Scientists have engineered a new, more accurate protein for CRISPR-Cas9 gene editing called HypaCas9, which cuts at target sites with higher accuracy and reduces the chances of unintentional externalities.
The discovery, led by researchers from UC Berkeley, Massachusetts General Hospital and Harvard Medical School, may lead to enhanced research capabilities and improved clinical applications for gene therapy in humans.
CRISPR stands for “clustered regularly interspaced short palindromic repeats” and is a family of DNA sequences that are part of the bacterial defense system. Jennifer Doudna, a UC Berkeley professor of molecular biology and chemistry, co-invented CRISPR-Cas9, the gene-editing tool built upon CRISPR.
Doudna’s lab on campus specializes in the exploration of molecular mechanisms of RNA-mediated gene regulation.
“Cas9 is an RNA-guided nuclease that can be programmed to bind and cut any matching DNA sequence, thereby enabling mechanisms for genome editing,” said Janice Chen, a UC Berkeley graduate student in Doudna’s lab and co-first author of the paper, in an email.
The researchers published a paper in the journal Nature, detailing their discovery of how REC3, a region in the Cas9 protein, plays a role in recognizing “on-target sites” in the DNA to activate Cas9 cutting. This discovery led to the creation of HypaCas9.
“By altering this region within Cas9, we were able to tune the balance between on-target activity and improved specificity such that the engineered protein can avoid cutting a large number of off-targets yet remain sufficiently active at the intended target site,” Chen said.
The ability to selectively remove and insert genes also opens the possibility of going into a patient’s DNA and correcting mutations that might cause a genetic disease.
Acquired and hereditary diseases that could be cured by a perfected gene-therapy treatment might include cystic fibrosis, cardiovascular diseases, severe combined immunodeficiency (SCID), AIDS and potentially even cancer.
The concern with gene therapy in humans, however, is that accidental cuts in the DNA could have unintended negative consequences, according to Benjamin Kleinstiver, instructor of pathology at Harvard Medical School and co-first author of the paper.
The creation of HypaCas9 is a step towards reducing errors in gene-editing technologies.
“Targeting accuracy (being able to distinguish between on- and off-target sites) is of paramount importance for clinical translation of genome editing tools,” Kleinstiver said in an email.
The creation of HypaCas9 is a result of an improved understanding of the nature of Cas9. By conducting experiments and further refining the accuracy of these gene-editing tools, a future where fatal genetic diseases can be fixed by the insertion or removal of a gene may be within reach.
“By developing tools with improved specificity, we can mitigate the risk of cleaving and mutating the genome in unwanted or unintended locations,” Kleinstiver said.