The last decade has witnessed major advancements in the gene-editing field that have applications in an array of scientific fields, from medicine and drug development to agriculture.
The CRISPR-Cas 9 system has seemingly garnered the most attention from both the scientific community and the media; however, an array of other gene-editing techniques are being utilized in laboratories across the globe.
Take zinc finger nucleases (ZFNs), for example. These are engineered DNA binding proteins that can be targeted to bind to specific sequences in the genome. They can be used to introduce a double stranded break at a desired location, thus enabling gene-editing. Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be used to cut specific sequences of DNA at a desired location. Recombinant adeno-associated virus (rAAV) based genome engineering utilizes rAAV vectors that allow for insertion, deletion or substitution of DNA sequences into the genomes of live cells.
The challenge with current approaches to gene-editing is that certain techniques are unable to target critical areas of DNA known as "dark" non-coding DNA. We now know that only one percent of the genome actually contains protein-encoding DNA. The other ~99 %, initially deemed "junk DNA", plays a crucial role in the regulation of gene expression, encoding promoters, enhancers, silencers and insulators. Having the ability to edit this region of the genome opens the door for scientists to adopt further control over gene expression.
The genome-editing toolbox continues to grow
Salk Institute Researchers have added to the gene-editing toolbox through their novel technique known as intercellular linearized Single homology Arm donor mediated intron-Targeting Integration, or SATI for short.
"We sought to create a versatile tool to target these non-coding regions of the DNA, which would not affect the function of the gene, and enable the targeting of a broad range of mutations and cell types," says Mako Yamamoto, co-first author on the paper and a postdoctoral fellow in the Izpisua Belmonte lab. "As a proof-of-concept, we focused on a mouse model of premature aging caused by a mutation that is difficult to repair using existing genome-editing tools." Their results are published in the journal Cell Research.
The novel technique is a knock-in method that can target specific mutated genes in vivo. A normal copy of the problematic gene is inserted into the non-coding region of the DNA before the mutation site. The new gene is then integrated into the genome alongside the mutated gene via DNA repair pathways. As a result, the organism is relieved of the detrimental effects of the mutated gene but does not run the risk of additional adverse effects incurred by fully replacing it.
Extending life span in a progeria mouse model
SATI was tested for validity in a mouse model of progeria, a rare autosomal dominant disorder that is caused by a mutation in the lamin A (LMNA) gene. Children affected with progeria sadly live on average 14 years, as the disorder causes premature aging and severe cardiac dysfunction resulting from hardening of the arteries. The mouse model of Hutchinson–Gilford progeria syndrome (HGPS) carrying the Lmna G609G gene presents with the same physiological changes and symptoms as observed in humans.
The researchers inserted a normal copy of LMNA in the progeria mice's non-coding DNA and observed diminished features of aging in several tissues, including the skin and spleen. They also found that gene-editing via this technique produced an extended life span of 45 % compared to control models. The scientists say that this period of time in the mouse's life translated to an extension of approximately ten years in human lifespan.
"This study has shown that SATI is a powerful tool for genome editing," says Juan Carlos Izpisua Belmonte, a professor in Salk's Gene Expression Laboratory and senior author of the paper. "It could prove instrumental in developing effective strategies for target-gene replacement of many different types of mutations and opens the door for using genome-editing tools to possibly cure a broad range of genetic diseases."
The next steps for the scientists involve improving the efficiency of SATI by increasing the number of cells that uptake the new DNA: "Specifically, we will investigate the details of the cellular systems involved in DNA repair to refine the SATI technology even further for better DNA correction," says Reyna Hernandez-Benitez, co-first author on the paper and a postdoctoral fellow in the Izpisua Belmonte lab.
Reference: Suzuki et al. 2019. Precise in vivo genome editing via single homology arm donor mediated intron-targeting gene integration for genetic disease correction. Cell Research. DOI: https://doi.org/10.1038/s41422-019-0213-0.