DNA Template Creation – Part 1

Hello! This week, I researched creating a DNA template for homology-directed repair (HDR). I focused my research on the importance of HDR and DNA templates in the context of CRISPR-Cas9n genetic therapy and how I worked to design my template sequences. 

HDR and DNA Templates in the Context of CRISPR-Cas9n 

When cells identify DNA damage in the nucleus, they initiate homology-directed repair mechanisms to fix the DNA damage. Homology-directed repair occurs when double-strand breaks (DSBs) are present. When enzymes identify these breaks, a template sequence complementary to the CRISPR-Cas9n system is copied and inserted into the double-strand break. This is done by the homology arm (200 bp of the native DNA sequence before and after the DNA template [correct form of mutated DNA sequence]) of the template binding to the blunt end of the DSB to rejoin cleaved DNA, and by DNA polymerase working to add base pairs complementary to the template sequence, as seen in the diagram below. Eventually, after replicating the entire template sequence, the homology arms and the desired insert are then incorporated into the DNA of the cell. 

A DNA template consists of 3 main components: 2 homology arms, a backbone, and the desired insert sequence. If the desired insert sequence is between 500-1000 bp, then it is good to have homology arms of up to 100-200 base pairs to ensure template stability. It is also good to have a plasmid backbone to ensure the proper transport of all template elements and incorporate other elements like antibiotic resistance for testing and proof-of-concept experiments.   

Image taken from this link:  https://sites.tufts.edu/crispr/genome-editing/homology-directed-repair/  

Mapping Mutation Clusters 

Before I get to template sequence design, I would like to get into how I determined where the template sequence should be located in the first place. To determine this, I had mentioned mapping mutation clusters onto protein structure diagrams for both MLH1 and MSH2. These mutation clusters can be seen in the following diagram:  

As you can see in the diagram above, more mutation locations (represented by red rectangles) are clustered near the ATPase domain of MLH1 and the MutL homolog interaction domain of MSH2. Using this information I worked to design the template sequences around these protein domains.  

Template Sequence Design 

Based on the mutation locations, I then revisited the UCSC Genome Browser to obtain the proper genetic sequence for MLH1 and MSH2. One mistake I made here was that I had obtained just the exon sequence, rather than the canonical sequence for MLH1 and MSH2. I then had to change the settings to get the correct sequence and proceed with making the template. The ATPase binding domain and MutL homolog interaction domain turned out to be 416 and 413 bp, respectively. Homology arms of 200 bp before and after the target sequence were designed to induce HDR in cancer cells and increase the stability of the template sequence. I also identified a plasmid to incorporate the template into, the psPAX2 plasmid backbone. However, I am still looking into how to incorporate the template into the plasmid backbone, which will be something I’ll be researching in the following week. 

Thank you for reading my blog and stay tuned for more information!