title: Modified bases enable high-efficiency oligonucleotide-mediated allelic replacement via mismatch repair evasion.
abstract: Genome engineering using single-stranded oligonucleotides is an efficient method for generating small chromosomal and episomal modifications in a variety of host organisms. The efficiency of this allelic replacement strategy is highly dependent on avoidance of the endogenous mismatch repair (MMR) machinery. However, global MMR inactivation generally results in significant accumulation of undesired background mutations. Here, we present a novel strategy using oligos containing chemically modified bases (2'-Fluoro-Uridine, 5-Methyl-deoxyCytidine, 2,6-Diaminopurine or Iso-deoxyGuanosine) in place of the standard T, C, A or G to avoid mismatch detection and repair, which we tested in Escherichia coli. This strategy increases transient allelic-replacement efficiencies by up to 20-fold, while maintaining a 100-fold lower background mutation level. We further show that the mismatched bases between the full length oligo and the chromosome are often not incorporated at the target site, probably due to nuclease activity at the 5' and 3' termini of the oligo. These results further elucidate the mechanism of oligo-mediated allelic replacement (OMAR) and enable improved methodologies for efficient, large-scale engineering of genomes.
authors: Wyss Institute for Biologically Inspired Engineering, Harvard University, Department of Genetics, Harvard Medical School, Harvard University, Boston, MA 02115, USA. hhwang@genetics.med.harvard.edu
authors: Harris H
authors: HH
authors: Wang
authors: George
authors: G
authors: Xu
authors: Ashley J
authors: AJ
authors: Vonner
authors: George
authors: G
authors: Church
references: Chem Biol. 2002 Oct;9(10):1073-84
references: 12401492
references: Annu Rev Genet. 1991;25:229-53
references: 1812808
references: Proc Natl Acad Sci U S A. 2003 Dec 9;100(25):14822-7
references: 14634210
references: J Gene Med. 2005 Dec;7(12):1534-44
references: 16025558
references: J Mol Biol. 2009 Aug 21;391(3):586-98
references: 19527729
references: BMC Mol Biol. 2010 Jul 29;11:54
references: 20670401
references: J Gene Med. 2006 Feb;8(2):217-28
references: 16142817
references: Genetics. 2010 Nov;186(3):791-9
references: 20813883
references: Methods Enzymol. 2007;421:171-99
references: 17352923
references: Trends Biotechnol. 2005 Aug;23(8):399-406
references: 15982766
references: Gene Ther. 2004 May;11(10):838-46
references: 14961068
references: Genetics. 1992 Aug;131(4):811-9
references: 1325385
references: Genetics. 2003 Feb;163(2):527-38
references: 12618392
references: Methods Enzymol. 2011;498:409-26
references: 21601688
references: Mutat Res. 1996 Nov 11;372(1):87-96
references: 9003535
references: Biotechniques. 2007 Nov;43(5):617-8, 620, 622 passim
references: 18072591
references: Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7207-12
references: 12771385
references: Science. 1996 Sep 6;273(5280):1386-9
references: 8703073
references: Proc Natl Acad Sci U S A. 1987 Sep;84(17):6220-4
references: 3306672
references: Annu Rev Biochem. 2005;74:681-710
references: 15952900
references: Annu Rev Genet. 2002;36:361-88
references: 12429697
references: Proc Natl Acad Sci U S A. 2000 May 23;97(11):5978-83
references: 10811905
references: BMC Mol Biol. 2003 Jan 16;4(1):1
references: 12530927
references: Nucleic Acids Res. 2011 Apr;39(8):3482-95
references: 21183463
references: Mol Microbiol. 2010 Jan;75(1):138-48
references: 19943907
references: Proc Natl Acad Sci U S A. 1999 Jul 20;96(15):8768-73
references: 10411950
references: Proc Natl Acad Sci U S A. 2003 Dec 23;100(26):15748-53
references: 14673109
references: Genetics. 2000 Mar;154(3):959-70
references: 10757746
references: Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5
references: 10829079
references: Protein Sci. 2010 May;19(5):967-73
references: 20217896
references: Gene Ther. 2001 Mar;8(5):391-9
references: 11313816
references: J Mol Biol. 2011 Mar 18;407(1):45-59
references: 21256136
references: Proc Natl Acad Sci U S A. 1988 Jan;85(2):524-8
references: 2829192
references: Proc Natl Acad Sci U S A. 2001 Jun 5;98(12):6742-6
references: 11381128
references: Nat Genet. 1998 Oct;20(2):123-8
references: 9771703
references: Gene Ther. 2007 Dec;14(24):1675-80
references: 17972921
references: Chem Rev. 2006 Feb;106(2):302-23
references: 16464007
references: Proc Natl Acad Sci U S A. 1999 Jul 20;96(15):8774-8
references: 10411951
references: Curr Mol Med. 2004 Aug;4(5):445-63
references: 15267219
references: J Mol Biol. 2002 Nov 22;324(2):297-307
references: 12441108
references: Nat Protoc. 2009;4(2):206-23
references: 19180090
references: Nature. 2009 Aug 13;460(7257):894-898
references: 19633652
references: J Bacteriol. 2003 Feb;185(4):1459-61
references: 12562818