Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST

Yaojun Tong, Christopher M. Whitford, Helene L. Robertsen, Kai Blin, Tue S. Jørgensen, Andreas K. Klitgaard, Tetiana Gren, Xinglin Jiang, Tilmann Weber*, Sang Yup Lee

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

Streptomycetes serve as major producers of various pharmacologically and industrially important natural products. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSBs) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide–resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem), which comprises a cytidine (CRISPR-cBEST) and an adenosine (CRISPR-aBEST) deaminase-based base editor. Specifically targeted by an sgRNA, CRISPR-cBEST can efficiently convert a C:G base pair to a T:A base pair and CRISPR-aBEST can convert an A:T base pair to a G:C base pair within a window of approximately 7 and 6 nucleotides, respectively. CRISPR-BEST was validated and successfully used in different Streptomyces species. Particularly in nonmodel actinomycete Streptomyces collinus Tü365, CRISPR-cBEST efficiently inactivated the 2 copies of kirN gene that are in the duplicated kirromycin biosynthetic pathways simultaneously by STOP codon introduction. Generating such a knockout mutant repeatedly failed using the conventional DSB-based CRISPR-Cas9. An unbiased, genome-wide off-target evaluation indicates the high fidelity and applicability of CRISPR-BEST. Furthermore, the system supports multiplexed editing with a single plasmid by providing a Csy4-based sgRNA processing machinery. To simplify the protospacer identification process, we also updated the CRISPy-web (https://crispy.secondarymetabolites.org), and now it allows designing sgRNAs specifically for CRISPR-BEST applications.

Original languageEnglish
JournalProceedings of the National Academy of Sciences of the United States of America
Volume116
Issue number41
Pages (from-to)20366-20375
ISSN0027-8424
DOIs
Publication statusPublished - 2019

Keywords

  • Adenosine deaminase
  • CRISPR base editor
  • Cytidine deaminase
  • Genome editing
  • Streptomycetes

Cite this

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title = "Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST",
abstract = "Streptomycetes serve as major producers of various pharmacologically and industrially important natural products. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSBs) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide–resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem), which comprises a cytidine (CRISPR-cBEST) and an adenosine (CRISPR-aBEST) deaminase-based base editor. Specifically targeted by an sgRNA, CRISPR-cBEST can efficiently convert a C:G base pair to a T:A base pair and CRISPR-aBEST can convert an A:T base pair to a G:C base pair within a window of approximately 7 and 6 nucleotides, respectively. CRISPR-BEST was validated and successfully used in different Streptomyces species. Particularly in nonmodel actinomycete Streptomyces collinus T{\"u}365, CRISPR-cBEST efficiently inactivated the 2 copies of kirN gene that are in the duplicated kirromycin biosynthetic pathways simultaneously by STOP codon introduction. Generating such a knockout mutant repeatedly failed using the conventional DSB-based CRISPR-Cas9. An unbiased, genome-wide off-target evaluation indicates the high fidelity and applicability of CRISPR-BEST. Furthermore, the system supports multiplexed editing with a single plasmid by providing a Csy4-based sgRNA processing machinery. To simplify the protospacer identification process, we also updated the CRISPy-web (https://crispy.secondarymetabolites.org), and now it allows designing sgRNAs specifically for CRISPR-BEST applications.",
keywords = "Adenosine deaminase, CRISPR base editor, Cytidine deaminase, Genome editing, Streptomycetes",
author = "Yaojun Tong and Whitford, {Christopher M.} and Robertsen, {Helene L.} and Kai Blin and J{\o}rgensen, {Tue S.} and Klitgaard, {Andreas K.} and Tetiana Gren and Xinglin Jiang and Tilmann Weber and Lee, {Sang Yup}",
year = "2019",
doi = "10.1073/pnas.1913493116",
language = "English",
volume = "116",
pages = "20366--20375",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
publisher = "The National Academy of Sciences of the United States of America",
number = "41",

}

Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST. / Tong, Yaojun; Whitford, Christopher M.; Robertsen, Helene L.; Blin, Kai; Jørgensen, Tue S.; Klitgaard, Andreas K.; Gren, Tetiana; Jiang, Xinglin; Weber, Tilmann; Lee, Sang Yup.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 116, No. 41, 2019, p. 20366-20375.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST

AU - Tong, Yaojun

AU - Whitford, Christopher M.

AU - Robertsen, Helene L.

AU - Blin, Kai

AU - Jørgensen, Tue S.

AU - Klitgaard, Andreas K.

AU - Gren, Tetiana

AU - Jiang, Xinglin

AU - Weber, Tilmann

AU - Lee, Sang Yup

PY - 2019

Y1 - 2019

N2 - Streptomycetes serve as major producers of various pharmacologically and industrially important natural products. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSBs) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide–resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem), which comprises a cytidine (CRISPR-cBEST) and an adenosine (CRISPR-aBEST) deaminase-based base editor. Specifically targeted by an sgRNA, CRISPR-cBEST can efficiently convert a C:G base pair to a T:A base pair and CRISPR-aBEST can convert an A:T base pair to a G:C base pair within a window of approximately 7 and 6 nucleotides, respectively. CRISPR-BEST was validated and successfully used in different Streptomyces species. Particularly in nonmodel actinomycete Streptomyces collinus Tü365, CRISPR-cBEST efficiently inactivated the 2 copies of kirN gene that are in the duplicated kirromycin biosynthetic pathways simultaneously by STOP codon introduction. Generating such a knockout mutant repeatedly failed using the conventional DSB-based CRISPR-Cas9. An unbiased, genome-wide off-target evaluation indicates the high fidelity and applicability of CRISPR-BEST. Furthermore, the system supports multiplexed editing with a single plasmid by providing a Csy4-based sgRNA processing machinery. To simplify the protospacer identification process, we also updated the CRISPy-web (https://crispy.secondarymetabolites.org), and now it allows designing sgRNAs specifically for CRISPR-BEST applications.

AB - Streptomycetes serve as major producers of various pharmacologically and industrially important natural products. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSBs) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide–resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem), which comprises a cytidine (CRISPR-cBEST) and an adenosine (CRISPR-aBEST) deaminase-based base editor. Specifically targeted by an sgRNA, CRISPR-cBEST can efficiently convert a C:G base pair to a T:A base pair and CRISPR-aBEST can convert an A:T base pair to a G:C base pair within a window of approximately 7 and 6 nucleotides, respectively. CRISPR-BEST was validated and successfully used in different Streptomyces species. Particularly in nonmodel actinomycete Streptomyces collinus Tü365, CRISPR-cBEST efficiently inactivated the 2 copies of kirN gene that are in the duplicated kirromycin biosynthetic pathways simultaneously by STOP codon introduction. Generating such a knockout mutant repeatedly failed using the conventional DSB-based CRISPR-Cas9. An unbiased, genome-wide off-target evaluation indicates the high fidelity and applicability of CRISPR-BEST. Furthermore, the system supports multiplexed editing with a single plasmid by providing a Csy4-based sgRNA processing machinery. To simplify the protospacer identification process, we also updated the CRISPy-web (https://crispy.secondarymetabolites.org), and now it allows designing sgRNAs specifically for CRISPR-BEST applications.

KW - Adenosine deaminase

KW - CRISPR base editor

KW - Cytidine deaminase

KW - Genome editing

KW - Streptomycetes

U2 - 10.1073/pnas.1913493116

DO - 10.1073/pnas.1913493116

M3 - Journal article

VL - 116

SP - 20366

EP - 20375

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 41

ER -