Efficient genome editing by CRISPR/Cas9 with a tRNA-sgRNA fusion in the methylotrophic yeast Ogataea polymorpha

Minori Numamoto, Hiromi Maekawa, Yoshinobu Kaneko

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

The methylotrophic yeast Ogataea polymorpha (syn. Hansenula polymorpha) is an attractive industrial non-conventional yeast showing high thermo-tolerance (up to 50°C) and xylose assimilation. However, genetic manipulation of O. polymorpha is often laborious and time-consuming because it has lower homologous recombination efficiency relative to Saccharomyces cerevisiae. To overcome this disadvantage, we applied the CRISPR/Cas9 system as a powerful genome editing tool in O. polymorpha. In this system, both single guide RNA (sgRNA) and endonuclease Cas9 were expressed by a single autonomously-replicable plasmid and the sgRNA portion could be easily changed by using PCR and In-Fusion cloning techniques. Because the mutation efficiency of the CRISPR/Cas9 system was relatively low when the sgRNA was expressed under the control of the OpSNR6 promoter, the tRNACUG gene was used for sgRNA expression. The editing efficiency of this system ranged from 17% to 71% of transformants in several target genes tested (ADE12, PHO1, PHO11, and PHO84). These findings indicate that genetic manipulation of O. polymorpha will be more convenient and accelerated by using this CRISPR/Cas9 system.

Original languageEnglish
Pages (from-to)487-492
Number of pages6
JournalJournal of Bioscience and Bioengineering
Volume124
Issue number5
DOIs
Publication statusPublished - Nov 1 2017
Externally publishedYes

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Guide RNA
Transfer RNA
RNA
Yeast
Fusion reactions
Genes
Yeasts
Xylose
Pichia
Cloning
Endonucleases
Homologous Recombination
Saccharomyces cerevisiae
Organism Cloning
Plasmids
Polymerase Chain Reaction
Mutation
Gene Editing

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Efficient genome editing by CRISPR/Cas9 with a tRNA-sgRNA fusion in the methylotrophic yeast Ogataea polymorpha. / Numamoto, Minori; Maekawa, Hiromi; Kaneko, Yoshinobu.

In: Journal of Bioscience and Bioengineering, Vol. 124, No. 5, 01.11.2017, p. 487-492.

Research output: Contribution to journalArticle

@article{536c5f556fb94dffa71b8d21e0ff82c0,
title = "Efficient genome editing by CRISPR/Cas9 with a tRNA-sgRNA fusion in the methylotrophic yeast Ogataea polymorpha",
abstract = "The methylotrophic yeast Ogataea polymorpha (syn. Hansenula polymorpha) is an attractive industrial non-conventional yeast showing high thermo-tolerance (up to 50°C) and xylose assimilation. However, genetic manipulation of O. polymorpha is often laborious and time-consuming because it has lower homologous recombination efficiency relative to Saccharomyces cerevisiae. To overcome this disadvantage, we applied the CRISPR/Cas9 system as a powerful genome editing tool in O. polymorpha. In this system, both single guide RNA (sgRNA) and endonuclease Cas9 were expressed by a single autonomously-replicable plasmid and the sgRNA portion could be easily changed by using PCR and In-Fusion cloning techniques. Because the mutation efficiency of the CRISPR/Cas9 system was relatively low when the sgRNA was expressed under the control of the OpSNR6 promoter, the tRNACUG gene was used for sgRNA expression. The editing efficiency of this system ranged from 17{\%} to 71{\%} of transformants in several target genes tested (ADE12, PHO1, PHO11, and PHO84). These findings indicate that genetic manipulation of O. polymorpha will be more convenient and accelerated by using this CRISPR/Cas9 system.",
author = "Minori Numamoto and Hiromi Maekawa and Yoshinobu Kaneko",
year = "2017",
month = "11",
day = "1",
doi = "10.1016/j.jbiosc.2017.06.001",
language = "English",
volume = "124",
pages = "487--492",
journal = "Journal of Bioscience and Bioengineering",
issn = "1389-1723",
publisher = "The Society for Biotechnology, Japan",
number = "5",

}

TY - JOUR

T1 - Efficient genome editing by CRISPR/Cas9 with a tRNA-sgRNA fusion in the methylotrophic yeast Ogataea polymorpha

AU - Numamoto, Minori

AU - Maekawa, Hiromi

AU - Kaneko, Yoshinobu

PY - 2017/11/1

Y1 - 2017/11/1

N2 - The methylotrophic yeast Ogataea polymorpha (syn. Hansenula polymorpha) is an attractive industrial non-conventional yeast showing high thermo-tolerance (up to 50°C) and xylose assimilation. However, genetic manipulation of O. polymorpha is often laborious and time-consuming because it has lower homologous recombination efficiency relative to Saccharomyces cerevisiae. To overcome this disadvantage, we applied the CRISPR/Cas9 system as a powerful genome editing tool in O. polymorpha. In this system, both single guide RNA (sgRNA) and endonuclease Cas9 were expressed by a single autonomously-replicable plasmid and the sgRNA portion could be easily changed by using PCR and In-Fusion cloning techniques. Because the mutation efficiency of the CRISPR/Cas9 system was relatively low when the sgRNA was expressed under the control of the OpSNR6 promoter, the tRNACUG gene was used for sgRNA expression. The editing efficiency of this system ranged from 17% to 71% of transformants in several target genes tested (ADE12, PHO1, PHO11, and PHO84). These findings indicate that genetic manipulation of O. polymorpha will be more convenient and accelerated by using this CRISPR/Cas9 system.

AB - The methylotrophic yeast Ogataea polymorpha (syn. Hansenula polymorpha) is an attractive industrial non-conventional yeast showing high thermo-tolerance (up to 50°C) and xylose assimilation. However, genetic manipulation of O. polymorpha is often laborious and time-consuming because it has lower homologous recombination efficiency relative to Saccharomyces cerevisiae. To overcome this disadvantage, we applied the CRISPR/Cas9 system as a powerful genome editing tool in O. polymorpha. In this system, both single guide RNA (sgRNA) and endonuclease Cas9 were expressed by a single autonomously-replicable plasmid and the sgRNA portion could be easily changed by using PCR and In-Fusion cloning techniques. Because the mutation efficiency of the CRISPR/Cas9 system was relatively low when the sgRNA was expressed under the control of the OpSNR6 promoter, the tRNACUG gene was used for sgRNA expression. The editing efficiency of this system ranged from 17% to 71% of transformants in several target genes tested (ADE12, PHO1, PHO11, and PHO84). These findings indicate that genetic manipulation of O. polymorpha will be more convenient and accelerated by using this CRISPR/Cas9 system.

UR - http://www.scopus.com/inward/record.url?scp=85021728831&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85021728831&partnerID=8YFLogxK

U2 - 10.1016/j.jbiosc.2017.06.001

DO - 10.1016/j.jbiosc.2017.06.001

M3 - Article

C2 - 28666889

AN - SCOPUS:85021728831

VL - 124

SP - 487

EP - 492

JO - Journal of Bioscience and Bioengineering

JF - Journal of Bioscience and Bioengineering

SN - 1389-1723

IS - 5

ER -