The MX4blaster cassette: repeated and cleanSaccharomyces cerevisiaegenome modification using the genome-wide deletion collection

Abstract

The kanMX4 resistance marker is widely used for Saccharomyces cerevisiae gene deletion and has been used to create a genome-wide deletion mutant collection. Transfer of PCR-amplified marker loci from collection mutants is a very efficient way of introducing mutations into other S. cerevisiae strains of interest. An important limitation of this strategy is that the kanMX4 marker is not easily removed, impairing the construction of multiple gene deletion strains. The MX4blaster is a novel MX4-compatible cassette that facilitates clean or scarless kanMX4 removal. The MX4blaster cassette efficiently replaces the kanMX4 cassette due to shared promoter and terminator sequences. The MX4blaster cassette is removed by the induction of an internal double-stranded break and transformation with a DNA fragment designed to recombine on either side of the cassette in combination with counter selection. Simple and inexpensive media are used for induction and counter selection, mitigating the use of expensive chemicals. Routinely, thirty percent of transformants successfully removed the MX4blaster cassette. The resulting mutants contain no trace of the MX4blaster cassette. The unique properties of the MX4blaster cassette make it an important addition to S. cerevisiae genetic tools, especially in combination with the genome-wide deletion collection.

Yeast genetic interaction screens in the age of CRISPR/Cas

“…Although other methods have been used to make markerless deletions from the deletion collection, they also remove the deletion barcodes (Carvalho et al 2013; Soreanu et al 2018). Similarly, one could swap the kanMX marker from the deletion strains with other MX markers.…”

“…Using CRISPR/Cas to edit deletion strain collections, one could remove the kanMX marker from the deletion strains and replace it with markerless donor DNA fragments homologous to the two ends of the kanMX cassette. Although other methods have been used to make markerless deletions from the deletion collection, they also remove the deletion barcodes (Carvalho et al 2013; Soreanu et al 2018). Similarly, one could swap the kanMX marker from the deletion strains with other MX markers.…”

Yeast metabolic chassis designs for diverse biotechnological products

“…The modularized chassis designs proposed here offer an excellent starting point for experimental validation. The proposed deletion strains can be engineered using the broad gene disruption toolbox available for yeast, including tools for simultaneous introduction of multiple gene deletions 48 49 50 . On the pathway front, new tools enable large pathways to be assembled and integrated in a high-throughput fashion 51 52 53 .…”

Saccharomyces cerevisiae Shuttle vectors

“…Nuclease‐stimulated and scarless marker removal could also be applied to the marker recycling of YIps. An example of such a marker cassette is the MX4blaster, where a marker, the inducible I‐SceI nuclease, a recognition site and an inducible counterselection is integrated (Carvalho et al, ). The cassette is recycled by transformation with a DNA sequence recombining on each side of the cassette and induction of the counterselectable gene.…”