Abstract
CRISPR advances genome engineering by directing endonuclease sequence specificity with a guide RNA molecule (gRNA). For precisely targeting a gene for modification, each genetic construct requires a unique gRNA. By generating a gRNA against the flippase recognition target (FRT) site, a common genetic element shared by multiple genetic collections, CRISPR-FRT circumvents this design constraint to provide a broad platform for fast, scarless, off-the-shelf genome engineering.
Highlights
CRISPR advances genome engineering by directing endonuclease sequence specificity with a guide RNA molecule
CRISPR-flippase recognition target (FRT) (Fig. 1), which directs a guide RNA molecule (gRNA) to a FRT sequence present in each knockout mutant of the E. coli Keio collection[8]
CRISPR-FRT circumvents this aspect of rescue DNA template design by removing all traces of the FRT sites targeted by the gRNA
Summary
CRISPR advances genome engineering by directing endonuclease sequence specificity with a guide RNA molecule (gRNA). Even though the mechanism of Cas9-based gene editing is still incompletely understood[2], the incorporation of a rescue template into the genome by homologous recombination likely prevents Cas9-gRNA from cutting its target sequence, thereby providing a selection wherein engineered clones survive by preventing a futile cycle of lethal double-strand DNA breakage and repair. The λ-red recombinase genes encoded on the pKDsgRNA-FRT4 or the pCas[3] plasmid can be used to precisely replace the new target gene by a FRT-flanked KanR cassette. In this approach, a PCR-amplified oligo from the appropriate Keio clone is used as a template for homologous recombination in the
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