The CRISPR gene editing system allows scientists to make edits to DNA – removing damaged or malfunctioning genes and inserting new genetic material into the DNA.
The CRISPR gene editing system can be used to make highly specific edits to double stranded DNA without off-target effects. However, while the system has great potential for the treatment of many genetic diseases, the system does not always work. Approximately 15% of the time the Cas9 enzyme component of the system which is responsible for making cuts to the DNA fails.
To improve the safety and efficiency of the CRISPR gene editing system, scientists need to determine why these failures occur and tweak the system to improve the success rate. Researchers at the University of Illinois have done just that and are the first to describe why the CRISPR gene editing system does not work 100% of the time.
When cuts are made to the DNA by the Cas9 enzyme component of the system, the cells repair mechanisms rejoin the cut DNA. If this fails to happen the cell dies. The University of Illinois researchers determined that the reason why the two strands of DNA are not glued back together is due to the persistent binding of the Cas9 enzyme at the cut site.
With the Cas9 enzyme still in place, the enzymes responsible for rejoining the strands of DNA cannot perform their repair function. Further, that means the Cas9 enzyme cannot then go on to make further DNA cuts, reducing the efficiency of the system.
The researchers also found that translocating RNA polymerase enzymes in cells can knock the Cas9 enzyme off of the DNA, but that this is only possible if they approach the Cas9 enzyme from a particular direction. To improve the efficiency of the CRISPR gene editing system, the guide RNA used to find the correct gene sequence where the cut is made needs to be developed so that it binds to the strand of DNA used by the RNA polymerase enzymes. By developing the guide RNA to ensure that it binds to the correct strand, the team was able to ensure that the RNA polymerase enzymes could easily knock off the Cas9 enzyme.
“I was shocked that simply choosing one DNA strand over the other had such a powerful effect on genome editing,” said Ryan Clarke, lead author of the paper. “Uncovering the mechanism behind this phenomenon helps us better understand how Cas9 interactions with the genome can cause some editing attempts to fail and that, when designing a genome editing experiment, we can use that understanding to our benefit.”
Since the interaction between the Cas9 enzyme and the DNA is the rate limiting step in the gene editing process, being able to control how long the Cas9 enzyme binds to the DNA has the greatest potential to improve the efficiency, safety and effectiveness of the system.
“If we can reduce the time that Cas9 interacts with the DNA strand, which we now know how to do with an RNA polymerase, we can use less of the enzyme and limit exposure,” said Merrill. “This means we have more potential to limit adverse effects or side effects, which is vital for future therapies that may impact human patients.”
The research is detailed in the paper – Enhanced bacterial immunity and mammalian genome editing via RNA-polymerase-mediated dislodging of Cas9 from double-stand DNA breaks – which was recently published in the journal Molecular Cell. DOI: doi.org/10.1016/j.molcel.2018.06.005