivery to Cas9 mice using adeno-associated virus (AAV) and lipid nanoparticles (LNPs). To show proof of
concept, we initially targeted leptin, as successful knockout is easily observed by an obese phenotype.
Currently, we are expanding this approach to demonstrate its utility to understand the role of cold-inducible
RNA binding protein in adipocyte cold adaptation. Ultimately, this approach can transform how we study
adipose tissue and uncover novel therapeutic targets for treating obesity and its associated morbidities.
Terms: <21+ years old><ACRP30 protein><Address><Adeno-Associated Viruses><Adipocytes><Adipose Cell><Adipose tissue><Adult><Adult Human><Adult-Onset Diabetes Mellitus><Apo-E><ApoE><Apolipoprotein E><Assay><Bioassay><Biologic Assays><Biological Assay><Biology><Body Composition><Body Temperature Regulation><Body Thermoregulation><Body Tissues><Body Weight><Budgets><CRISPR><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/Cas system><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cancers><Cardiac Diseases><Cardiac Disorders><Cardiovascular Diseases><Cas nuclease technology><Cell Body><Cell Culture Techniques><Cells><Cessation of life><Cloning><Clustered Regularly Interspaced Short Palindromic Repeats><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><Consumption><DNA><Data><Death><Deoxyribonucleic Acid><Dependoparvovirus><Dependovirus><Diabetes Mellitus><Diet><Disease model><Eating><Embryo><Embryonic><Engineering><Ensure><Fat Cells><Fats><Fatty Tissue><Fatty acid glycerol esters><Food Intake><Gene Proteins><Gene Targeting><Genes><Genetic Alteration><Genetic Change><Genetic defect><Genetics-Mutagenesis><Genome><Guide RNA><Healthcare><Heart Diseases><Heat Production><In Vitro><Incubated><Individual><Intermediary Metabolism><Investigation><Ketosis-Resistant Diabetes Mellitus><Knock-in><Knock-out><Knockout><Leptin><Lethal Genes><Life Style><Lifestyle><Lipids><Lipocytes><Malignant Neoplasms><Malignant Tumor><Mature Lipocyte><Mature fat cell><Maturity-Onset Diabetes Mellitus><Measures><Metabolic Pathway><Metabolic Processes><Metabolism><Methodological Studies><Methods><Mice><Mice Mammals><Modeling><Monitor><Monounsaturated Fatty Acids><Morbidity><Morbidity - disease rate><Murine><Mus><Mutagenesis><Mutagenesis Molecular Biology><Mutation><NIDDM><Non-Insulin Dependent Diabetes><Non-Insulin-Dependent Diabetes Mellitus><Noninsulin Dependent Diabetes><Noninsulin Dependent Diabetes Mellitus><Ob Gene Product><Ob Protein><Obese Gene Product><Obese Protein><Obesity><Over weight><Overweight><Pathway interactions><Peptides><Phenotype><Physiologic><Physiologic Thermoregulation><Physiological><Physiology><Play><Prevalence><Protein Gene Products><Proteomics><QOL><Quality of life><RNA Seq><RNA delivery><RNA sequencing><RNA-Binding Proteins><RNAseq><Risk><Role><Saturated Fatty Acids><Site><Slow-Onset Diabetes Mellitus><Specificity><Stable Diabetes Mellitus><Stearate Desaturase><Stearoyl-CoA Desaturase><Stearyl-CoA Desaturase><System><T2 DM><T2D><T2DM><Targeted Resequencing><Technology><Testing><Thermogenesis><Thermoregulation><Time><Tissues><Transgenic Mice><Transgenic Model><Transgenic Organisms><Tropism><Type 2 Diabetes Mellitus><Type 2 diabetes><Type II Diabetes Mellitus><Type II diabetes><VEGF><VEGFs><Vascular Endothelial Growth Factors><adeno associated virus group><adipocyte complement-related protein 30-kDa><adipocyte development><adipocyte differentiation><adipocyte, C1q and collagen domain containing protein><adipogenesis><adiponectin><adipose><adiposity><adult onset diabetes><adulthood><apM-1 protein><apM1 (adipose-specific) protein><cancer type><cardiovascular disorder><cell culture><cold temperature><combat><corpulence><corpulency><corpulentia><delta-9 Desaturase><design><designing><diabetes><dietary><disorder model><gRNA><gene function><genome mutation><genome scale><genome-wide><genomewide><global health><health care><heart disorder><improved><in vivo><innovate><innovation><innovative><interest><ketosis resistant diabetes><knock-down><knockdown><knockin><knockout gene><lipid biosynthesis><lipid nanoparticle><lipogenesis><low temperature><malignancy><maturity onset diabetes><mouse model><murine model><nano particle delivery><nanoparticle delivered><nanoparticle delivery><neoplasm/cancer><new drug target><new druggable target><new pharmacotherapy target><new technology><new therapeutic target><new therapy target><novel><novel drug target><novel druggable target><novel pharmacotherapy target><novel technologies><novel therapeutic target><novel therapy target><obese><obese people><obese person><obese population><off-target site><pandemic><pandemic disease><pathway><prediction algorithm><predictive algorithm><predictor algorithm><premature><prematurity><response><screening><social role><therapeutic target><transcriptome sequencing><transgenic><transgenic trait><type 2 DM><type II DM><type two diabetes><white adipose tissue><yellow adipose tissue>