How to switch on genes with CRISPR/Cas9? (2018)

Knockout technologies and the application of siRNA made it possible to turn off genes or to diminish their activity. The opposite application was much harder so far. Directed switching on of endogenous genes has been virtually impossible. One option of overexpression of a gene of interest is to bring the complete genetic information of that gene into the desired target cell. Researchers used viruses or plasmids shuttling the DNA into the cell to accomplish this. For large genes, this is rather a difficult art as the size capacity of the lentiviral systems is limited. New developments in the field of molecular biology have revolutionized genetic engineering recently. A number of new tools that allow directed modification of genetic information are available now. An example is the CRISPR/Cas9 system which is used to modify genetic code in cells or animals.1 Briefly, the basic principle here is that using a so-called target-specific guide RNA that contains a 21-nt-long complementary sequence to the DNA target site, together with a cleaving Cas9 enzyme that binds the guide RNA, is “dragged” to the desired locus in the genome where Cas9 can cleave the DNA. In other words, this system provides a kind of programmable restriction enzyme. Once the DNA is cut, the endogenous repair mechanisms in the cell get active. By providing short pieces of DNA or RNA that consist of homologous sequences at both ends and the desired modification in-between the cell uses those templates for repair. By this procedure, it is possible to modify the genome from single base exchanges up to large deletions or insertion of new large pieces of DNA. In a recent work by Konermann et al2 the authors use a modified CRISPR/Cas9 system to switch on genes. The basic idea behind this is that the cleaving property of the Cas9 is turned off by mutations in the active cleaving sites of the enzyme. In addition to this modification, the altered Cas9 is fused to a transcription activator complex that consists of known transcription factors such as VP64, the NF-κB subunit p65 and the activation domain of HSF1. These elements constitute a programmable transcription factor complex called synergistic activation mediator (SAM) (Figure 1). If one adds a Cas9-binding guide RNA that has an additional specific target sequence, one can “drag” the activating SAM to almost any location in the genome. The authors show that cell lines that express this complex and specific guide RNAs that target promoter regions of a defined gene this SAM complex can activate that defined gene (Figure 1). Why do we care about turning on genes? Switching on genes is useful for a number of purposes, for example for the reprogramming of somatic cells to induced pluripotent stem cells.3 In this application, an activation of about 4 transcription factors is necessary depending on the cell type to bring somatic cells into a pluripotent state. Renin as a key enzyme in long-term blood pressure regulation is in the focus of hypertension research.4 For the study of renin in cell culture, only few cell lines are available. Examples are the human CALU-6 cell line5 (ACC 734) and the mouse AS4.16 (ATCC® CRL2193). However, in both cases, the expression level of renin cannot be controlled. Using the SAM system, we tested the efficiency of controlling the levels of human renin in HEK293 cells that typically do not express renin. We have generated HEK293 cells that stably express the SAM complex. By addition of renin promoter-specific guide RNAs, the cells showed a marked expression of renin mRNA (Figure 2) and renin protein measured in ELISA (data not shown). The level of renin expression was dependent on the concentration of the added guide construct. Other applications of the SAM technology would refer to the study of gene function and gene regulation, for example to test the hypothesis whether a cell function depends on the activity of a specific gene of interest. Further, the SAM system may be used for genomewide screening assays, where a library of guide RNAs covering all the promoter regions of human genes is brought to individual cells that in turn are screened for a specific phenotype. By this approach, it is potentially possible to find upstream regulators of that specific phenotype. This may help to decipher regulatory genetic networks in a systematic manner. This specific SAM technology is another fascinating application derived from the CRISPR/Cas9 technology that will greatly enhance our toolbox for deciphering gene regulation and ultimately to understand better physiological processes. None. Bundesministerium für Bildung und Forschung (BMBF) iPS-Profiler, FKZ: 01EK1612B; Bundesministerium für Bildung und Forschung SysToxChip FKZ: 031A303A. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.