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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 In the arrayed Keio collection of 3,884 deletion mutants, each non-essential E. coli gene has been replaced by a kanamycin-resistance (KanR) cassette flanked on each end by FRT sites.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.

They found that a Keio strain targeted with a CRISPR-Cas9 complex with the gRNA-FRT experiences a lethal double-strand DNA break at the FRT sites. And since E. coli naturally lacks non-homologous end joining, its survival depends on escaping a cycle of homology-directed repair and recutting by the Cas9-gRNA-FRT complex. "Escape from the Cas9-gRNA-FRT complex can occur by recombination of a homologous rescue DNA template lacking an FRT site," the authors wrote.

In order to produce specific changes to genes in their native locus, the researchers utilized mutated forms of the genes corresponding to their Keio knockouts. They amplified the mutated genes of interest, along with about 200 to 500 base pairs of homology flanking the FRT-KanR-FRT cassette, and then introduced the gRNA-FRT and Cas9. Recombination of the rescue DNA template resulted in replacement of the FRT-flanked kanamycin-resistance cassette by the gene containing the mutation of interest.

"Consequently, this protocol allows one to easily introduce specific mutations in the ancestral Keio background, without any scars. Other E. coli strains can be engineered by first transferring the KanR cassette using P1vir transduction and then proceeding with the CRISPR-FRT protocol," the authors added. "Likewise, multiple mutations can be constructed in the same strain by consecutive cycles of P1vir transduction of a new gene deletion from the Keio collection followed by another round of CRISPR-FRT."

The method, they noted, eliminates the need for cloning of new gRNAs and the design of specific rescue DNA constructs for each desired mutation. It also provides a ready-to-use method for reconstruction of mutations in the E. coli Keio collection. However, they added, it is also applicable to other collections with insertion sequences that can be targeted using a similar strategy.