Rapid, efficient and precise allele replacement in the fission yeast Schizosaccharomyces pombe

Abstract

Gene targeting provides a powerful tool to modify endogenous loci to contain specific mutations, insertions and deletions. Precise allele replacement, with no other chromosomal changes (e.g., insertion of selectable markers or heterologous promoters), maintains physiologically relevant context. Established methods for precise allele replacement in fission yeast employ two successive rounds of transformation and homologous recombination and require genotyping at each step. The relative efficiency of homologous recombination is low and a high rate of false positives during the second round of gene targeting further complicates matters. We report that pop-in, pop-out allele replacement circumvents these problems. We present data for 39 different allele replacements, involving simple and complex modifications at seven different target loci, that illustrate the power and utility of the approach. We also developed and validated a rapid, efficient process for precise allele replacement that requires only one round each of transformation and genotyping. We show that this process can be applied in population scale to an individual target locus, without genotyping, to identify clones with an altered phenotype (targeted forward genetics). It is therefore suitable for saturating, in situ, locus-specific mutation screens (e.g., of essential or non-essential genes and regulatory DNA elements) within normal chromosomal context.

Fig. 1

Streamlined approach for rapid and precise allele replacement. This overview of methodology and work flow also illustrates key advantages of the system: the process requires only one round each of transformation and genotyping. The streamlined approach has been validated using multiple different gene targeting vectors (nine successes and zero failures) with an observed allele replacement efficiency range of 24%–58%.

Fig. 2

Precise allele replacement by pop-in, pop-out gene targeting. Alleles are different shades for visual reference and differ by discrete modifications (open vs. filled circle). (a) The targeting vector contains a wild-type ura4⁺ gene and target DNA with desired changes (e.g., yfg1-M), but lacks an ARS (origin of replication). The target genome lacks the ura4 gene (ura4-D18). A restriction endonuclease is used to cut the vector within the region of homology (DSB) to promote homologous recombination (×) with the endogenous target. For efficient allele replacement, subregions of homology (a, b and c) should each be at least 250 bp in length. (b) Stable Ura⁺ transformants arise by homologous recombination with the target to produce tandem copies, one wild-type and one modified, or by non-homologous integration elsewhere (not shown). (c–d) Homologous recombination (×) between tandem repeats excises the plasmid from the genome. Because the plasmid lacks an ARS it cannot replicate and is lost during cell divisions. Removal of selection for uracil prototrophy allows pop-out cells to survive, and subsequent plating on media with FOA selects for cells that have lost the plasmid. Plasmid excision/loss from recombination to one side of the modification leaves that modification in the genome (c), whereas excision from recombination to the other side leaves the wild-type allele in the genome (d).

Fig. 3

Confirmation of pop-in and pop-out genotypes. (a) Successful pop-in gene targeting produces a tandem integrant with one wild-type and one modified version of the target locus. These are readily identified by PCR with one primer outside of the region of homology used for targeting and one primer within non-homologous portions of the targeting vector (top, primer pairs 1–2, 3–4, or both). In rare cases, the locus can have two wild-type or two modified alleles (see text for details). These can be identified by the presence or absence of an RFLP in the diagnostic PCR products (top) or by RFLP analysis of PCR products amplified, simultaneously in one reaction, from both copies using primers inside the region of homology (bottom, primer pair 5–6). (b) Pop-out of the targeting vector leaves a single copy of the locus, which is confirmed by PCR using primers located outside the region of homology used for targeting (primer pair 1–4). The single copy can be either wild-type or modified and these can be distinguished by the presence or absence of the diagnostic RFLP.

Fig. 4

Success rates of precise allele replacement are proportional to length ratios of homology. For pop-out recombination events, the fraction of homology in which “productive” events occur [Fig. 2c, (length a)/(total length)] is predicted to influence the frequency with which the modified allele is left in the genome. Plot shows the Expected and Observed Efficiencies derived from data in Table 1. Linear regression analysis confirmed the predicted correlation (slope = 0.95 ± 0.17; intercept = 7.7 ± 6.5; R² = 0.45; P < 0.0001). Scatter of data points is attributable, at least in part, to low n for individual measurements (Table 1, last column).

Fig. 5

Precisely targeted, saturating mutagenesis in situ. Diagram shows homology between targeting vector and genome (bold line), a hypothetical region of interest (box), and positions of DSBs used to promote pop-in recombination. Plot displays calculated frequency distribution of mutations left in the genome after pop-in, pop-out recombination (sum of mutations introduced using DSB1 and DSB2 in separate batches). Plot assumes that: (A) population size is infinite; (B) mutations are randomly distributed in the gene targeting vector (region of homology); and (C) all mutations at a DSB are lost to gene conversion, with the effect diminishing over distance from DSB in the “DSB zone” (shaded). Recombination events initiating from DSB2 compensate partially for conversion (loss) of mutations near DSB1 and vice versa. Thus, by linearizing the gene targeting vector in two batches (DSB1 and DSB2), one can generate and recover genomic mutations spanning the element of interest, including within the zones of gene conversion.