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Simple-to-use CRISPR-SpCas9/SaCas9/AsCas12a vector series for genome editing in Saccharomyces cerevisiae

Satoshi Okada, Goro Doi, Shitomi Nakagawa, Emiko Kusumoto, Takashi Ito
doi: https://doi.org/10.1101/2021.05.07.443192
Satoshi Okada
Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
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  • For correspondence: stsokada@med.kyushu-u.ac.jp tito@med.kyushu-u.ac.jp
Goro Doi
Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
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Shitomi Nakagawa
Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
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Emiko Kusumoto
Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
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Takashi Ito
Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
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  • For correspondence: stsokada@med.kyushu-u.ac.jp tito@med.kyushu-u.ac.jp
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Abstract

Genome editing using the CRISPR/Cas system has been implemented for various organisms and becomes increasingly popular even in the genetically tractable budding yeast Saccharomyces cerevisiae. Since each CRISPR/Cas system recognizes only the sequences flanked by its unique protospacer adjacent motif (PAM), a certain single system often fails to target a region of interest due to the lack of PAM, thus necessitating the use of another system with a different PAM. Three CRISPR/Cas systems with distinct PAMs, namely SpCas9, SaCas9, and AsCas12a, have been successfully used in yeast genome editing and their combined use should expand the repertoire of editable targets. However, currently available plasmids for these systems were individually developed under different design principles, thus hampering their seamless use in the practice of genome editing. Here we report a series of Golden Gate Assembly-compatible backbone vectors designed under a unified principle to exploit the three CRISPR/Cas systems in yeast genome editing. We also created a software to assist the design of genome-editing plasmids for individual target sequences using the backbone vectors. Genome editing with these plasmids demonstrated practically sufficient efficiency in both insertion of gene fragments to essential genes and complete deletion of an open reading frame. The backbone vectors with the software would thus provide a versatile toolbox to facilitate the seamless use of SpCas9, SaCas9, and AsCas12a in various types of genome manipulation, especially those that are difficult to perform with conventional techniques in yeast genetics.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • https://github.com/poccopen/Genome_editing_plasmid_for_budding_yeast

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted May 08, 2021.
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Simple-to-use CRISPR-SpCas9/SaCas9/AsCas12a vector series for genome editing in Saccharomyces cerevisiae
Satoshi Okada, Goro Doi, Shitomi Nakagawa, Emiko Kusumoto, Takashi Ito
bioRxiv 2021.05.07.443192; doi: https://doi.org/10.1101/2021.05.07.443192
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Simple-to-use CRISPR-SpCas9/SaCas9/AsCas12a vector series for genome editing in Saccharomyces cerevisiae
Satoshi Okada, Goro Doi, Shitomi Nakagawa, Emiko Kusumoto, Takashi Ito
bioRxiv 2021.05.07.443192; doi: https://doi.org/10.1101/2021.05.07.443192

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