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Updated: Jan 28, 2026

Substrate Generation for Endonucleases of CRISPR/Cas Systems
Published on: September 8, 2012
Delta Integration CRISPR-Cas (Di-CRISPR) in Saccharomyces cerevisiae
Shuobo Shi1, Youyun Liang1, Ee Lui Ang1
1Metabolic Engineering Research Laboratory, Science and Engineering Institutes, Agency for Science, Technology and Research, Singapore, Singapore.
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).
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