Using new gene-editing tools, researchers have developed a way to both eliminate a mutated gene sequence and influence how the gene is expressed and regulated.
A team of researchers from the University of Illinois at Urbana-Champaign has adapted a new CRISPR technique to cause a cell’s internal machinery to skip over a small portion of a gene when transcribing it into a template for protein building.
By targeting specific genes, researchers believe they could treat genetic diseases caused by mutations in the genome like Duchenne’s muscular dystrophy, Huntington’s disease and some forms of cancer.
CRISPR technologies usually turn genes off by breaking their DNA at the start of a targeted gene to induce mutations when the DNA binds back together. However, this can often cause DNA to break in places outside of the intended target that reattaches to different chromosomes.
The new technique—dubbed CRISPR-SKIP—alters a single point in the targeted DNA sequence rather than breaking entire DNA strands.
“Given the problems with traditional gene editing by breaking the DNA, we have to find ways of optimizing tools to accomplish gene modification,” Illinois bioengineering professor Pablo Perez-Pinera said in a statement. “This is a good one because we can regulate a gene without breaking genomic DNA.”
In mammal cells, genes are broken up into segments called exons that are interspersed with regions of DNA that do not appear to code for anything. After the cell’s machinery transcribes a gene into RNA to be translated into a protein, there are signals in the DNA sequence that indicates which portions are exons and which are not part of the gene.
The cell splices together the RNA transcribed from the coding portions to get one continuous RNA template that is used to make proteins.
The new CRISPR system alters just a single base before the beginning of an exon to cause the cell to read it as a non-coding portion.
“When the cell treats the exon as non-coding DNA, that exon is not included in mature RNA, effectively removing the corresponding amino acids from the protein,” Michael Gapinske, a bioengineering graduate student and first author of the paper, said in a statement.
The researchers found that by skipping exons in proteins that are missing a few amino acids, the truncated proteins would retain partial or full activity—which could be enough to restore function in some genetic diseases.
Other approaches to skip exons or eliminate amino acids only provide a temporary benefit because they do not permanently alter DNA and often require repeated administrations over the lifetime of the patient.
“By editing a single base in genomic DNA using CRISPR-SKIP, we can eliminate exons permanently and, therefore, achieve a long-lasting correction of the disease with a single treatment,” Alan Luu, a physics graduate student and co-first author of the study, said in a statement. “The process is also reversible if we would need to turn an exon back on.”
The researchers tested their new method in multiple cell lines of healthy and cancerous mice and humans.
“We tested it in three different mammalian cell lines to demonstrate that it can be applied to different types of cells,” Illinois physics professor Jun Song said in a statement. “We also demonstrated it in cancer cell lines because we wanted to show that we could target oncogenes. We haven’t used it in vivo; that will be the next step.”
They found that CRISPR-SKIP could target specific bases, skip exons with high efficiency and skip multiple exons in one gene if necessary.
“In Duchenne’s muscular dystrophy, for example, just correcting 5 to 10 percent of the cells is enough to achieve a therapeutic benefit,” Perez-Pinera said. “With CRISPR-SKIP, we have seen modification rates of more than 20 to 30 percent in many of the cell lines we have studied.”
The study was published in Genome Biology.