MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional activator, elicits gene expression in nematodes, mice and human cells

CRISPR-mediated gene activation (CRISPRa) is a promising therapeutic gene editing strategy without inducing DNA double-strand breaks (DSBs). However, in vivo implementation of these CRISPRa systems remains a challenge. Here, we report a compact and robust miniCas9 activator (termed miniCAFE) for in vivo activation of endogenous target genes. The system relies on recruitment of an engineered minimal nuclease-null Cas9 from Campylobacter jejuni and potent transcriptional activators to a target locus by a single guide RNA. It enables robust gene activation in human cells even with a single DNA copy and is able to promote lifespan of C. elegans through activation of longevity-regulating genes. As proof-of-concept, delivered within an all-in-one adeno-associated virus (AAV), miniCAFE can activate Fgf21 expression in the liver and regulate energy metabolism in adult mice. Thus, miniCAFE holds great therapeutic potential against human diseases. Overall design: using RNA-seq, we tested the specificity of gene activation with S-D system. Compared with the control group transfected with all the plasmids except the sgRNA, the expression level of untargeted genes in the groups co-transfected with S-D system and sgRNA2 targeting MYOD promoter region was not broadly affected, and the expression of MYOD was significantly increased (FDR<0.05). To check whether a truncated 15-nt sgRNA or WT CjCas9 could affect the specificity of gene activation, we profiled genome-wide gene expression by RNA-seq using IL1RN as the target gene in HEK293T cells. Similar to V-D co-transfected with a 22-nt sgRNA1, V-D or V-WT co-transfected with a 15-nt sgRNA1 showed comparable high specificity. And we also noted that miniCAFE/sgRNA (F) system showed more potent capability to activate gene expression in mouse cells at each ratio. Again, RNA-seq revealed high specificity of the miniCAFE/sgRNA (F)system.