کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
6494485 | 44809 | 2015 | 39 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae
دانلود مقاله + سفارش ترجمه
دانلود مقاله ISI انگلیسی
رایگان برای ایرانیان
کلمات کلیدی
gRNAROX1UASPAMDSBMevalonateORFCRISPRCRISPR/Cas9 - CRISPR / Cas9squalene synthase - اسکالن سنتازclustered regularly interspaced short palindromic repeat - به طور منظم تکرار پیلندرومی کوتاه مدت میان دو طرف تقسیم می شودupstream activating sequence - دنباله فعال سازی بالادستguide RNA - راهنمای RNATranscription activator-like effector nuclease - رونویسی مانند actuator nucleaseTALEN - زبان هاdouble strand break - شکست دو رشتهopen reading frame - قاب خواندن بازYeast - مخمرprotospacer adjacent motif - موتیف مجاور protospacergeranylgeranyl pyrophosphate synthase - گرانیل گرانیل پیرو فسفات سنتاز
موضوعات مرتبط
مهندسی و علوم پایه
مهندسی شیمی
بیو مهندسی (مهندسی زیستی)
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
CRISPR/Cas9 is a simple and efficient tool for targeted and marker-free genome engineering. Here, we report the development and successful application of a multiplex CRISPR/Cas9 system for genome engineering of up to 5 different genomic loci in one transformation step in baker's yeast Saccharomyces cerevisiae. To assess the specificity of the tool we employed genome re-sequencing to screen for off-target sites in all single knock-out strains targeted by different gRNAs. This extensive analysis identified no more genome variants in CRISPR/Cas9 engineered strains compared to wild-type reference strains. We applied our genome engineering tool for an exploratory analysis of all possible single, double, triple, quadruple and quintuple gene disruption combinations to search for strains with high mevalonate production, a key intermediate for the industrially important isoprenoid biosynthesis pathway. Even though we did not overexpress any genes in the mevalonate pathway, this analysis identified strains with mevalonate titers greater than 41-fold compared to the wild-type strain. Our findings illustrate the applicability of this highly specific and efficient multiplex genome engineering approach to accelerate functional genomics and metabolic engineering efforts.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Metabolic Engineering - Volume 28, March 2015, Pages 213-222
Journal: Metabolic Engineering - Volume 28, March 2015, Pages 213-222
نویسندگان
Tadas JakoÄiÅ«nas, Ida Bonde, Markus HerrgÃ¥rd, Scott J. Harrison, Mette Kristensen, Lasse E. Pedersen, Michael K. Jensen, Jay D. Keasling,