title: Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.
abstract: An efficient genome-scale editing tool is required for construction of industrially useful microbes. We describe a targeted, continual multigene editing strategy that was applied to the Escherichia coli genome by using the Streptococcus pyogenes type II CRISPR-Cas9 system to realize a variety of precise genome modifications, including gene deletion and insertion, with a highest efficiency of 100%, which was able to achieve simultaneous multigene editing of up to three targets. The system also demonstrated successful targeted chromosomal deletions in Tatumella citrea, another species of the Enterobacteriaceae, with highest efficiency of 100%.
authors: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Shanghai Research Center of Industrial Biotechnology, Shanghai, China.
authors: Yu
authors: Y
authors: Jiang
authors: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Shanghai Research Center of Industrial Biotechnology, Shanghai, China.
authors: Biao
authors: B
authors: Chen
authors: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors: Chunlan
authors: C
authors: Duan
authors: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Shanghai Research Center of Industrial Biotechnology, Shanghai, China.
authors: Bingbing
authors: B
authors: Sun
authors: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Shanghai Research Center of Industrial Biotechnology, Shanghai, China.
authors: Junjie
authors: J
authors: Yang
authors: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Shanghai Research Center of Industrial Biotechnology, Shanghai, China Shanghai Collaborative Innovation Center for Biomanufacturing Technology, Shanghai, China syang@sibs.ac.cn.
authors: Sheng
authors: S
authors: Yang
mesh_terms: D064113:CRISPR-Cas Systems; D004755:Enterobacteriaceae / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D005827:Genetics, Microbial / Q000379:methods*; D016680:Genome, Bacterial; D008967:Molecular Biology / Q000379:methods*; D011995:Recombination, Genetic; D013297:Streptococcus pyogenes / Q000201:enzymology / Q000235:genetics
references: Appl Environ Microbiol. 1997 Jan;63(1):71-6
references: 8979341
references: Nat Biotechnol. 2013 Aug;31(8):686-8
references: 23929338
references: Proc Natl Acad Sci U S A. 1997 Jul 22;94(15):8168-72
references: 9223333
references: Nat Biotechnol. 2013 Mar;31(3):233-9
references: 23360965
references: Nat Chem Biol. 2011 Jul;7(7):445-52
references: 21602812
references: Nature. 2009 Aug 13;460(7257):894-8
references: 19633652
references: Cell. 2013 Sep 12;154(6):1380-9
references: 23992846
references: Appl Environ Microbiol. 2014 Jul;80(13):3826-34
references: 24747889
references: Nature. 2011 Mar 31;471(7340):602-7
references: 21455174
references: Nat Genet. 1998 Oct;20(2):123-8
references: 9771703
references: Cell Res. 2013 Dec;23(12):1414-6
references: 24165890
references: Curr Opin Biotechnol. 2003 Oct;14(5):454-9
references: 14580573
references: Nat Biotechnol. 2001 Dec;19(12):1162-7
references: 11731786
references: Nucleic Acids Res. 2013 Nov;41(20):9584-92
references: 23939622
references: Mol Syst Biol. 2013;9:685
references: 24002656
references: Biotechnol Bioeng. 1998 Apr 5;58(2-3):204-14
references: 10191391
references: Proc Natl Acad Sci U S A. 2003 Dec 23;100(26):15748-53
references: 14673109
references: Proc Natl Acad Sci U S A. 2000 May 23;97(11):5978-83
references: 10811905
references: Nat Biotechnol. 2010 Aug;28(8):856-62
references: 20639866
references: Appl Microbiol Biotechnol. 2005 Nov;69(1):1-8
references: 16195792
references: Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1541-6
references: 12563033
references: ACS Synth Biol. 2015 Jun 19;4(6):723-8
references: 25458909
references: Genetics. 1988 Nov;120(3):621-3
references: 2852134
references: Metab Eng. 2001 Oct;3(4):289-300
references: 11676565
references: Nucleic Acids Res. 1999 Nov 15;27(22):4409-15
references: 10536150
references: Nucleic Acids Res. 2008 Aug;36(14):e84
references: 18567910
references: Genetics. 2013 Sep;195(1):289-91
references: 23833182
references: Nat Protoc. 2009;4(2):206-23
references: 19180090
references: Gene. 1988 Aug 15;68(1):159-62
references: 2851489
references: Methods Enzymol. 2011;498:409-26
references: 21601688
references: J Biol Eng. 2008 Apr 14;2:5
references: 18410688
references: Nature. 2010 Jan 28;463(7280):559-62
references: 20111002
references: Genome Biol. 2013;14(4):R40
references: 23628424
references: Trends Biochem Sci. 2003 Feb;28(2):62-6
references: 12575991
references: Nature. 2014 Sep 25;513(7519):569-73
references: 25079318
references: J Bacteriol. 1995 Jul;177(14):4121-30
references: 7608087
references: Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):1846-51
references: 10051557
references: Metab Eng. 2002 Oct;4(4):273-84
references: 12646322
references: J Bacteriol. 2000 Apr;182(8):2230-7
references: 10735866
references: Nature. 2008 Jan 3;451(7174):86-9
references: 18172501
references: Science. 2013 Feb 15;339(6121):819-23
references: 23287718
references: Nat Biotechnol. 2013 Sep;31(9):822-6
references: 23792628
references: Science. 2013 Feb 15;339(6121):823-6
references: 23287722
references: Mol Syst Biol. 2013;9:641
references: 23340847
references: Proc Natl Acad Sci U S A. 2010 Feb 2;107(5):2141-6
references: 20133858
references: Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5
references: 10829079
references: Gene. 1988 Sep 30;69(2):301-15
references: 3069586
references: Nucleic Acids Res. 2013 Apr;41(7):4336-43
references: 23460208
references: Cell. 2013 Feb 28;152(5):1173-83
references: 23452860
references: Nat Biotechnol. 2003 Jul;21(7):796-802
references: 12778056
references: Science. 2012 Aug 17;337(6096):816-21
references: 22745249
references: DNA Repair (Amst). 2007 Oct 1;6(10):1413-24
references: 17560174
references: Cell. 2013 Sep 26;155(1):27-38
references: 24074859