A highly efficient single-step, markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae

Shi, Shuobo; Li, Tiehai; Zhang, Mingzi M.; Ang, Ee Lui; Zhao, Huimin

doi:10.1016/j.ymben.2015.10.011

Cited by 178 publications

(151 citation statements)

References 38 publications

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“…These tools have enabled single-gene and simultaneous multigene editing [1,14,34]. The CRISPR-Cas9 system used in S. cerevisiae was successfully extended to K. lactis [14].…”

mentioning

confidence: 99%

Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system

Gao

Tong

Wen

et al. 2016

Journal of Industrial Microbiology and Biotechnology

155

143

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Yarrowia lipolytica is categorized as a generally recognized as safe (GRAS) organism and is a heavily documented, unconventional yeast that has been widely incorporated into multiple industrial fields to produce valuable biochemicals. This study describes the construction of a CRISPR-Cas9 system for genome editing in Y. lipolytica using a single plasmid (pCAS1yl or pCAS2yl) to transport Cas9 and relevant guide RNA expression cassettes, with or without donor DNA, to target genes. Two Cas9 target genes, TRP1 and PEX10, were repaired by non-homologous end-joining (NHEJ) or homologous recombination, with maximal efficiencies in Y. lipolytica of 85.6 % for the wild-type strain and 94.1 % for the ku70/ku80 double-deficient strain, within 4 days. Simultaneous double and triple multigene editing was achieved with pCAS1yl by NHEJ, with efficiencies of 36.7 or 19.3 %, respectively, and the pCASyl system was successfully expanded to different Y. lipolytica breeding strains. This timesaving method will enable and improve synthetic biology, metabolic engineering and functional genomic studies of Y. lipolytica.

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“…These tools have enabled single-gene and simultaneous multigene editing [1,14,34]. The CRISPR-Cas9 system used in S. cerevisiae was successfully extended to K. lactis [14].…”

mentioning

confidence: 99%

Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system

Gao

Tong

Wen

et al. 2016

Journal of Industrial Microbiology and Biotechnology

155

143

View full text Add to dashboard Cite

show abstract

“…More recently, several studies have highlighted significant improvements in genome editing in plants using DNA-free CRISPR/Cas9 ribonucleoproteins [65,98]. Besides its multiplexing qualities, CRISPR has also shown great efficiency to integrate large pathways and Simultaneous downregulation of 6 genes for fatty alcohol production [48] libraries [38,88]. For example, Shi et al specifically designed gRNAs to target multiple delta sites in the yeast genome, ultimately achieving 18-copy genomic integrations of a 24 kb combined xylose utilization and (R,R)-2,3-butanediol (BDO) production pathway in a single step, in S. cerevisiae [88].…”

Section: Genome Engineeringmentioning

confidence: 99%

“…Besides its multiplexing qualities, CRISPR has also shown great efficiency to integrate large pathways and Simultaneous downregulation of 6 genes for fatty alcohol production [48] libraries [38,88]. For example, Shi et al specifically designed gRNAs to target multiple delta sites in the yeast genome, ultimately achieving 18-copy genomic integrations of a 24 kb combined xylose utilization and (R,R)-2,3-butanediol (BDO) production pathway in a single step, in S. cerevisiae [88]. DNA libraries, such as error-prone PCRs derived or double-stranded fragments obtained from DNA synthesizing companies, can be genomically integrated to find variants of a studied enzyme with enhanced catalytic activities or optimal level of expression [64,83].…”

Section: Genome Engineeringmentioning

confidence: 99%

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

Ferreira

David

Nielsen

2018

Journal of Industrial Microbiology and Biotechnology

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Clustered regularly interspaced short palindromic repeats (CRISPR) is poised to become one of the key scientific discoveries of the twenty-first century. Originating from prokaryotic and archaeal immune systems to counter phage invasions, CRISPRbased applications have been tailored for manipulating a broad range of living organisms. From the different elucidated types of CRISPR mechanisms, the type II system adapted from Streptococcus pyogenes has been the most exploited as a tool for genome engineering and gene regulation. In this review, we describe the different applications of CRISPR/Cas9 technology in the industrial biotechnology field. Next, we detail the current status of the patent landscape, highlighting its exploitation through different companies, and conclude with future perspectives of this technology.

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“…Ген белка Cas9 экспрессировали под контролем конститутивных промо-торов разной силы. Наиболее часто используемый про-мотор -TEF1 Zhang et al, 2014;Bao et al, 2015;Jakočiūnas et al, 2015;Mans et al, 2015;Shi et al, 2016;Jensen et al, 2017;Liu et al, 2017;Ng, Dean, 2017), второй по частоте использования промотор -ADH1 (Gao, Zhao, 2014;Jacobs et al, 2014;Fernandez, Berro, 2016;Reider Apel et al, 2017), далее следуют RNR2 , FBA1 (Horwitz et al, 2015), TDH3 (Laughery et al, 2015), PGK1 (Lee et al, 2015) и др.…”

Section: Crispr/casunclassified

“…4. В исследовании (Shi et al, 2016) был получен штамм-продуцент (R,R)-2,3-бутандиола, способный к коферментации глюкозы и ксилозы, а также проведено многокопийное клонирование 24 кб фрагмента, несущего необходимые последователь-ности.…”

Section: примеры использования Crispr/cas в работе с дрожжамиunclassified

Methods of yeast genome editing

Розанов¹,

Шляхтун²,

Текутьева³

et al. 2017

Vestn. VOGiS

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Yeasts are a convenient model eukaryote used for genome studies and genome editing. Saccharomyces cerevisiae is the species most widely employed in bio technology, since it is easily cultivated in bioreactors and is absolutely safe. The last decade saw a significant development of methods of yeast genetic engineer ing and the creation of novel instruments adapted from other fields, which allowed one to significantly accelerate the construction of new strains. The most prominent examples are the proteins used for directed DNA editing. For a long time, yeast genome engineer ing was based on the yeasts' system of homologous recombination. It was sufficient for several decades before the development of highthroughput methods. Many highthroughput methods were developed in the second decade of the XXI century, including those used in genomics, transcriptomics, proteomics, metabolomics, interactomics, etc. Modern bioinfor matic databases now allow one to rapidly process the increasing flow of information and model cellular pro cesses. As a result, the rate of analysis and prediction of targets for genome editing is currently higher than the rate of genome editing, which led to the develop ment of new methods of genetic engineering. This process was particularly pronounced for microorgan isms. Modern tasks require tens, hundreds, sometimes even thousands of genome modifications, which made researchers to look for new techniques. As a result, the instruments used for more complex objects, such as animals, plants, and cell lines, were adapted for yeasts. Modern methods for yeast genome editing allow introducing several modifications into the genome in a single step. In this study, we review the methods of directed genome editing and their applications and perspectives for yeasts.Key words: yeast; Saccharomyces cerevisiae; genetic engineering; ZFN; zinc fingers; TALENS; CRISPR/Cas; Argonaut; NgAgo.Дрожжи являются модельным эукариотическим организмом, на котором отрабатываются многие предположения о работе генома, а также методы его редактирования. Наиболее часто в исследо вательских работах используют Saccharomyces cerevisiae, которые очень хорошо приспособлены физиологически к культивированию в условиях биореактора и признаны абсолютно безопасными. В по следнее десятилетие методы генетической инженерии дрожжей претерпели значительные изменения. Появились новые инстру менты, которые пришли из смежных направлений и позволили значительно ускорить процесс получения новых штаммов. Прежде всего это белки для направленного внесения изменений в после довательность ДНК. Длительное время методы редактирования генома дрожжей базировались на использовании их собственной системы гомологичной рекомбинации. Она удобна и удовлетво ряла потребности исследователей на протяжении нескольких де сятилетий, до того времени, когда на первый план стали выходить высокопроизводительные методы. Во втором десятилетии XXI века произошло бурное развитие высокопроизводительных подходов, в первую очередь методов анализа в биологии: геномики, транс крипто...

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A highly efficient single-step, markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae

Cited by 178 publications

References 38 publications

Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system

Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

Methods of yeast genome editing

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