Researchers Create CRISPR Method That Avoids Need for Unique Guide RNAs
NEW YORK (GenomeWeb) – A team of researchers in Belgium and Texas have developed a CRISPR editing approach that takes advantage of a common genetic element present in Escherichia coli, making the genome editing process faster and scarless.
Genome editing normally requires the creation of CRISPR constructs that include unique guide RNAs (gRNA), which target a gene for modification. But by generating a gRNA against this common genetic element, called the flippase recognition target (FRT) site, the CRISPR-FRT circumvents this design constraint and obviates the need for unique gRNAs, according to the researchers.
"In E. coli, efficient genome editing using CRISPR and homology-directed repair requires induction of the CRISPR components (Cas9 and gRNA), induction of λ phage recombinase genes, and a rescue DNA template with the desired mutation," the authors, led by the University of Leuven's Jan Michiels and Baylor College of Medicine's Olivier Lichtarge, wrote in Nature Communications today. "Here we make CRISPR more accessible and standardized with a simple solution that simultaneously avoids cloning of new gRNAs, circumvents complex design of rescue templates, and provides an easy phenotypic screen for positive clones."
The team showed that the CRISPR-FRT method directs a gRNA to an FRT sequence present in each knockout mutant of the E. coli strains in the Keio collection. In the arrayed Keio collection of 3,884 deletion mutants, each non-essential E. coli gene has been replaced by a kanamycin-resistance (KanR) cassette flanked on each end by FRT sites. Instead of designing and cloning a unique gRNA for each gene, a single gRNA-FRT can target any gene in this collection, the researchers said.