Gene insertion in Saccharomyces cerevisiae using the CRISPR/Cas9 system

Guo, Xuan; Wang, Yuehua; Wu, Meixiao; Hu, Jianbing; Wang, Xuefei; Yu, Minli; Tang, Hui

doi:10.1007/s13205-021-02648-4

Cited by 1 publication

(1 citation statement)

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“…In S. cerevisiae , 25–60 base pairs of homologous sequences are sufficient for chromosomal integration [ 62 ]. Other relevant studies showed that a homology arm of 50 base pairs was sufficient for genomic integration into S. cerevisiae [ 63 , 64 ]. Studies reported that the minimal homology arm for efficient, targeted chromosomal integration of GFP was 24 base pairs in yeast strain PR109 where the integration efficiency 15% with dsDNA [ 65 ].…”

Section: Discussionmentioning

confidence: 99%

Easy efficient HDR-based targeted knock-in in Saccharomyces cerevisiae genome using CRISPR-Cas9 system

et al. 2022

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During the last two decades, yeast has been used as a biological tool to produce various small molecules, biofuels, etc., using an inexpensive bioprocess. The application of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein (Cas) techniques in yeast genetic and metabolic engineering has made a paradigm shift, particularly with a significant improvement in targeted chromosomal integration using synthetic donor constructs, which was previously a challenge. This study reports the CRISPR-Cas9-based highly efficient strategy for targeted chromosomal integration and in-frame expression of a foreign gene in the genome of Saccharomyces cerevisiae ( S. cerevisiae ) by homology-dependent recombination (HDR); our optimized methods show that CRISPR-Cas9-based chromosomal targeted integration of small constructs at multiple target sites of the yeast genome can be achieved with an efficiency of 74%. Our study also suggests that 15 bp microhomology flanked arms are sufficient for 50% targeted knock-in at minimal knock-in construct concentration. Whole-genome sequencing confirmed that there is no off-target effect. This study provides a comprehensive and streamlined protocol that will support the targeted integration of essential genes into the yeast genome for synthetic biology and other industrial purposes. Highlights • CRISPR-Cas9 based in-frame expression of foreign protein in Saccharomyces cerevisiae using Homology arm without a promoter. • As low as 15 base pairs of microhomology (HDR) are sufficient for targeted integration in Saccharomyces cerevisiae . • The methodology is highly efficient and very specific as no off-targeted effects were shown by the whole-genome sequence.

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