NHEJ Introduction And Proof-Of-Concept Experiment 4

Hello! Today, we will continue discussing the proof-of-concept experiments that are part of my theoretical approach. This week, I researched how to assess the efficacy of gene treatment by analyzing levels of homology-directed repair (HDR) and non-homologous end joining (NHEJ). I also finished the introduction section of the paper, added this experiment to the theoretical approach section of it, and started on the discussion section. I separated the proof-of-concept experiments and gRNA findings based on my mentor’s guidance, into two separate sections for clarity.

NHEJ Introduction 

Before we jump into the experiment, I would like to explain NHEJ a little bit. Similar to HDR, NHEJ is also a DNA repair mechanism that is used by cells when DNA is damaged. This mechanism is also the most common and most efficient DNA mechanism used to fix DNA compared to HDR. The reason behind this efficiency is the lack of a DNA template and precise editing, allowing cells to use enzymes to repair the DNA quickly. When NHEJ occurs, the damaged part of the DNA is cut out, and the two blunt ends left behind are ligated together by DNA ligase. Although this may seem simple enough, it actually can be futile for the cells due to their increased potential for cancer development. If the gene cut out through NHEJ is a tumor-suppressor gene, then the cell will lose control of replication leading to uncontrolled cell proliferation and cancer. This is the reason why I am looking into whether NHEJ or HDR occurs in cells as the goal of the gene treatment is to reduce the effects of Lynch syndrome. IF NHEJ occurs in cells because of the gene treatment, cancer could escalate into a higher stage, leading to severe complications.   

Figure 1: NHEJ schematic diagram. Source: https://www.addgene.org/crispr/cut/  

Assessing Efficacy of HDR-Mediated Restoration Function of Gene Treatment 

Analyzing the negative impacts of NHEJ led me to design a proof-of-concept experiment to find out the occurrence levels of HDR and NHEJ. To determine these levels, I plan to use a modified version of PCR called droplet-digital PCR (ddPCR), as described by the Gladstone Institute of Cardiovascular Disease. To come to that conclusion, I had to analyze whether qPCR or ddPCR is the correct approach to solving this problem. The main drawback with qPCR is the way data is presented (as a linear graph). This graph will be used to track the expression of genes in cells. The problem with that is we know the genes will be expressed both before and after the gene treatment is administered in cells. Instead of tracking expression, we are tracking how the expression is taking place within cells in the first place, which led me to use ddPCR.  

As you can see in Figure 2 below, ddPCR separates 1 PCR reaction into 20000 oil droplets. Each oil droplet contains multiple copies of the genomic target. After the PCR reaction is complete, the droplets will be analyzed for HDR and NHEJ probes within each droplet. The color coding will look similar to what is seen in Figure 3 below. The FAM and HEX probes are fluorescent dyes used to identify if HDR or NHEJ is used. The Ref (FAM) probe will be used as a control to determine whether probes can attach to the DNA or not. The wild-type allele with no modification will have the Ref, Dark, and NHEJ (HEX) probes binding to the DNA sequence, referenced by the status (FAM+, HEX+). The HDR allele will have the Ref, HDR (FAM), and NHEJ probes binding to the DNA sequence, denoted by the status (FAM++, HEX+). The NHEJ allele will have only the Ref probe binding to the DNA sequence due to the presence of an indel, which will be represented by the status (FAM+, HEX-).   

Figure 2: ddPCR Schematic Diagram. Source: https://www.mdpi.com/1422-0067/23/9/4802  

Figure 3: ddPCR Color Coding. Source: https://link.springer.com/protocol/10.1007/978-1-4939-7778-9_20 

 In the image above, we can see the assay results with one NHEJ probe. Although NHEJ and HDR are not that predominant in the results graph, I predict that HDR and NHEJ will be more concentrated than what we see on the graph because this system will be applied to HEK293 cells with knock-in exon 2 MLH1 mutations and exon 12 MLH2 mutations in vivo. This means that there is no chance for a wild-type sequence to begin with. The system will also be applied to the genome DNA collected from the colonic epithelia of HNPCC mouse models, which will be another proof-of-concept experiment for my next blog.  

Thank you so much for reading and stay tuned for more information!