Delta Integration CRISPR-Cas (Di-CRISPR) in Saccharomyces cerevisiae

Despite the advances made in genetic engineering of Saccharomyces cerevisiae, the multicopy genomic integration of large biochemical pathways remains a challenge. Here, we developed a Di-CRISPR (delta integration CRISPR-Cas) platform based on cleavage of the delta sites by Clustered Regularly Inters...

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Bibliographic Details
Published inMethods in molecular biology (Clifton, N.J.) Vol. 1927; p. 73
Main Authors Shi, Shuobo, Liang, Youyun, Ang, Ee Lui, Zhao, Huimin
Format Journal Article
LanguageEnglish
Published United States 01.01.2019
Subjects
CRISPR-Cas Systems
Flow Cytometry
Gene Order
Genetic Engineering
Genetic Loci
Genome, Fungal
Genomics - methods
Plasmids - genetics
Real-Time Polymerase Chain Reaction
Saccharomyces cerevisiae - genetics
Transformation, Genetic
CRISPR-Cas
Genome engineering
Synthetic biology
Saccharomyces cerevisiae
Delta integration
Genome integration
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Summary:Despite the advances made in genetic engineering of Saccharomyces cerevisiae, the multicopy genomic integration of large biochemical pathways remains a challenge. Here, we developed a Di-CRISPR (delta integration CRISPR-Cas) platform based on cleavage of the delta sites by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas) to enable unprecedented high-efficiency, multicopy, markerless integrations of large biochemical pathways into the S. cerevisiae genome. Detailed protocols are provided on the entire workflow which includes pDi-CRISPR plasmid and donor DNA construction, Di-CRISPR-mediated integration and analysis of integration efficiencies and copy numbers through flow cytometry and quantitative polymerase chain reaction (qPCR).
ISSN:1940-6029
DOI:10.1007/978-1-4939-9142-6_6