Relevance Of Project And Proof-Of-Concept Experiment 2 And 3
Hello everyone! Today, we will continue the theoretical approach to creating a gene editing therapy for Lynch syndrome. This week I researched how to clone a plasmid containing the IDLV viral vector, gRNA, and SpCas9n, followed by how to produce IDLVs containing the plasmids. I also began the introduction and methods section of my research paper, the end goal of my research. I will also explain the relevance of my project in this blog as well.
Molecular Cloning of an Integrase-Deficient Lentiviral (IDLV) Packaging Plasmid Containing gRNA and SpCas9n
To find out how to conduct this experiment, I conducted a literature review search on the usage of IDLVs with CRISPR-Cas9 treatments. From this review, I found a source with a protocol regarding IDLV production combined with CRISPR-Cas9 elements. The link to this source is the following: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5755554/
This protocol contains the steps required to create high-efficiency IDLVs with CRISPR-Cas9 elements. However, this protocol had to be modified to incorporate the gRNA and convert Cas9 to Cas9 nickase. To do this, annealed oligos were created using the gRNA sequences flanked with BbsI restriction enzyme sequence overhangs. I then plan to incorporate the annealed oligos into the pLentiCRISPR/v2 plasmid, the plasmid that contains both the CRISPR-Cas9 system and the viral elements of the IDLV. To ensure successful incorporation, this plasmid will be digested with BbsI to allow the incorporation of annealed oligos. Finally, the Cas9 catalytic domain (part of the protein that initiates DNA cuts) will be converted into Cas9 nickase by creating a D10A amino acid substitution through thymine to guanine point mutation. This will be done by making use of PCR-based site-directed mutagenesis in that location. BsrGI restriction enzymes will digest the resulting PCR product and create the sticky ends necessary for the Cas9n sequence to be incorporated into the pLentiCRISPR/v2 plasmid.
Figure 1: Map of the pLentiCRISPR/v2 plasmid
IDLV Packaging and Lentiviral Production
To produce IDLVs containing all elements of the gene treatment, the pLentiCRISPR/v2 plasmid and the psPAX2-HDRtemplate plasmid would be transfected into HEK293 (human embryonic kidney 293) cells. These cells were chosen because they have easier maintenance overall, are efficient at producing proteins, and are more accessible for transfection compared to other cell lines. These cells would be transfected with both plasmids once they reach 80% confluency in the dish they are grown in. 80% confluency means that 80% of the space on the bottom of a cell plate is filled with cells and 20% of the space has no cells in it. 8 hours after the transfection takes place, these cells should be replenished with fresh media, and 24 hours later, the supernatant (the liquid above the cells) should be centrifuged and concentrated into pellets for future use.
Relevance of Research Project
The genetic treatment I am creating in this project is for Lynch syndrome, a hereditary disorder impacting multiple organs such as the colon, endometrium, pancreas, etc. There are no cures for Lynch syndrome, and the only standard method of care used for patients is colectomy (removal of parts of the colon or the entire colon in severe cases) and chemotherapy if tumor development takes place. This syndrome is caused by gene mutations in the DNA MMR (mismatch repair) pathway, which can lead to tumorigenic mutations and microsatellite instability. Within Lynch syndrome, one of the most debilitating diseases is hereditary non-polyposis colorectal cancer (HNPCC), which is also the most common form of hereditary colon cancer. There are no genetic treatments available for HNPCC as well.
Adding on to this, according to NPR Health, creating gene therapies for colon cancer is also important because of the increase in cases among individuals under the age of 50. Ever since the mid-1990s, colon cancer cases for individuals above the age of 50 have increased by 50%, making it one of the deadliest cancers in its age group. Also, around 20000 people in the US are projected to be diagnosed with colon cancer in this year alone, and ~3750 people are projected to pass away from it as well. These dismal statistics show the importance of finding new treatments for colon cancer and the necessity to conduct screenings for colon cancer as well.