Folie 1
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Folie 1
Student Members: M. Agne, I. Blank, A. Emhardt, C. Gäbelein, F. Gawlas, N. Gillich, P. Gonschorek, T. Juretschke, S. Krämer, N. Louis, A. Müller, A. Rudorf, L. Schäfer, M. Scheidmann, L. Schmunk, P. Schwenk, M. Stammnitz, P. Warmer Contact: [email protected] Supervisors: A. Fischer, B. Kaufmann, H. Wagner, J. Kaiser, G. Radziwill, W. Weber Our team developed a universal toolkit, termed uniCAS, that enables customizable gene regulation in mammalian cells. Therefore, we engineered the recently understood and highly promising Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system. The CRISPR-associated catalytically inactive dCas9 is the heart of our toolkit: A protein that enables multiple and sequence - We fused dCas9 to the transactivation domain of VP16. This fusion protein was successfully used to activate gene expression of the secreted embryonic alkaline phosphatase (SEAP) reporter in mammalian cells in presence of appropriate crRNAs. We achieved over 25fold upregulation of SEAP expression (see Figure 1 C). A specific DNA binding. The key components for binding dCas9 to its targets are two small, non-coding RNAs: The CRISPR-RNA (crRNA) and the transactivating crRNA (tracrRNA). The RNA complex guides dCas9 to the DNA sequences of interest. By fusing several effector domains to dCas9, we constructed novel engineered proteins for gene regulation, even controllable by light stimulus. Specific chromatin modification was achieved by fusing the histone methyltransferase G9a to dCas9 and thereby resulting in an epigenetic BioBrick. G9a primarily methylates histone 3. Different endogenous loci in the vascular endothelial growth factor (VEGF) gene were targeted in mammalian cells. This resulted in an up to 50% repression (*, p<0.05) (see Figure 2 C). A C C Target sites -337 The greatest advantage of the CRISPR/Cas9 system is that only one protein is required for targeting various DNA sequences. The only component which needs to be replaced is the CRISPR-RNA (crRNA). We therefore designed an RNA plasmid termed the RNAimer. It provides the backbone for easy insertion of the crRNA sequences. -215 B Figure 7: Plasmid design of the RNAimer. Transactivating crRNA is transcribed by H1 promoter and crRNA by U6 promoter. The crRNA can be exchanged by cutting the plasmid with Bbs1. Me B VEGF SEAP Figure 1. Activation of SEAP in mammalian cells using the fusion protein dCas9-VP16. (A) Plasmid design for activating gene expression. (B) Schematic representation of dCas9-VP16 activation system. (C) Plasmids were cotransfected into HEK-293T cells and expression of SEAP reporter was measured after 24 h. The numbers characterizing the crRNAs represent the distance from the translation start site. Figure 2. Repression of the endogenous VEGF gene via histone modification by dCas9-G9a. (A) Plasmid design for histone modification. (B) Schematic representation of dCas9-G9a repression system. (C) Plasmids shown in A were cotransfected into HEK-293T cells and the amount of VEGF expression was measured. VEGF-8 corresponds to a region in the VEGF locus at position -8 bp from the transcription start site (TSS) and VEGF-573 corresponds to a region -573 bp from the TSS. The transcriptional repressor domain Krüppel-associated box (KRAB) was fused to dCas9 resulting in a fusion protein with the ability to repress gene expression. The device was tested in mammalian cells by targeting different sites in the endogenous VEGF locus. Up to 50% repression was achieved (Figure 3). A For multiple targeting, different crRNAs can be combined into one RNAimer plasmid using the BioBrick standard assembly method. Gene regulation in mammalian cells worked even more efficiently when using multiple targets (see Figure 1 C). We aimed to control our system with light thereby allowing for gene regulation with high spatiotemporal resolution. We engineered a system for induction by red, UVB and blue light. The blue light system is based on the light-triggered interaction of CRY2 and CIB1. CRY2 was fused to dCas9 which can upon light-stimulus recruit the CIB1-VP16 fusion protein to any DNA sequence of interest. D B VEGF C GFP A GFP dCas9-KRAB Target sites Figure 3: Repression of GFP & the endogenous VEGF gene expression via dCas9-KRAB. (A) Plasmid design for repression. (B) Schematic representation of the dCas9-KRAB repression system. (C) Repression of GFP in HeLa cells via dCas9-KRAB analyzed by fluorescence microscopy. (D) HEK-293T cells were cotransfected with plasmids shown in A and the amount of VEGF expression was measured. The numbers characterizing the crRNAs represent the distance from the transcription start site. We used a thermodynamic approach to model our system for dCas9-VP16 gene activation. Figure 4: Behavior of the different components. By minimizing the least square error between the time resolved dCas9 and SEAP data (purple) and the model prediction (cyan) the best parameters for our model were found. Using these parameters a potential behavior for measured and unmeasured (tracr/crRNA & gene recognition complex) components can be predicted. Expression of our fusion proteins seems to represent the bottleneck part of the system. -636 -411 -337 -215 B We developed a novel and innovative ELISA-based method to quantify the binding efficiencies of our dCas9 fusion proteins: The uniCAS Binding Assay (uniBAss). For this purpose, biotinylated oligos were coated on 96-well ELISA plates via the interaction with streptavidin. A B SEAP C Figure 5. The uniCAS Binding Assay (uniBAss). (A) Schematic overview of uniBAss. (B) Binding affinity of dCas9 fusion constructs. HEK-293T cells were cotransfected with the indicated dCas9 constructs and a crRNA plasmid. Samples were normalized to dCas9 expression levels that were quantified by Western Blot. Background level of wells without coated oligos was substracted of depicted values. Gene regulation was achieved based on a modified CRISPR/Cas9 system. We engineered a toolkit which offers efficient and customizable gene activation and repression via effector domains fused to the dCas9 protein. Combining uniCAS with multiple targeting, light induction and the uniBAss we generated a universally 1 Figure 6. Light controlled activation of SEAP reporter via dCas9-CRY2 and CIB1-VP16. (A) Plasmid design for light control. (B) Schematic representation of the dCas9-CRY2 & CIB1-VP16 light system. (C) Transfection of plasmids (Figure 6 A) in NIH/3T3 cells and illumination with 460 nm. A 5-fold upregulation of SEAP reporter was achieved (*, p<0.05). dCas9-UVR8 was targeted to four DNA regions upstream of the promoter. applicable toolkit which allows regulation of any gene of interest. Our toolbox of standardized parts of the CRISPR/Cas9 system offers broad application in research fields such as tissue engineering, stem cell reprogramming and fundamental research.