Delta integration CRISPR-Cas (Di-CRISPR) in saccharomyces cerevisiae

Shuobo Shi, Youyun Liang, Ee Lui Ang, Huimin Zhao

Research output: Chapter in Book/Report/Conference proceedingChapter

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).

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages73-91
Number of pages19
DOIs
StatePublished - Jan 1 2019

Publication series

NameMethods in Molecular Biology
Volume1927
ISSN (Print)1064-3745

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Keywords

  • CRISPR-Cas
  • Delta integration
  • Genome engineering
  • Genome integration
  • Saccharomyces cerevisiae
  • Synthetic biology

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Shi, S., Liang, Y., Ang, E. L., & Zhao, H. (2019). Delta integration CRISPR-Cas (Di-CRISPR) in saccharomyces cerevisiae. In Methods in Molecular Biology (pp. 73-91). (Methods in Molecular Biology; Vol. 1927). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-9142-6_6