Design Principles for CRISPR Transcriptional Activation

Clustered regularly interspaced palindromic repeats (CRISPR) technology has advanced rapidly in recent years, but this explosion of interest has generated a multitude of methodologies and left some mechanistic underpinnings of the system unexplored. One such example is CRISPR-based transcriptional activation. In order to systematically investigate this technique we analyzed several components of the system including identity of target gene, location of sgRNA target sites, quantity of sgRNAs delivered, mass of dCas9/gRNA plasmid transfected, transfection vs. transduction, temporal kinetics of activation, and the Synergistic Activation Mediator (SAM) versus the VP64-p65-Rta (VPR) activation domain.

Using an sgRNA-aptamer binding system, we transiently transfected human embryonic kidney (HEK) cells with plasmids expressing the sgRNA, deactivated Cas9 (dCas9), and the aptamer-binding activation domain. We transfected multiple guides in varying combinations targeted to activate Cyp3a4, albumin, OSM, ZGPAT, Fah, and ATF5. High (200ng) and low (25ng) levels of sgRNA and dCas9 were tested in addition to both the SAM and VPR activation domains along with variants of the Cas9 protein (M2, M4, P300 fusion). The expression levels of albumin and Fah following activation were tracked for seven days post-transfection.

Increased expression was achieved for all genes tested, but certain genes could be induced much more than others, perhaps due to varying endogenous expression; albumin expression was increased by more than 2400-fold, whereas Fah was increased approximately 10-fold. Factors found to improve the expression levels included sgRNA target sites on distinct DNA strands, delivery of low levels of Cas9 and sgRNA, and use of the SAM activation system. Interestingly, in investigation of temporal changes in expression, target transcript levels were found to spike at four days post-transfection. Identifying the effects of each of these variables provides a more robust framework to approach experimental design and optimize standard operating procedures.