Taylor Brown and Dr. Jonathan Alder, Physiology and Developmental Biology
DNA contains all the information a cell needs to grow, divide, differentiate, and survive. It also dictates how and when a cell should die. DNA damage (particularly double strand breaks i.e. DSBs) may lead to cell death and/or disease. To circumvent DNA breaks, cells use various DNA repair mechanisms including non-homologous end joining (NHEJ). My ORCA research project focused on identifying novel genes involved in NHEJ. I did this by performing a forward genetic screen (FGS) that tested every known gene in the genome for involvement in NHEJ.
To perform my FGS I used iMEF cells that had a conditional allele of Trf2. Trf2 normally protects the telomere ends of each chromosome, but if deleted, the exposed telomere is recognized as a DNA DSB and NHEJ occurs. With this, NHEJ proteins fuse chromosome ends together causing cell senescence and apoptosis. The massive cell death cause by Trf2 deletion provided an excellent way to screen for NHEJ mutations. Theoretically, if NHEJ were disrupted, a cell with dysfunctional Trf2 could still grow and avoid senescence and/or apoptosis.
To trigger telomere fusion via NHEJ I transduced my iMEF cell line with CreER. This form of Cre would delete the conditional allele of Trf2 in the presence of 4HT.
To disrupt potential NHEJ genes I used CRISPR/Cas9 technology. Cas9 deletes DNA sequences when provided with an sgRNA. Using a gecko sgRNA library that provided 6 different guides to each gene in the genome I was able to knock out each gene at least once.
Once I had selected for iMEF cells with CreER, Cas9 and the sgRNA library, the set up for my FGS was complete. I then made two groups, one that deleted Trf2 and another that did not. Once enough cells grew after Trf2 deletion, I then collected and sequence their DNA (via next generation deep sequencing) to find out what mutations disrupted the NHEJ pathway.
DNA sequences from the iMEF control group revealed that each gene was deleted equally. This suggests the gecko library and experimental procedures consistently tested each gene.
Deletion of Trf2 resulted in massive cell death in the experimental group. When the few surviving cells grew out, their DNA was harvested. Hundreds of genes were identified from the sequencing results, but the deletion of three genes was found in most surviving experimental cells. These genes I annotated as C1, K1 and G1. To determine if these genes really were involved in NHEJ, I deleted them from original iMEF cells and then deleted Trf2. Interestingly the deletion of these genes (even in combination) did not rescue the iMEF cells as was expected.
Following these results, Dr. Alder and I hypothesized that more than one gene may need to be deleted for NHEJ disruption. I then performed the same FGS as before, but added enough library sgRNAs so each cell would delete approx. 5 genes each. This time all 40 experimental plates had an average of 6 surviving colonies each. After examining the genome of each colony, we did not find any overlap in the genes deleted and none matched our first FGS results.
Although my FGS did not uncover any novel genes, most likely there are still more NHEJ genes to be discovered. Strong evidence for this is the fact that this FGS did not uncover any known NHEJ genes.
While the experimental set-up may have been sound, perhaps the timing and harvesting of the surviving colonies disrupted the results. In the first FGS, I waited to harvest DNA until the surviving experimental cells took over the plate. Most likely a few mutated iMEF cells resistant to the deletion of Trf2 took over the plate, masking the few cells that actually survived the screen. In the second FGS I tried to correct this by harvesting all the cells once small colonies formed. However, I harvested the DNA of half the plates all at once while I put the rest in 12 well plates for the rest to see if they would grow out. It is likely the deletion of NHEJ genes makes the cell more susceptible to damage and sickness, and the longer the wait before harvest, the less likely it is to find surviving cells.
We did not identify any novel genes nor did we find any genes that have already been characterized. We did, however, demonstrate that upon the deletion of Trf2, NHEJ does occur. Other experiments could potentially be used to identify novel genes involved in NHEJ. One might be to take known NHEJ proteins and perform a pull-down assay. Proteins that are “pulled down” with known NHEJ proteins may be involved in NHEJ. This FGS screen could work, but before performing it again, I would test the set-up by deleting Ku70/80 and/or DNA-PKc. These genes are known for their heavy involvement in NHEJ. If the deletion of these genes cannot save the cells from Trf2 deletion, perhaps there is more to the specific process of telomere fusion that goes beyond the typical realm of DNA NHEJ.
Determining what genes are involved in NHEJ is key to better understanding normal cell function and in unlocking the key to various genetic diseases and abnormalities caused by NHEJ mutations. Opening the door to these mysteries, in tandem with the advances in gene therapy could help improve and save the lives of hundreds of thousands of people currently living with diseases like ataxia telangiectasia, werner syndrome, SCID and others.