Construction of Vectors for the Genome Editing of Saccharomyces Yeast Using CRISPR-Cas9 System

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Abstract

New vectors for the yeast genome editing using CRISPR/Cas9 were constructed. A system for cloning of new targets using the standard methods (PCR‒restriction‒ligation) was developed and successfully applied. The constructed vectors allowed us to obtain the sup35-25 mutants, deletion of the PSH1 gene and disruption of the NAM7 (UPF1). A convenient method for identifying plasmids with a new target was tested. A detailed description of the cloning technique used and selection of plasmids with the new targets is provided.

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About the authors

A. G. Matveenko

St Petersburg State University

Email: g.zhuravleva@spbu.ru
Russian Federation, St Petersburg, 199034

A. S. Mikhailichenko

St Petersburg State University

Email: g.zhuravleva@spbu.ru
Russian Federation, St Petersburg, 199034

G. A. Zhouravleva

St Petersburg State University; Laboratory of Amyloid Biology SPbU

Author for correspondence.
Email: g.zhuravleva@spbu.ru
Russian Federation, St Petersburg, 199034; St Petersburg, 199034

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2. Fig. 1. Creation of a convenient target cloning system into a vector for editing the Saccharomyces cerevisiae genome using CRISPR-Cas9. (a) — A diagram of the location of the sup35-25 mutation and targets S35(-25) A and S35(-25) B in the SUP35 gene. (b) — A map of the YEplac181GC9H-sgS35 plasmid(-25) B; created using the SnapGene Viewer program. (c) — A scheme for obtaining sup35-25 mutants. (d) — Colony growth of strain 74-D694 transformed by plasmids in these combinations. (e) is a scheme for cloning new targets into the YEplac181GC9H-sgS35(-25) B vector and selecting E. coli colonies carrying a correctly constructed new vector.

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