Although CRISPR systems may be programmed to run various DNA-editing plays, these plays sometimes end in fumbles, depending on which parts of the genomic gridiron are being targeted. In some genomic regions, the lack of protospacer adjacent motifs (PAMs) means that CRISPR is bound to lose its grip. Sometimes, CRISPR is best kept on the sidelines. A substitute genome editing system, however, may enter the genome editing game. This system, which is based on the protein Argonaut, may even add new pages to the DNA-editing playbook.
At Purdue University, researchers led by Kevin Solomon, PhD, assistant professor of agricultural & biological engineering, created a genome editing method that uses the Argonaut from Natronobacterium gregoryi (NgAgo). NgAgo, the researchers report, may be supplied DNA as a guide to enable modification of any genomic location, providing new options to potentially improve manufacturing, disease treatment, drug discovery, and crop production.
The researchers described their method April 4 at the National Meeting of the American Chemical Society in Orlando, FL. In their presentation, the researchers explain that their approach can overcome a problem that has kept Argonaut on the bench. Basically, all currently characterized pAgos are thermophilic, making them infeasible for use in common mesophilic model organisms. The Purdue team, however, has been working with a mesophilic pAgo, one that shows promise as a genome editing tool.
“Computational structure modeling revealed that our pAgo candidate consists of canonical N-terminal, PAZ, MID, and PIWI domains found in other catalytically active pAgos, such as the programmable restriction enzyme PfAgo,” the researchers noted in the abstract for their presentation. “However, our mesophilic protein also contains a putative single-stranded DNA binding or repA domain.”
The repA domain, phylogenetic analysis of pAgos revealed, is characteristic of a new class of mesophilic pAgos currently unrecognized in the literature.
“Our pAgo candidate nicks and cuts plasmid DNA nonspecifically in the absence of guide in in vitro assays, presumably due to the presence of single-stranded DNA that co-purifies with the protein,” the abstract continued. “Truncation and substitution mutants suggest that this cleavage may be independently attributed to the repA and PIWI domains. In vivo, we are able to program our pAgo candidate with 5′-phosphorylated DNA guides to cleave both plasmid and chromosomal targets to reduce survival under selective conditions in E. coli.”
The researchers observed this effect regardless of target gene function. According to the researchers, this finding suggests that lethality is due to unrepaired double-stranded DNA breaks. “In the presence of homologous donor DNA and pAgo,” they emphasized, “homologous recombination is increased by up to 50% at targeted gene loci, suggesting that gene editing applications are feasible.”
“While there is still work to do, we have shown that these molecular scissors can edit regions of DNA previously inaccessible by current technologies,” said Michael Mechikoff, a master’s student working on the project.
“One of my best friends died from a cancer caused by a genetic variant several years ago,” added Kok Zhi Lee, a doctoral student who works on the research team in Solomon’s lab. “I always dreamed of a different scenario for my friend—living in an era where genetic engineering is a regular and safe option to correct genetic disorders. With the potential of our technology, I anticipate a future where genetic disease is history for human beings.”
The team has worked with the Purdue Research Foundation Office of Technology Commercialization to file a utility patent on the technology. They are looking for partners and others interested in developing and licensing it.