The MX4blaster cassette: repeated and clean Saccharomyces cerevisiae genome modification using the genome-wide...

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.