Researchers at the University of Illinois have adapted to new CRISPR gene-editing technology that causes the cell's internal machinery to skip over a small portion of a gene during transcription; giving researchers an opportunity to eliminate a mutated gene sequence which influences how a gene is expressed and regulated. Such gene-editing technology can prove useful someday for treating genetic diseases including Duchenne's muscular dystrophy, Huntington's disease, and some cancers. The new technology is referred to as the CRISPR-SKIP technique which was described in the journal Genome Biology, works to alter a single point in the targeted DNA sequence.
"Given the problems with traditional gene editing by breaking the DNA, we have to find ways of optimizing tools to accomplish gene modification. This is a good one because we can regulate a gene without breaking genomic DNA," says Illinois bioengineering professor Pablo Perez-Pinera.
The CRISPR-SKIP specifically alters a single base before the beginning of an exon forcing the cell to read the specific sequence as non-coding.
"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," says bioengineering graduate student and first author of the paper, Michael Gapinske.
Although there are other approaches to skipping exons or eliminating amino acids, they don't work to permanently alter the DNA and only allow temporary benefit with 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," explains Alan Luu, a physics graduate student and co-first author of the study. "The process is also reversible if we would need to turn an exon back on."
Researchers tested the CRISPR-SKIP technology on multiple cell lines from mice and humans either diseased or healthy. "We tested it in three different mammalian cell lines to demonstrate that it can be applied to different types of cells. We also demonstrated it in cancer cell lines because we wanted to show that we could target oncogenes," said Jun Song, a physics professor. "We haven't used it in vivo; that will be the next step."
Investigators are hopeful in testing the technology in live animals for assessing its therapeutic potential and efficacy. "In Duchenne's muscular dystrophy, for example, just correcting 5 to 10 percent of the cells is enough to achieve a therapeutic benefit. With CRISPR-SKIP, we have seen modification rates of more than 20 to 30 percent in many of the cell lines we have studied," Perez-Pinera said.
Additionally, researchers developed a web tool that allows other researchers to investigate whether an exon could be targeted using the CRISPR-SKIP technique while reducing any chances of it binding to similar regions in the genome. "Biology is complex. The human genome is more than three billion bases. So the chance of landing at a location that's similar to the intended region is not negligible and is something to be aware of with any gene editing technique," explains Song. "The reason we spent so much time sequencing extensively to look for off-target mutations is that it could be a major barrier to medical applications. We hope that future improvements to gene editing technologies will increase the specificity of CRISPR-SKIP so we can begin to address some of the problems that have kept gene therapy from being widely applied in the clinic."
