The findings were reported in the study, “Improved Cas9 activity by specific modifications of the tracrRNA,” published in Scientific Reports.
CRISPR/Cas9 is a gene-editing tool that can be used to inactivate certain genes by removing a portion of their DNA sequence.
It is based on a “cut-and-paste” mechanism, in which the enzyme Cas9 is guided toward the DNA sequence that needs to be removed to inactivate a certain gene with the help of a small guide RNA (sgRNA) that specifically recognizes that DNA sequence.
Scientists made several attempts over the years to improve the technology’s cutting ability and efficiency. However, those attempts were based on costly procedures, such as chemical modifications, that were not straightforward.
In this study, researchers from the City of Hope described a new method that allowed them to improve CRISPR/Cas9 cutting ability in a much easier and cost-effective way. Their idea was to modify certain parts of the sgRNA used by Cas9 to recognize the DNA sequence of the gene that needs to be removed to optimize the tool’s cutting ability and overall efficiency.
“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,” Tristan Scott, PhD, said in a press release. Scott is lead author of the study and a staff research scientist at City of Hope’s Center for Gene Therapy,
“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 said.
In their experiments, they modified one of the components of the sgRNA called the “trans-activating CRISPR RNA” (also known as “tracrRNA”) and then tested the efficiency of the CRISPR/Cas9 gene-editing tool in different cell types.
They found that the new modified tracrRNA improved the ability of Cas9 to cut and inactivate the CCR5 gene in immune T-cells. CCR5 is being investigated as a therapeutic target to increase people’s resistance to HIV in clinical trials.
In addition, they found this modified version of CRISPR/Cas9 was more efficient than the standard technology at restoring the normal activity of HBB and BCL11A — two genes that have been tied to SCD and are being studied as therapeutic targets for the disorder — by inactivating a portion of their DNA sequence that normally shuts down the production of fetal hemoglobin.
“Overall, the data presented here suggests that novel facile tracrRNA sequence changes could potentially be integrated with current (…) technology, and open up the possibility for the development of sequence modified tracrRNAs to improve Cas9 (…) activity,” the investigators said.
The team also has filed a patent for this modified CRISPR/Cas9 technology, which, they claim, may increase the activity of Cas9 up to two-times, depending on which specific DNA sequence is being targeted. They noted that more research will be needed to explore the full potential of this new technology.
“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.