title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham Street (slot 516), Little Rock, AR, 72205-7199, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Gao
authors.forename: Fengling
authors.initials: F
authors.lastname: Kan
authors.forename: Jacy L
authors.initials: JL
authors.lastname: Wagnon
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Storey
authors.forename: Reine U
authors.initials: RU
authors.lastname: Protacio
authors.forename: Mari K
authors.initials: MK
authors.lastname: Davidson
authors.forename: Wayne P
authors.initials: WP
authors.lastname: Wahls
mesh_terms: D000483:Alleles; D004247:DNA / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005838:Genotype; D009154:Mutation; D011995:Recombination, Genetic; D012568:Schizosaccharomyces / Q000235:genetics*
chemical_list: D004247:DNA
references.citation: Yeast. 1998 Jul;14(10):943-51
references.pmid: 9717240
references.citation: Biotechniques. 2001 Jul;31(1):88-90, 92
references.pmid: 11464525
references.citation: Mol Gen Genet. 1988 Dec;215(1):87-93
references.pmid: 3241625
references.citation: Gene. 2008 Jan 15;407(1-2):63-74
references.pmid: 18054176
references.citation: Genetics. 2000 Feb;154(2):599-607
references.pmid: 10655214
references.citation: Nat Protoc. 2007;2(4):924-32
references.pmid: 17446874
references.citation: Curr Genet. 2012 Feb;58(1):59-64
references.pmid: 22198627
references.citation: J Bacteriol. 1983 Jan;153(1):163-8
references.pmid: 6336730
references.citation: Nucleic Acids Res. 1990 Dec 25;18(24):7349-55
references.pmid: 2175433
references.citation: Biotechniques. 2010 Jun;48(6):463-5
references.pmid: 20569222
references.citation: Nat Cell Biol. 2010 Feb;12(2):111-8
references.pmid: 20098417
references.citation: Nucleic Acids Res. 2008 May;36(9):2838-51
references.pmid: 18375981
references.citation: Mol Gen Genet. 1988 Dec;215(1):81-6
references.pmid: 3241624
references.citation: Nucleic Acids Res. 2011 Mar;39(4):1460-72
references.pmid: 21030440
references.citation: EMBO J. 1991 Aug;10(8):2157-63
references.pmid: 2065658
references.citation: Mol Gen Genet. 1994 Jul 25;244(2):111-9
references.pmid: 8052229
references.citation: Cell. 2010 Sep 17;142(6):868-78
references.pmid: 20850009
references.citation: J Mol Biol. 1988 Dec 20;204(4):917-25
references.pmid: 3221399
references.citation: Yeast. 2006 Feb;23(3):173-83
references.pmid: 16498704
references.citation: Cell Chromosome. 2002 Sep 19;1(1):1
references.pmid: 12437782
references.citation: Yeast. 1996 Nov;12(14):1393-405
references.pmid: 8948095
references.citation: Genetics. 2011 Feb;187(2):385-96
references.pmid: 21098718
references.citation: Nucleic Acids Res. 2004 Aug 17;32(14):4400-10
references.pmid: 15316103
references.citation: Curr Genet. 1987;12(7):527-34
references.pmid: 2834100
references.citation: Genetics. 2009 Jun;182(2):459-69
references.pmid: 19363124
references.citation: Yeast. 1999 Sep 30;15(13):1419-27
references.pmid: 10509024
references.citation: FEMS Microbiol Rev. 2004 May;28(2):201-23
references.pmid: 15109785
references.citation: Eukaryot Cell. 2010 Apr;9(4):626-33
references.pmid: 20139237
references.citation: Mol Gen Genet. 1993 Apr;238(1-2):26-32
references.pmid: 8479431
references.citation: Genesis. 2006 Oct;44(10):465-76
references.pmid: 16981199
references.citation: Methods Enzymol. 2010;470:759-95
references.pmid: 20946835