Nav: Home

Researchers identify drugs that block CRISPR-Cas9 genome editing

May 02, 2019

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

By developing a suite of high-throughput biochemical and cell-based assays, the researchers screened a diverse collection of small molecules to identify compounds that disrupt the binding of SpCas9 to DNA and thereby interfere with its ability to cut DNA. These first small-molecule CRISPR-Cas9 inhibitors readily enter cells and are much smaller than the previously discovered anti-CRISPR proteins. The new compounds allow for reversible and dose-dependent control of SpCas9-based technologies, including its applications for gene editing, base editing, and epigenetic editing in mammalian cells.

"These studies lay the foundation for the rapid identification and use of small-molecule inhibitors against both SpCas9 and next-generation CRISPR-associated nucleases," says senior author Amit Choudhary of the Broad Institute, Harvard Medical School, and Brigham and Women's Hospital. "Small-molecule inhibitors targeting CRISPR-associated nucleases have the potential for widespread use in basic, biomedical, and defense research, as well as in biotechnological applications."

Currently, SpCas9 is being developed as a gene therapy agent for multiple conditions, including HIV, vision disorders, muscular dystrophy, and other hereditary disorders. But these therapeutic applications would greatly benefit from precise control over the dose and timing of SpCas9 activity to reduce off-target effects. Controlling these aspects of SpCas9 activity could also benefit other applications, such as efficiently editing the DNA of model organisms to model and study disease, and the use of gene drives in genetically engineered mosquitoes engineering mosquitoes to curb the spread of malaria and other mosquito-borne diseases.

The need for dose and temporal control of SpCas9 has created a demand for anti-CRISPR molecules. Although anti-CRISPR proteins that target SpCas9 exist, they are large and impermeable to cells, irreversible in action, can be chewed up by proteases, and may pose the risk of adverse immune reactions in the body. By contrast, small-molecule inhibitors are proteolytically stable, reversible, and generally non-immunogenic and can easily be delivered to cells through passive diffusion. In addition, they can be synthesized on a large scale at low cost with little batch-to-batch variability.

In the new study, Choudhary and his team introduced a robust, sensitive, and scalable platform for the rapid and cost-efficient identification and validation of small-molecule inhibitors of SpCas9. Measuring CRISPR-Cas9 activity in a high-throughput way that would allow for drug screening has been challenging due to the properties of the SpCas9 enzyme. In the new paper, Choudhary and colleagues developed high-throughput primary and secondary assays for SpCas9-DNA binding and SpCas9 DNA-cutting activity, respectively. For the primary assay, 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 assay, they used automated microscopy to measure fluorescence changes induced by SpCas9-mediated DNA cleavage of a reporter gene in cells.

Using these assays, the researchers first screened representative members of multiple classes of small molecules to identify the class whose members frequently inhibited SpCas9. The team identified two lead compounds that disrupt the ability of SpCas9 to bind DNA and inhibit SpCas9-mediated DNA cleavage in a dose-dependent manner in mammalian cells. Since they block DNA binding by the enzyme, these molecules also inhibit catalytically-impaired technologies of SpCas9, including those for transcriptional activation, and are stable in human plasma.

"These results lay the foundation for precise chemical control over CRISPR-Cas9 activities, enabling the safe use of such technologies," Choudhary says. "However, these molecules are not ready for applications in humans and not tested for efficacy in organisms."

In future studies, the researchers plan to identify the inhibitors' binding sites on the SpCas9:gRNA complex, examine their mechanism of action, and optimize their potency. They will also determine whether the molecules interact with other targets in mammalian cells, and assess their specificity toward other CRISPR-associated nucleases. Early results included in the Cell paper indicate that the molecules are quite specific for their target, as they have no effect on a distantly-related CRISPR enzyme, Cas12a.
Funding from DARPA's Safe Genes Program is acknowledged. The Broad Institute has filed a patent application including work described herein. Two co-authors have relationships with companies that use genome editing.

Cell, Maji et al.: "A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9"

Cell (@CellCellPress), the flagship journal of Cell Press, is a bimonthly journal that publishes findings of unusual significance in any area of experimental biology, including but not limited to cell biology, molecular biology, neuroscience, immunology, virology and microbiology, cancer, human genetics, systems biology, signaling, and disease mechanisms and therapeutics. Visit: To receive Cell Press media alerts, contact

Cell Press

Related Dna Articles:

Zigzag DNA
How the cell organizes DNA into tightly packed chromosomes. Nature publication by Delft University of Technology and EMBL Heidelberg.
Scientists now know what DNA's chaperone looks like
Researchers have discovered the structure of the FACT protein -- a mysterious protein central to the functioning of DNA.
DNA is like everything else: it's not what you have, but how you use it
A new paradigm for reading out genetic information in DNA is described by Dr.
A new spin on DNA
For decades, researchers have chased ways to study biological machines.
From face to DNA: New method aims to improve match between DNA sample and face database
Predicting what someone's face looks like based on a DNA sample remains a hard nut to crack for science.
Self-healing DNA nanostructures
DNA assembled into nanostructures such as tubes and origami-inspired shapes could someday find applications ranging from DNA computers to nanomedicine.
DNA design that anyone can do
Researchers at MIT and Arizona State University have designed a computer program that allows users to translate any free-form drawing into a two-dimensional, nanoscale structure made of DNA.
DNA find
A Queensland University of Technology-led collaboration with University of Adelaide reveals that Australia's pint-sized banded hare-wallaby is the closest living relative of the giant short-faced kangaroos which roamed the continent for millions of years, but died out about 40,000 years ago.
DNA structure impacts rate and accuracy of DNA synthesis
DNA sequences with the potential to form unusual conformations, which are frequently associated with cancer and neurological diseases, can in fact slow down or speed up the DNA synthesis process and cause more or fewer sequencing errors.
Changes in mitochondrial DNA control how nuclear DNA mutations are expressed in cardiomyopathy
Differences in the DNA within the mitochondria, the energy-producing structures within cells, can determine the severity and progression of heart disease caused by a nuclear DNA mutation.
More DNA News and DNA Current Events

Trending Science News

Current Coronavirus (COVID-19) News

Top Science Podcasts

We have hand picked the top science podcasts of 2020.
Now Playing: TED Radio Hour

Climate Mindset
In the past few months, human beings have come together to fight a global threat. This hour, TED speakers explore how our response can be the catalyst to fight another global crisis: climate change. Guests include political strategist Tom Rivett-Carnac, diplomat Christiana Figueres, climate justice activist Xiye Bastida, and writer, illustrator, and artist Oliver Jeffers.
Now Playing: Science for the People

#562 Superbug to Bedside
By now we're all good and scared about antibiotic resistance, one of the many things coming to get us all. But there's good news, sort of. News antibiotics are coming out! How do they get tested? What does that kind of a trial look like and how does it happen? Host Bethany Brookeshire talks with Matt McCarthy, author of "Superbugs: The Race to Stop an Epidemic", about the ins and outs of testing a new antibiotic in the hospital.
Now Playing: Radiolab

Speedy Beet
There are few musical moments more well-worn than the first four notes of Beethoven's Fifth Symphony. But in this short, we find out that Beethoven might have made a last-ditch effort to keep his music from ever feeling familiar, to keep pushing his listeners to a kind of psychological limit. Big thanks to our Brooklyn Philharmonic musicians: Deborah Buck and Suzy Perelman on violin, Arash Amini on cello, and Ah Ling Neu on viola. And check out The First Four Notes, Matthew Guerrieri's book on Beethoven's Fifth. Support Radiolab today at