A step toward advancement in regenerative medicine by improving the commonly used gene repair technique was made possible by researchers at the University of Wisconsin-Madison’s Morgridge Institute for Research and Northwestern University.
Using a nuclease, or an enzthat cleaves nucleotide strands in DNA, from meningitis bacteria and human pluripotent stem cells, the researchers discovered a gene repair technique more efficient than the traditional method in regenerative medicine. The latest technology in gene repair, CRISPR (clustered regularly interspaced short palindromic repeats) was then used to specifically target and repair defective genes.
According to first author Zhonggang Hou from the Morgridge Institute for Research, research concerning gene repair and simplification of technology has been going on for over 40 years and this new technique has been a ground-breaking discovery.
In the CRISPR technique, engineered ribonucleic acids guide a nuclease to create a sequence specific double strand break in target DNA. In simple terms, a nuclease breaks a particular or target region of the DNA.
There are three types (I, II and III) of CRISPR pathways and their presence was first found in bacteria. The pathways act like a component of the bacteria’s immune system against viruses and plasmid DNA. Spacers or short DNA sequences are “remembered” at CRISPR loci (the location of a given gene within the chromosome) in the initial infection and during a re-infection and the matching sequence is found with high specificity. This allows the nuclease to create a double-strand break in the foreign DNA. Among the three types, type II is the basis for CRISPR gene repair technology.
In this study, the scientists focused on Neisseria meningitidis bacteria because of the specific nuclease availability for cutting target gene repair sequences. The biggest change stems from the ease of breaking these nucleotide chains at the correct loci.
Regenerative medicine involves replacing, repairing or regenerating damaged mechanisms. Gene repair is a critical technique of this field and it defines the process of correcting errors in DNA sequences.
For a major part, cells within our body will recognize and correct any errors in the chromosome such as mutations. One method our body uses to correct these errors is homologous recombination. Homologous recombination is the exchange of nucleotide sequences between two similar strands of DNA. The cell will use this recombination to repair a double-stranded break in target DNA.
“Most of the time, the cell will repair the mutation or error by using another DNA molecule homologous to the break, cell will use that as a template to repair the break in the host DNA,” Hou said.
Researchers can take advantage of homologous recombination by replacing a DNA locus with an engineered DNA casette with regions homologous to the host DNA.
The study looks at how to promote homologous recombination and to find an efficient way to cut DNA with specificity. As of now, three methods exist—Zinc finger nuclease, TALENs (transcription-activator-like effector nucleases) and CRISPR.
Both Zinc finger nucleases and TALENs have the same nuclease domains. In the study, research was conducted with CRISPR.
“CRISPR is the latest technology and is better than TALEN and Zinc finger nuclease because for the both of them, for each locus, a different protein must be engineered. And making proteins is time-consuming and difficult,” Hou said.
Both Zinc finger nucleases and TALENs involve protein-mediating mechanisms. CRISPR, however, uses RNA-based guiding systems in order for the nuclease to reach targeted loci. Therein lies the major advantage of utilizing this technology — the simplicity and correctness with which DNA can be repaired.
While the researchers were not the only ones working on CRISPR, their findings helped further regenerative medicine.
“As of now, the success of the method is case-by-case. We were able to get a 60 percent efficiency for a seamless repair... As it is still in the early stage for the technique, it is not a very extensive study but rather case-by-case where some cases are efficient and others inefficient. The cause for this variation is still unknown,” Hou said.
The research UW-Madison conducted, however, has a very large impact.
“[The technique] does not yet affect the clinical setting because it is too early to say, but it affects how people deal with gene repair in the research setting right now,” Hou said.