CasPER: A CRISPR/Cas9-Based Method for Directed Evolution in Genomic Loci in Saccharomyces cerevisiae

Tadas Jakočiūnas, Michael K. Jensen*, Jay D. Keasling

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

Abstract

Here, in this chapter, we describe a detailed protocol for the method named Cas9-mediated protein evolution reaction or short CasPER. CasPER is based on the generation of large 300–600-bp mutagenized linear DNA fragments by error-prone PCR which are used as a donor for repair of double-strand break mediated by Cas9 and subsequently integrated to the genome. This method can be efficiently used for directed evolution of desired essential or nonessential genes in the genome and most importantly can be multiplexed. Altogether, the described method allows for heterogeneous DNA integration with successful transformation efficiencies of 98–100% for both single and multiplex targeting.
Original languageEnglish
Title of host publicationYeast Metabolic Engineering : Methods and Protocols
Number of pages15
Volume2513
PublisherSpringer
Publication date2022
Pages23-37
DOIs
Publication statusPublished - 2022
SeriesMethods in Molecular Biology
ISSN1064-3745

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