CRISPR – Cas

Overview of CRISPR

CRISPR is a technology that enables researchers to edit parts of a genome. It works via the enzyme Cas9 and a guide RNA strand. Cas9 acts as a pair of ‘molecular scissors’ that can cut the two strands of DNA at a specific location. Guided RNA is designed to find and bind to a specific sequence to ensure that the Cas9 enzyme cuts at the right point in the genome.

HPV: Why Is It Important?

HPV is a very prevalent disease in the US with more than 42 million Americans infected with some type of HPV.  If not treated these cells can, over time, become cancer cells. According to the Minnesota Department of Health: More than 90 percent of sexually active men and 80 percent of sexually active women will be infected with HPV in their lifetime. Around 50 percent of these infections involve certain high-risk types of HPV, which can cause cancer cells to divide uncontrollably and is the second-leading cause of death in the world. Human papillomavirus (HPV) is the most common sexually transmitted infection (STI). 

Within HPV are the genes, E6 and E7, which are genes that have the potential to transform into tumor cells. E6 degrades a cancer suppressor protein known as P53 that controls the cell cycle. Under normal conditions, if a cell were to fail the checkpoint in the cell cycle, it would terminate itself. However, the E6 gene represses this function and allows cancerous cells to pass the checkpoints  Similarly the E7 gene deactivates another defense mechanism called the retinoblastoma protein, which is a tumor suppressor.

Previous research has involved repression of the HPV E2 protein via ectopic expression. Scientists have also tested the effect of interfering with the mRNA encoding of the genes to stop its production. However, people continue to study HPV, because both of the previous methods still leave the oncogenes intact and allow them to regenerate. Thus, our approach aims to inactivate the viral HPV E6 and E7 genes permanently.

Main Purpose

One of the main purposes of the research article was to demonstrate how the CRISPR/Cas (Clustered Regulatory Interspaced Short Palindromic Repeats) RNA-guided endonuclease can be reprogrammed to target and destroy the E6 and/or E7 genes that are found in the cervical carcinoma cells transformed by HPV (Human Papillomavirus). The elimination of these genes is said to result in cell cycle arrest, followed by cancer cell death. 

Experimental Results

To test if these guides were working correctly, researchers inserted sequences from the E6 and E7 genes into a system where, if cut effectively, it would stop the production of a green fluorescent protein. The results showed a significant reduction in fluorescence, meaning the guides successfully targeted and cut the DNA at the intended spots. The cells with treatment had the lowest green fluorescent protein percentage, indicating cell death within cancerous genes targeted with the signal RNA. Cells receiving the E6 or E7 edits greatly reduced in number over time, suggesting most were dying off. 

There’s the possibility of mutations when using CRISPR. So, researchers took the pieces of DNA they had targeted in their experiments and lots of copies of them so they could reread sequences very precisely. They recorded lots of mutations and In this case, the mutations were good because they disrupted E6 and E7. 

Final Summary

To briefly summarize the study’s findings, repressing E6 or E7 expression in cervical carcinoma cells by activating p53 (tumor-suppressing protein) may induce cell cycle arrest, senescence (stopping of cell multiplication), or apoptosis (programmed cell death), all of which are functions that serve to regulate cell growth and prevent cancers. This can be done by expressing Cas9/sgRNA combinations specific for HPV E6 or E7, consequently leading to the targeted elimination of HPV-transformed cells. To ensure that the activation of p53 and that enhanced Rb expression (retinoblastoma (RB) tumor ) in cancer cells was induced by the Cas9 protein and not a result of off-target effects like DNA damage in mutant forms of the HPV E6 gene that blocked sgRNA cleavage was introduced. p53 activation was still blocked, which strongly suggested that the induction of p53 resulted from losing E6 expression in the presence of Cas9 and E6 sgRNA.

Research Article: 

Kennedy, Edward M, et al. “Inactivation of the Human Papillomavirus E6 or E7 Gene in Cervical Carcinoma Cells by Using a Bacterial CRISPR/CAS RNA-Guided Endonuclease.” Journal of Virology, U.S. National Library of Medicine, Oct. 2014, www.ncbi.nlm.nih.gov/pmc/articles/PMC4178730/.