In a significant advancement, engineers at Duke University have devised a new method that greatly enhances the capability of CRISPR gene editing technology.
While the original CRISPR tool was limited to targeting only 12.5% of the human genome, this innovation broadens access to nearly every gene. This breakthrough holds promise for treating a wider array of diseases.
The findings, published in the journal Nature Communications, were the result of collaborative efforts between Duke University, Harvard University, Massachusetts Institute of Technology, University of Massachusetts Medical School, University of Zurich, and McMaster University.
Assistant Professor of Biomedical Engineering at Duke, Pranam Chatterjee, said,
“With this new tool, we can target nearly 100% of the genome with far more precision.”
CRISPR-Cas, initially discovered as a bacterial immune system, enables bacteria to utilize RNA molecules and CRISPR-associated (Cas) proteins to identify and dismantle the DNA of invading viruses. Since its inception, scientists have been striving to enhance CRISPR systems for applications in gene therapy and genome engineering.
The research team explored the therapeutic potential of this novel tool for genetic diseases previously deemed untreatable with conventional CRISPR methods. Their investigations initially focused on Rett syndrome, a progressive neurological disorder primarily affecting young females, caused by one of eight mutations in a specific gene.
Additionally, they examined Huntington’s disease, a rare inherited neurological disorder resulting in brain neuron degeneration. Using the new technology, the team succeeded in targeting previously inaccessible mutations, thereby opening up potential therapeutic avenues for both conditions.
How Does it Work?
To modify the genome, Cas proteins employ an RNA molecule to guide the enzyme to a targeted DNA sequence, along with a protospacer adjacent motif (PAM), a short DNA sequence immediately following the target DNA sequence, necessary for Cas protein binding.
Once the guide RNA identifies its complementary DNA sequence and the Cas enzyme binds to the adjacent PAM, the enzyme acts as molecular scissors, cleaving the DNA and initiating the desired genome alterations. The most widely used CRISPR-Cas system, Cas9 from Streptococcus pyogenes bacteria (SpCas9), necessitates a PAM sequence of two guanine bases (GG) in succession.
Previously, Chatterjee and colleagues utilized bioinformatics tools to identify and modify new Cas9 proteins, including Sc+ +, which requires only a single guanine base PAM for DNA cleavage. This innovation expanded the scope of DNA sequences amenable to editing by nearly 50%.
Concurrently, collaborators at Harvard, under the leadership of Benjamin Kleinstiver, engineered another variant known as SpRY. Unlike its predecessors, SpRY exhibited a robust affinity for adenine and guanine, although it could bind to any of the four DNA bases comprising the PAM.
Recognizing the limitations of both systems, the researchers combined the most effective attributes of each into a novel variant named SpRyc. While SpRyc demonstrated slower DNA cleavage compared to traditional enzymes, it surpassed them in efficacy and precision. Despite its broader target range, SpRyc exhibited greater accuracy than its predecessor, SpRY.
“There is a lot of potential with SpRYc, whether it’s exploring how to translate it into the clinic or finding ways to make it even more efficient. We look forward to exploring the full capabilities of our tool,” said Chatterjee.
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