CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae

被引：130

作者：

Jakociunas, Tadas ^{[1
]}

Rajkumar, Arun S. ^{[1
]}

Zhang, Jie ^{[1
]}

Arsovska, Dushica ^{[1
]}

Rodriguez, Angelica ^{[1
]}

Jendresen, Christian Bille ^{[1
]}

Skjodt, Mette L. ^{[1
]}

Nielsen, Alex T. ^{[1
]}

Borodina, Irina ^{[1
]}

Jensen, Michael K. ^{[1
]}

Keasling, Jay D. ^{[1
,2
,3
,4
,5
]}

机构：

[1] Tech Univ Denmark, Novo Nordisk Fdn, Ctr Biosustainabil, DK-2800 Lyngby, Denmark

[2] Joint BioEnergy Inst, Emeryville, CA 94608 USA

[3] Univ Calif Berkeley, Lawrence Berkeley Natl Lab, Phys Biosci Div, Berkeley, CA 94720 USA

[4] Univ Calif Berkeley, Dept Chem & Biomol Engn, Berkeley, CA 94720 USA

[5] Univ Calif Berkeley, Dept Bioengn, Berkeley, CA 94720 USA

来源：

ACS SYNTHETIC BIOLOGY | 2015年 / 4卷 / 11期

关键词：

DNA assembly; CRISPR/Cas9; double-strand break; metabolic engineering; CHROMOSOMAL INTEGRATION; YEAST TRANSFORMATION; CHORISMATE MUTASE; ESCHERICHIA-COLI; GENE; CLONING; REPAIR; CONSTRUCTION; MUTAGENESIS; SELECTION;

D O I：

10.1021/acssynbio.5b00007

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Homologous recombination (HR) in Saccharomyces cerevisiae has been harnessed for both plasmid construction and chromosomal integration of foreign DNA. Still, native HR machinery is not efficient enough for complex and marker-free genome engineering required for modern metabolic engineering. Here, we present a method for marker-free multiloci integration of in vivo assembled DNA parts. By the use of CRISPR/Cas9-mediated one-step double-strand breaks at single, double and triple integration sites we report the successful in vivo assembly and chromosomal integration of DNA parts. We call our method CasEMBLR and validate its applicability for genome engineering and cell factory development in two ways: (i) introduction of the carotenoid pathway from 15 DNA parts into three targeted loci, and (ii) creation of a tyrosine production strain using ten parts into two loci, simultaneously knocking out two genes. This method complements and improves the current set of tools available for genome engineering in S. cerevisiae.

引用

页码：1226 / 1234

页数：9

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