CRISPR technology has been made more accessible and standardized by a group of international researchers that says it was able to simplify the technology’s complex implementation. The simpler, faster CRISPR, called CRISPR-FRT, which is described in a Nature Communications paper published today, facilitates off-the shelf genome engineering, according to the team.
In addition to avoiding cloning of new gRNAs, CRISPR-FRT circumvents complex design of rescue templates and provides an easy phenotypic screen for positive clones.
"Toon [Swings, a postdoctoral scientist in the Jan Michiels laboratory at VIB-KU Leuven Center for Microbiology] and I had a set of projects in which we had to construct many mutations and guide RNAs for different genes in the bacterium E. coli. We realized we wouldn't need a new guide RNA 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," explained co-first author David Marciano, instructor in the Olivier Lichtarge laboratory at Baylor College of Medicine.
CRISPR-FRT works by directing a gRNA to a FRT sequence present in each knockout mutant of the E. coli Keio collection. Each clone in the Keio collection has had one gene replaced by a kanamycin resistance gene. "We ended up repurposing this valuable resource by targeting the two FRT sites flanking the collection's kanamycin cassette. This works out nicely because it gives you two cuts, which is harder to escape," Swings said.
Their 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 guide RNA and simplifies the strategy for constructing a rescue template.
"This is a nice example of the power of bacterial genetics. This is where CRISPR was first discovered, and now again, a different bacterial technology may make it even more useful," said corresponding author Dr. Olivier Lichtarge, Cullen Chair of Molecular and Human Genetics, and professor of biochemistry and molecular biology and of pharmacology Baylor College of Medicine.