Engineered adipocytes implantation suppresses tumor progression in cancer models

CRISPRa browning of human white adipocytes To induce browning in human adipocytes, we used CRISPRa to upregulate UCP1, PPARGC1A or PRDM16, all known genes involved in BAT development and function. Using CRISPick36, we designed five guide RNAs (gRNAs) targeting each gene’s promoter and cloned them into an adeno-associated virus (AAV)-based expression vector. Differentiated adipocytes derived from human white preadipocytes were co-transfected with the gRNAs along with a Staphylococcus aureus endonuclease-deficient Cas9 (dCas9) fused to the VP64 transcriptional activator. We used an S. aureus dCas9 due to its smaller size and the VP64 transcriptional activator (which carries four copies of VP16, a herpes simplex virus type 1 transcriptional activator37) because it provides moderate gene upregulation and is small enough to fit into AAV, which has a 4.7 kb optimal packaging capacity38. To generate mature human adipocytes, preadipocytes were subjected to adipocyte differentiation using a co ... Read full article.