New CRISPR technique skips over portions of genes that can cause disease

Liz Ahlberg, Biomedical Sciences Editor, Illinois News Bureau
8/17/2018

Illinois researchers adapted CRISPR gene-editing technology to help a cell skip over mutated portions of genes. From left, professor Pablo Perez-Pinera, graduate student Alan Luu, professor Jun Song and graduate student Michael Gapinske.
Photo by L. Brian Stauffer, University of Illinois at Urbana-Champaign
Illinois researchers adapted CRISPR gene-editing technology to help a cell skip over mutated portions of genes. From left, professor Pablo Perez-Pinera, graduate student Alan Luu, professor Jun Song and graduate student Michael Gapinske. Photo by L. Brian Stauffer, University of Illinois at Urbana-Champaign
In a new study in cells, University of Illinois researchers have adapted CRISPR gene-editing technology to cause the cell’s internal machinery to skip over a small portion of a gene when transcribing it into a template for protein building. This gives researchers a way not only to eliminate a mutated gene sequence, but to influence how the gene is expressed and regulated.

Such targeted editing could one day be useful for treating genetic diseases caused by mutations in the genome, such as Duchenne’s muscular dystrophy, Huntington’s disease or some cancers.

CRISPR technologies typically turn off genes by breaking the DNA at the start of a targeted gene, inducing mutations when the DNA binds back together. This approach can cause problems, such as the DNA breaking in places other than the intended target and the broken DNA reattaching to different chromosomes.

The new CRISPR-SKIP technique, described in the journal Genome Biology, does not break the DNA strands but instead alters 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,” said Illinois bioengineering professor Pablo Perez-Pinera, who led the study with Illinois physics professor Jun Song. Both are affiliated with the Carl R. Woese Institute for Genomic Biology at the U. of I.

In mammal cells, genes are broken up into segments called exons that are interspersed with regions of DNA that don’t appear to code for anything. When the cell’s machinery transcribes a gene into RNA to be translated into a protein, there are signals in the DNA sequence indicating 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.

CRISPR-SKIP alters a single base before the beginning of an exon, causing 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,” said Michael Gapinske, a bioengineering graduate student and first author of the paper.

While skipping exons results in proteins that are missing a few amino acids, the resulting truncated proteins often retain partial or full activity – which may be enough to restore function in some genetic diseases, said Perez-Pinera, who also is a professor in the Carle Illinois College of Medicine.

There are other approaches to skipping exons or eliminating amino acids, but since they don’t permanently alter the DNA, they provide only a temporary benefit and require repeated administrations over the lifetime of the patient, the researchers said.

“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,” said 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.”

The researchers tested the technique in multiple cell lines from mice and humans, both healthy and cancerous.

“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,” Song said. “We haven’t used it in vivo; that will be the next step.”

They sequenced the DNA and RNA from the treated cells and found that the CRISPR-SKIP system could target specific bases and skip exons with high efficiency, and also demonstrated that differently targeted CRISPR-SKIPs can be combined to skip multiple exons in one gene if necessary. The researchers hope to test its efficiency in live animals – the first step toward assessing its therapeutic potential.

“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.

The group built a web tool allowing other researchers to search whether an exon could be targeted with the CRISPR-SKIP technique while minimizing chances of it binding to similar sites in the genome.  

Since the researchers saw some mutations at off-target sites, they are working to make CRISPR-SKIP even more efficient and specific.

“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,” Song said. “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.”

The National Institutes of Health, the National Science Foundation and the American Heart Association supported this work. The conclusions presented are those of the researchers and not necessarily those of the funding agencies.

The paper “CRISPR-SKIP: programmable gene splicing with single base editors” is available online.

Recent News

  • In the Media

There have been accusations for years that the Major League ball is “juiced,” thus accounting for the increasing power numbers.

MLB officials have categorically denied that, and last year, commissioned a study of the baseball and how it’s produced.

In the landmark 85-page independent report replete with color graphs, algorithms and hypotheses, a group of 10 highly-rated professors and scientists chaired by Alan Nathan determined that the ball is not livelier or “juiced.” Nathan is a professor emeritus of physics from the University of Illinois at Urbana Champaign.

The surge in home runs “seems, instead, to have arisen from a decrease in the ball’s drag properties, which cause it to carry further than previously, given the same set of initial conditions – exit velocity, launch and spray angle, and spin. So, there is indirect evidence that the ball has changed, but we don’t yet know how,” wrote Leonard Mlodinow, in the report’s eight-page executive summary.

  • In the Media

Growing up in Trinidad and Tobago, Kandice Tanner went to a school where she was one of only a dozen girls among 1200 pupils. She had switched from an all-girl school to avoid the distractions of socializing and to take the more advanced math classes offered at the boys’ school. “Being submerged in an all-male environment early on was beneficial to me,” Tanner says. “I felt comfortable with guys, and more important, I knew I could hold my own in a male-dominated environment.”

  • Research
  • Condensed Matter Physics

Illinois Physics Professor Philip Phillips and Math Professor Gabriele La Nave have theorized a new kind of electromagnetism far beyond anything conceivable in classical electromagnetism today, a conjecture that would upend our current understanding of the physical world, from the propagation of light to the quantization of charge. Their revolutionary new theory, which Phillips has dubbed “fractional electromagnetism,” would also solve an intriguing problem that has baffled physicists for decades, elucidating emergent behavior in the “strange metal” of the cuprate superconductors.

This research is published in an upcoming colloquium paper in Reviews of Modern Physics (arXiv:1904.01023v1).

  • Accolades
  • Student News

The BPS Art of Science Image Contest took place again this year, during the 63rd Annual Meeting in Baltimore. The image that won first place was submitted by Angela Barragan, PhD Candidate at the Beckman Institute UIUC. Barragan took some time to provide information about the image and the science it represents.