Imagine opening a catalog and selecting a genome engineering kit, not worrying about the contents, since you know it’s a one size fits all scenario. Well now, an international team of investigators has just gotten a few steps closer to that situation by making CRISPR technology more accessible and standardized through simplifying its complex implementation. Findings from the new study—published in Nature Communications, in an article entitled “CRISPR-FRT Targets Shared Sites in a Knock-Out Collection for Off-the-Shelf Genome Editing”—offer a broad platform for off-the-shelf genome engineering that may lower the barrier of entry for this powerful technology.
"CRISPR technologies can be programmed to target specific sequences of genetic code and to edit DNA at precise locations, thus allowing research scientists to permanently modify genes in living cells and model organisms to explore gene function in the laboratory, including genes associated with human disease," explains co-lead study investigator David Marciano, Ph.D., an instructor at Baylor College of Medicine.
"These technologies permit a ribonucleoprotein complex to cleave DNA at a specific sequence that base-pairs with a guide RNA (gRNA) in the complex,” adds co-lead study investigator Toon Swings, Ph.D., a postdoctoral scientist in at VIB-KU Leuven Center for Microbiology in Belgium. “Modularity of the nucleic acid/protein complex allows researchers to specify the gRNA sequence to target nearly any sequence. This greatly improves researchers' ability to edit DNA.”
Still, this approach presents some challenges, such as constraints on the sequences that can be targeted, the possibility of off-target effects, and the requirement of a unique gRNA for each target gene. The research team established an international collaboration that led to a simple solution that circumvents all these issues.
"Toon and I had a set of projects in which we had to construct many mutations and gRNAs for different genes in the bacterium E. coli,” Dr. Marciano noted. “We realized we wouldn't need a new gRNA for each gene if we just targeted a universal sequence found in gene knockout collections. The sequence we targeted is found in many genetic collections of medically important bacteria and is even in some fruit fly collections.”
For the current study, the investigators used a library of Escherichia coli clones, called the Keio collection. Each clone in this collection has had one gene replaced by a kanamycin resistance gene. The collection was made available in 2006 through an international collaboration between Keio University of Japan and Purdue University in the United States.
"We ended up repurposing this valuable resource by targeting the two flippase recognition target (FRT) sites flanking the collection's kanamycin cassette,” Dr. Swings stated. “This works out nicely because it gives you two cuts, which is harder to escape."
The researcher's approach avoids some technical aspects of CRISPR and makes it available as an off-the-shelf ingredient for genetic engineering. It removes the need to design and clone a gRNA and simplifies the strategy for constructing a rescue template. Also, the Keio collection can be found in laboratories across the globe, and individual clones are available for a nominal fee from centralized genetic stock centers.
"This is a nice example of the power of bacterial genetics,” commented co-senior study investigator Olivier Lichtarge, Ph.D., chair of molecular and human genetics and professor of biochemistry, molecular biology, and of pharmacology at Baylor College of Medicine. “This is where CRISPR was first discovered, and now again a different bacterial technology may make it even more useful."
This new work also presents the broad utility of the approach by showing it is possible to target genes that are essential to life, to make a large collection of organisms with different mutations in a single chromosomal gene and to append new sequences onto a gene, all in the gene's natural context. The method should complement existing techniques for genetic engineering of E. coli.
"Many model organisms, besides E. coli, have collections of gene replacements or insertions that could be targeted by a single gRNA in a similar manner," Dr. Swings remarked. "We hope our work provides a broad platform for a variety of genetic engineering approaches."
"The developed platform based on CRISPR technology will be valuable to many researchers in microbiology, allowing them to perform rapid single-nucleotide editing of their genes of interest or to generate chromosomal mutant collections, one of the first steps in understanding gene function," concluded co-senior study investigator Jan Michiels, Ph.D., group leader at VIB-KU Leuven Center for Microbiology and professor of biochemistry and molecular microbiology at the University of Leuven.