Modified design of the CRISPR Gene Editing may Improve Therapy for HIV and Sickle Cell Disease

City of Hope. Courtesy 2016 City of Hope, Duarte, CA.
City of Hope. Courtesy 2016 City of Hope, Duarte, CA.

City of Hope’s cell experiments show more effective genetic ‘cuts’ that could one day become the foundation of new and more effective gene therapies

Researchers at City of Hope, an independent biomedical research and treatment center focusing on cancer, may have found a way to sharpen the fastest, cheapest and most accurate gene editing technique, CRISPR/Cas9. Based on results from a study supported by the National Institute of Allergy and Infectious Diseases and the National Institute of Mental Health, the researchers believe that their approach can more successfully cut out undesirable genetic information.

Editing DNA
CRISPRs or Clustered Regularly Interspaced Short Palindromic Repeats are specialized regions of DNA with two distinct characteristics. The include the presence of nucleotide repeats, the building blocks of DNA, and spacers. The spacers are bits of DNA interspersed among the repeated sequences throughout CRISPR region.

Cas9 is an enzyme that acts like a pair of molecular scissors, capable of cutting strands of DNA.

CRISPR/Cas9, in essence, is simple, but powerful technique that allows scientists to easily make precision edits to DNA sequences, whether bacterial or human, and modify gene function. The technique has many applications include correcting genetic defects to treat and prevent diseases and even improving crops.

No, for the first time, scientist at City of Hope have improved cutting ability of CRISPR/Cas9 which could, one day ,fast-track potential therapies for HIV, sickle cell disease and, potentially, other immune conditions.

Butter knife vs. steak knife
“Our CRISPR/Cas9 design may be the difference between trying to cut a ribeye steak with a butter knife versus slicing it with a steak knife,” noted Tristan Scott, Ph.D., lead author of the study and a staff research scientist at City of Hope’s Center for Gene Therapy. He is also a staff research scientist at the University of Witwatersrand in South Africa).

“Other scientists have tried to improve CRISPR cutting through chemical modifications, but that’s an expensive process and is like diamond-coating a blade. Instead, we have designed a better pair of scissors you can buy at any convenience store,” Scott added.

The study, published in Scientific Reports on November 6, 2019 is the first time scientists have systematically gone through the guide RNA sequence to change it and improve CRISPR/Cas9 technology, Scott explained.

Patent
The Kevin Morris Lab at City of Hope has filed a patent application claiming this improved CRISPR/Cas9 design, which could result in a doubling of activity but the exact amount was dependent on the target site, Scott said.

This study is just one of the many ways City of Hope is helping to enable precision medicine. Its researchers are building foundational blocks that equip gene therapies with tools that work better.

“Knocked Out”
The downstream effects could be more “clean” results in cell and mouse model experiments aimed at developing new therapies because the target that was “knocked out” was more successfully removed.

More pronounced results could quicken new therapies from the laboratory to patients’ bedsides. In theory, the therapeutic product should have more successful cuts, which could translate into an improved therapy, but further research is needed. The exact mechanics of why this change to the CRISPR system improves gene editing still needs to be determined.

The researchers experimented on cells by making changes to the “trans-activating CRISPR RNA” (also known as “tracrRNA”), which is derived from Streptococcus pyogenes bacteria and is a part of the components used to guide the genetic scissors (Cas9) to the right gene sequence.

Streptococcus pyogenes Cas9 is the mostly widely used genetic scissor. The scientists used an RNA protein system because it gives a burst of activity that disappears about 12 hours after being introduced into the cell, which means there’s a decreased chance of accidentally editing the human genome later, after the “fix” has been made, Scott explained.

Modified tracrRNA
The researchers found that the modified tracrRNA improved the silencing of certain genes by increasing desirable mutations in the genetic material. In this study, the target was an essential component of HIV’s lifecycle, the protein CCR5 on immune CD4+ T-cells — a current target in clinical trials seeking to re-engineer a person’s immune system to be resistant to HIV. The modified tracrRNA improved cutting at this site and inactivation of CCR5, and hopefully that will translate into better protection for the immune system.

The new design was also better at improving activity at the HBB gene and the BCL11A site, both of which are tied to sickle cell disease and are being targeted in order to develop therapies for the currently incurable blood disease that causes intense pain and premature death.

“If this line of research remains consistent and we can dependably sharpen the genetic scissor, the result could eventually be new or improved genetic therapies,” Scott said, adding that his team is at the beginning of this long scientific process.

Reference
[1] Scott T, Urak R, Soemardy C, Morris KV. Improved Cas9 activity by specific modifications of the tracrRNA. Sci Rep 9, 16104 (2019) doi:10.1038/s41598-019-52616-5