Efficient and rapid exact gene replacement without selection

Abstract

We describe a highly efficient method for exact gene replacement in budding yeast. Induction of rapid and efficient recombination in an entire cell population results in at least 50% of the recombinants undergoing a specific switch of the endogenous copy to a specific mutated allele, with no remaining markers or remnant of foreign DNA, without selection. To accomplish this, a partial copy of the replacement allele, followed by an HO cut site, is installed adjacent to the wild-type locus, in a GAL-HO MATa-inc background. HO induction results in near-quantitative site cleavage and recombination/gene conversion, resulting in either regeneration of wild-type, or switch of the endogenous allele to the mutant, with accompanying deletion of intervening marker sequences, yielding an exact replacement. Eliminating the need for selection (over days) of rare recombinants removes concerns about second-site suppressor mutations, and also allows direct phenotypic analysis even of lethal gene replacements without the need of a method to make the lethality conditional, or to employ regulated promoters of unknown strength compared to the endogenous promoter. To test this method, we tried two known lethal gene replacements, substituting the non-essential CDH1 gene with a dominantly lethal version mutated for its Cdk phosphorylation sites, and substituting the essential CDC28 gene with two recessively lethal versions, one containing an early stop codon and another one inactivating Cdc28 kinase activity. We also tested a gene replacement of unknown phenotypic consequences: replacing the non-essential CLB3 B-type cyclin with a version lacking its destruction box.

Degradation of the Mitotic Cyclin Clb3 Is not Required for Mitotic Exit but Is Necessary for G1 Cyclin Control of the Succeeding Cell Cycle

“…Thus, removal of the D box from CLB3 could have similar results as with CLB2. We employed GAL-HO mediated exact gene replacement in a novel method to construct an exact endogenous replacement of the wild-type CLB3 allele with an allele lacking the D box sequence RVALSRVTN (Cross and Pecani 2011). This method allows recovery of CLB3Ddb or CLB3 in individual cells (depending on crossover point) without selection, and efficiently detects lethal recombinants.…”

“…All strains were derivatives of W303. All strains with CLB3Ddb were generated using HO-induced exact gene replacement of the CLB3 allele (Cross and Pecani 2011).…”

KNL1/Spc105 Recruits PP1 to Silence the Spindle Assembly Checkpoint

“…We generated two mutants of the Spc105 RVSF motif, a PP1-binding mutant, R A S A , and a potential phosphorylation site mutant, RV A F (see Figures S2A and B), by employing a method to rapidly introduce a site-specific mutation on the genome without selection ( H O-induced G ene R eplacement, or HGR) [6] (Figure 1A). At the promoter region of SPC105 , we first inserted a cassette ( spc105-NT ), containing a partial gene encoding the N-terminal region of Spc105 with desired mutations, followed by an HO endonuclease cut site (HOcs).…”

“…While several centromeric and kinetochore kinases, including Aurora B, regulate kinetochore-microtubule attachment and/or SAC activation [2–4], the molecular mechanism that translates bioriented attachment into SAC silencing remains unclear [5]. Employing a method to rapidly induce exact gene replacement in budding yeast [6], we show here that the binding of protein phosphatase 1 (PP1/Glc7) to the evolutionarily conserved RVSF motif of the kinetochore protein Spc105 (KNL1/Blinkin/CASC5) is essential for viability by silencing the SAC, while it plays an auxiliary nonessential role for physical chromosome segregation. Although Aurora B may inhibit this binding, persistent PP1-Spc105 interaction does not affect chromosome segregation and is insufficient to silence the SAC in the absence of microtubules, indicating that dynamic regulation of this interaction is dispensable.…”

A Tool for Multiple Targeted Genome Deletions that Is Precise, Scar-Free, and Suitable for Automation

“…Techniques for the deletion of multiple genes and other genomic regions are now a standard part of the modern biologist’s toolbox [ 1 – 6 ]. For example multiple genes must be removed from an organism to abolish traits controlled by several functionally overlapping genes [ 7 – 10 ], or to engineer strains that lack several unwanted functions [ 11 ].…”