Researchers identify drugs that block CRISPR-Cas9 genome editing

The discovery of the first small molecule inhibitors of Streptococcus pyogenes Cas9 protein (SpCas9) could allow more accurate control of genome editing based on CRISPR-Cas9, researchers report on May 2 in the journal Cell.

By developing a series of high-throughput biochemical and cellular assays, the researchers analyzed a diverse set of small molecules to identify compounds that disrupt SpCas9 binding to DNA and thereby interfere with its ability to cut off DNA. DNA. These first small molecule CRISPR-Cas9 inhibitors penetrate easily into cells and are much smaller than previously discovered anti-CRISPR proteins. The new compounds allow reversible and dose-dependent control of SpCas9-based technologies, including its applications for gene editing, basic editing, and epigenetic editing in mammalian cells .

"These studies lay the groundwork for the rapid identification and use of small molecule inhibitors against the CRISPR-associated nucleases of SpCas9 and the next generation," said Amit Choudhary, an experienced author. Broad Institute, Harvard Medical School and Brigham Hospital and Women's Hospital. "Small molecule inhibitors targeting CRISPR-associated nucleases could be widely used in basic, biomedical and defense research, as well as in biotechnological applications."

SpCas9 is currently under development as a gene therapy agent for many diseases, including HIV, vision disorders, muscular dystrophy and other hereditary disorders. But these therapeutic applications would greatly benefit from accurate control of the dose and timing of SpCas9 activity to reduce non-target effects. The control of these aspects of the activity of SpCas9 could also benefit other applications, such as the efficient editing of DNA model organisms to model and develop. 39, study the disease, and the use of genetic drives in genetically modified mosquitoes to reduce the spread of malaria and other mosquito-borne diseases. diseases.

The need to control the dose and time of SpCas9 has created a demand for anti-CRISPR molecules. Although anti-CRISPR proteins targeting SpCas9 do exist, they are bulky and impervious to cells, their action is irreversible, they can be gnawed by proteases and can lead to a risk of unwanted immune reactions in the body. In contrast, small molecule inhibitors are proteolytically stable, reversible and generally non-immunogenic and can readily be delivered to cells by passive diffusion. In addition, they can be synthesized on a large scale and at low cost, with low variability from one batch to the other.

In the new study, Choudhary and his team presented a robust, sensitive and scalable platform for the rapid and cost-effective identification and validation of small molecule inhibitors of SpCas9. Measuring high throughput CRISPR-Cas9 activities, which would allow drug screening, was difficult because of the properties of the SpCas9 enzyme. In the new article, Choudhary and his colleagues developed high throughput primary and secondary tests for SpCas9-DNA binding and SpCas9 DNA cleavage activity, respectively. For the main test, they used a biochemical technique called fluorescence polarization to monitor the interaction between SpCas9 and a fluorophore-labeled DNA segment containing PAM sequences. In the secondary test, they used automated microscopy to measure the fluorescence changes induced by SpCas9-induced DNA cleavage of a reporter gene in cells.

Using these tests, researchers first examined representative members of several small molecule classes to identify the class whose members frequently inhibited SpCas9. The team identified two major compounds that disrupt the ability of SpCas9 to bind to DNA and inhibit SpCas9-induced DNA cleavage in a dose-dependent manner in mammalian cells. Since they block the binding of DNA by the enzyme, these molecules also inhibit SpCas9 technologies with catalytic alteration, including those allowing activation of transcription, and are stable in human plasma.

"These results lay the groundwork for a precise chemical control over CRISPR-Cas9 activities, thus allowing the safe use of such technologies," Choudhary said. "However, these molecules are not ready for human applications and their effectiveness in organisms has not been tested."

In future studies, the researchers plan to identify inhibitor binding sites on the SpCas9: gARN complex, examine their mechanism of action, and optimize their potency. They will also determine if the molecules interact with other targets in mammalian cells and evaluate their specificity relative to other CRISPR-associated nucleases. The first results included in the Cell The paper indicates that the molecules are quite specific for their target because they have no effect on a remotely connected CRISPR enzyme, Cas12a.