Wetlab
Introduction
In our work, we have tried to develop a comprehensive approach to studying the activity of cas proteins. At the first stage, we studied the possibility of evaluating the interaction using biochemical approaches. At the second stage, we tried to create a simple assembly procedure for evaluating the interaction of cas proteins inside cells. Since in the future, within the framework of our project, it is planned to assess the impact of various combinations of targets or types of CAS systems to select the most optimal, we needed to develop a simple assembly method based on existing parts.
Results
In the course of our work, we used various plasmids (see Plasmid). To obtain these plasmids, we used E. coli Top 10 cells. After the plasmids were generated, we purified them using a commercial kit (Omega) . The purification results are shown to the image below
After obtaining the necessary plasmids, we transformed BL21 DE3 cells for subsequent purification of dCas proteins. During the work, we successfully purified these proteins in concentrations sufficient for further measurements.
To conduct analyses for the interactions of cas proteins with the DNA sequence, we prepared the DNA matrix and also synthesized sgRNA. The obtained components were successfully assembled and the interaction parameters were evaluated using EMSA analysis. After that, we obtained labeled DNA to measure the interaction using fluorescence polarization. We have successfully demonstrated the possibility of using a flatbed scanner to measure the parameters of the interaction of DNA complexes with Cas proteins.
To study the processes of interaction of cas proteins with DNA inside cells, we have proposed an assembly system, a detection system from existing parts. In the course of our work, we have identified and characterized the use of sequencing the parts that are used in our project. Next, we proposed new 5’ and 3’ UTR parts to be able to assemble parts from various collections. 5’ and 3’ UTR were chosen to pair different elements because they have a small length. Thus, to create new parts, it is enough to order the necessary oligonucleotides and anneal them on top of each other to obtain a double-stranded DNA molecule. Schematically, these parts are shown in the figure.
We have created new parts. To do this, we ordered DNA sequences in the form of oligonucleotides. These nucleotides were collected into double-stranded DNA fragments and used for subsequent cloning. Their sequence is described in detail in BBa_K4911000, BBa_K4911001, BBa_K4911002, BBa_K4911003.
In addition, we have characterized the existing parts BBa_J428064, BBa_J428060, BBa_J428063. The sequence of this part was verified using sequencing. The availability of the necessary sites was checked using restriction analysis (BsaI). The part can be cloned using the RFC1000 standard.
Possible improvements
At this stage, we used simple vectors for the assembly procedure. In the future, we believe that it is necessary to use vectors suitable for viral transfection. In addition, to determine the activity of two types of systems in one cell, we must consider the potential toxicity of Cas proteins to the cell. Such an approach can significantly impact the potential outcome. Our project is aimed at assessing the efficiency of editing systems, however, the existing biosensor variant won't allow for measuring the editing activity of proteins. Comparison is only possible for CRISPi and CRISPa systems. We believe that it is necessary to add the capability to evaluate the editing activity of CRISPR systems into the system.