|M.Sc Student||Massri Rami|
|Subject||Development of new tools for studying TCR signaling, through|
the application of TALENs/CRISPR-based strategies
for precise genome editing in the T cell
|Department||Department of Medicine||Supervisor||Dr. Deborah Yablonski|
|Full Thesis text|
Mutant cell lines are an important tool for studying T-Cell Receptor (TCR) signaling. Site-specific mutations may be induced using genome editing technologies. In this study, we tried to apply and optimize these technologies in the Jurkat cell line in order to elevate the frequency of mutant cell lines production.
Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are the most advanced techniques developed in the last decade for genome editing. These techniques have significantly improved genome editing efficiency in comparison to their previous counterparts; nonetheless, inducing mutations using these techniques is still challenging, and needs to be optimized.
In order to optimize TALENs approach, we attempted to facilitate the screening process. A major challenge is to identify the cells that received two TALEN plasmids, which are both required for TALEN-induced mutagenesis. Our proposed solution was to create a two-component selection system, so that each component sits on a different TALEN plasmid. Co-transfection of both plasmids drives the expression of a surface protein (H-2Kk), which serves as a selectable marker.
We constructed and tested the system in the Jurkat cell line, an established model for studying the TCR signaling pathway. As expected, co-transfection of both plasmids increased H-2Kk expression, however, the system suffered from leakiness, so that one component (encoding for H-2Kk) was sufficient for moderate H-2Kk expression.
We next attempted to apply CRISPR in Jurkat cells. CRISPR-mediated double strand breaks are repaired by error prone Non-Homologous End joining (NHEJ), or precise Homology Directed Repair (HDR) pathways, which can be used for creating knock-out or knock-in mutations, respectively. HDR requires the use of donor template, which could be either double-strand DNA or single strand Oligonuleotide (ssODN).
Using the CRISPR system, we succeed to create a clean LAT-deficient cell line, and proved that this cell line has lost its ability to mediate a T cell response. In addition, we successfully used HDR to knock-in YFP, to create LAT-YFP fusion protein, using double-strand DNA as donor template, and proved that this cell line retaines its functionality in mediating a T cell response. Finally, we created HDR-mediated knock-in using ssODN as donor template, to insert a tetracycteine-tag (12 amino acids sequence) into LAT. The isolation of homozygous tetracycteine- tagged LAT cell line and testing its functionality are still incomplete, but HDR success using ssODN has been proved.
In this study, we proved the feasibility of CRISPR in Jurkat cells, optimized the application of TALENs and CRISPR techniques, and created new Jurkat-derived cell lines (LAT-deficient, LAT-YFP, and LAT-tetracycteine-tagged) for studying the regulation of signal formation at LAT and the binding dynamics and kinetics between LAT and other adaptor proteins involved in the TCR signaling pathway.