August, 2020 – In new research from Wake Forest Institute for Regenerative Medicine (WFIRM), scientists working with gene editing technology have gained further understanding of CRISPR/Cas9-mediated gene editing by following footprints left behind from a previously overlooked effect: CRISPR/Cas9-mediated gene conversion.
CRISPR (clustered regularly interspaced short palindromic repeats) technology is used to alter DNA sequences and modify gene function. CRISPR/Cas9 is an enzyme that is used like a pair of scissors to cut two strands of DNA at a specific location to add, remove or repair bits of DNA. But CRISPR/Cas9 is not 100 percent accurate and could potentially cut unexpected locations, causing unwanted results.
Published in the journal of Gene Therapy,the latest study from WFIRM demonstrates that using CRISPR/Cas9 to target hemoglobin subunit beta (HBB) gene actually caused the inflow of hemoglobin subunit delta (HBD) footprints. The two genes are highly homologous -- both encode proteins carrying oxygen to the blood. The study further showed that using CRISPR/Cas9 to target HBD similarly caused the inflow of HBB footprints into HBD, hence the gene conversion.
"The study suggests that we have to pay attention to CRISPR/Cas9-mediated gene conversion between homologous genes when performing gene editing research," said senior study author Baisong Lu, PhD, an assistant professor of regenerative medicine at WFIRM.
To explain, Lu points to sickle cell disease, the most common inherited blood disorder affecting as many as 100,000 people in the United States. To correct the gene, CRISPR/Cas9 is used to cut the DNA strands in a specific location in research models. When that happens, the cells have two mechanisms to repair the damage. The two cut ends can come together and seal the damage; however, this is not always accurate and can leave the genetic mutation in place.
Or, the cells look for a template to repair themselves. WFIRM scientists found that in addition to using the provided exogenous template to repair the DNA damage, the cells may also use the endogenous homologous sequences, e.g., sequence from HBD, to repair the DNA damage. This explains how CRISPR/Cas9 causes gene conversion. Fortunately, the study found that in the conditions used, the exogenous template was preferably used as the template for repair by the cells.
Gaetan Burgio, PHD, a researcher at The Australian National University who is not involved in the study, pointed out in a comment paper published in the same journal, that by knowing the possibility of CRISPR/Cas9-mediated gene conversion, scientists can now better design gene editing studies to increase the accuracy of correcting gene mutations.
Anthony Atala, MD, WFIRM director and a co-author of the study, added, “This research is part of an ongoing effort to improve in vivo gene editing efficiency which will be useful in research and clinical applications by improving safety and avoiding possible immune responses. Prior to this study, this type of gene conversion had been overlooked. This understanding helps us as we work to design gene editing therapies that are most effective."
Co-authors include Parisa Javidi-Parsijani, Pin Lyu, Vishruti Makani, Walaa Mohamed Sarhan, Kyung Whan Yoo, and Lobna El-Korashi, all of WFIRM. The study is supported by North Carolina state grant 330054.