A Salk Institute team has created a form of CRISPR/Cas9 that can activate targeted genes without making breaks in the DNA. Eliminating the need to physically cut the DNA can cut down on the harmful side effects that might come with CRISPR.
Most CRISPR/Cas9 systems correct faulty genes by cutting two DNA strands and either deleting mutated genes or inserting healthy ones. But breaking the DNA makes it vulnerable to unintended, off-target mutations, which can lead to unwanted side effects.
Previous attempts to attach a "dead" form of Cas9 to molecules that activate targeted genes have seen little success. While these combinations can theoretically target specific genes without cutting them, they are too large to fit into the usual Cas9 delivery vehicle, an adeno-associated virus.
Led by Juan Carlos Izpisua Belmonte, the Salk scientists tested Cas9 or dead Cas9 with different molecular switches to find a combination that worked without being fused to one another. (For more, see video below, courtesy of Salk.)
The result was a two-part treatment—the enzyme was packaged into one virus, while the molecular switches and guide RNA were put into another virus.
"The components all work together in the organism to influence endogenous genes," said co-first author Hsin-Kai (Ken) Liao, a researcher in Izpisua Belmonte's lab.
The researchers engineered their system to target and activate endogenous genes that could potentially treat symptoms of acute kidney injury, Type 1 diabetes and a form of muscular dystrophy in mouse models. In the case of diabetes, for example, they designed the system to boost the expression of genes that could create insulin-producing cells. The treatment reduced blood glucose levels in a mouse model of diabetes.
"We were very excited when we saw the results in mice," said co-first author Fumiyuki Hatanaka. "We can induce gene activation and at the same time see physiological changes."
Scientists are addressing the question of safer CRISPR from a number of angles. Some, like a team from the UC Berkeley and UC San Francisco, focus a "kill switch," or a molecule that limits Cas9 activity once the desired editing is complete. Another Berkeley team is testing a nonviral delivery vehicle for CRISPR—gold nanoparticles—in the hope that this will tamp down on off-target effects.
Broad Institute scientists are focusing on fixing DNA mutations without "tampering with the genome." They devised a form of CRISPR that edits RNA to elicit reversible changes in DNA.
The Salk scientists plan to perfect their CRISPR system to improve its specificity and to try to apply it to a wider range of diseases.