Gene activation by a CRISPR-assistant trans enhancer

Abstract

15Gene activation is essential to the basic biological research and biomedicine. Therefore, various gene 16 activators such as activation domain-ZNF, TALE and CRISPR proteins have been developed for this end, in 17 which the CRISPR protein dead Cas9 (dCas9) is now most widely used. However, the current gene activators 18 are still limited by their inefficient gene activation activity. In this study, we developed a new strategy, 19 CRISPR-assistant trans enhancer, for activating gene expression in high efficiency by combining dCas9-20 VP64/sgRNA with a widely used strong enhancer, the CMV enhancer. In this strategy, a trans CMV enhancer 21 DNA was recruited to target gene by dCas9-VP64/sgRNA via annealing between 3′ end of sgRNA and CMV 22 enhancer. The trans enhancer activates gene transcription as the natural looped cis enhancer. The trans 23 enhancer could activate both exogenous reporter gene and variant endogenous genes in various cells, with 24 much higher activation efficiency than the current dCas9 activators. 25 Keywords: CRISPR/dCas9, CMV enhancer, trans enhancer, gene activation 26 27 28 2013; Perez-Pinera et al. 2013), VPR (VP64-p65-Rta) (Chavez et al. 2015), and KRAB (Zheng et al. 2018). 49 Additionally, the dCas9 protein was also fused with other functional domains with transcriptional regulatory 50 functions, such as p300(Hilton et al., 2015), LSD1(Kearns et al. 2015), Dnmt3a (Liu et al. 2016a; 51 Saunderson et al. 2017), and Tet1(Choudhury et al. 2016; Liu et al., 2016a). Based on these domains, 52 more elaborate activators have been developed for more potent activation of target genes in mammalian cells, 53 such as SunTag (dcas9-GCN4/sgRNA plus scFV-VP64) (Tanenbaum et al. 2014), and SPH (dCas9-54 GCN4/sgRNA plus scFV-p65-HSF1) (Zhou et al. 2018). More complex, some inducible dCas9 systems 55 have been developed to control activity of dCas9 activators in cells, such as light-activated CRISPR/Cas9 56 effector (Nihongaki et al. 2015; Polstein and Gersbach 2015), hybrid drug inducible CRISPR/Cas9 57 technology (HIT) (Lu et al. 2018), and CRISPR activator gated by human antibody-based chemically 58 induced dimerizers (AbCIDs) (Liu et al. 2018). A trouble of these typical dCas9 fusion proteins in their in 59vivo applications is that they are difficult to be accommodated by adeno-associated virus (AAV), a most 60 suitable type of non-integrating gene therapy vector, due to its limited packaging capacity. 61Besides dCas9 engineering, sgRNA has also been engineered to develop new dCas9-based activators. 62Compared with dCas9 engineering, sgRNA is more simple, flexible and efficient to redesign. Moreover, the 63 engineered sgRNA is more helpful for the in vivo application of dCas9-based activators due to its limited 64 length for virus packaging. The most widely used engineered sgRNA is the sgRNA fused with MS2 loops 65 at the 3′ end (sgRNA-MS aptamer), which can be bound by dimerized MS2 bacteriophage coat proteins 66 fused with transcription-activating domains VP64-HSF1 (MS2-VP64-HSF1, MPH)(Konermann et ...