CRISPR/Cas9 is a powerful genome editing tool that requires CRISPR RNA (crRNA) with an interchangeable 20 nucleotide complementary sequence to a target DNA site, and a trans-activating crRNA (tracrRNA) scaffold recognized by a catalytically active Cas9 protein. TracrRNA 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. The system uses guide RNA (gRNA) to facilitate target site activation and new gene insertion.

The research team identifies specific modifications to the gRNA that significantly enhance Cas9 ribonuclearprotein complex (RNP) activity. This is the first time that scientists have systematically gone through gRNA sequences to change it and improve the technology.

"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," said Tristan Scott, PhD, 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."

First, they screened U-modified tracrRNAs and identified nucleotide substitutions that improved Cas9 RNP knockdown of HIV reporter cell lines and observed this enhanced targeting of the long terminal repeat of HIV. The modified tracrRNAs improved knockout activity of an essential HIV co-receptor, C-C chemokine receptor type 5 (CCR5). CCR5 is a current target in clinical trials seeking to re-engineer a person's immune system to be resistant to HIV. Furthermore, the modified tracrRNA improved target accuracy and subsequently increased the inactivation of CCR5. Improved targeting and improved gene insertion were also observed 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.

The researchers hope that these results could lead to more "clean" gene editing in cell and mouse model experiments. More pronounced results could quicken new therapies from the laboratory to patients' bedsides. "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.

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