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

Methods in Molecular Biology
Shuobo ShiHuimin Zhao

Abstract

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).

Citations

Jun 15, 2019·Natural Product Reports·Jia Jia ZhangBradley S Moore
Jul 23, 2020·Frontiers in Bioengineering and Biotechnology·Wentao DingShuobo Shi

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