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DNA targeting by Clostridium cellulolyticum CRISPR-Cas9 Type II-C system

Fedorova, Iana ; Arseniev, Anatolii ; Selkova, Polina LU ; Pobegalov, Georgii ; Goryanin, Ignatiy ; Vasileva, Aleksandra ; Musharova, Olga ; Abramova, Marina ; Kazalov, Maksim and Zyubko, Tatyana , et al. (2020) In Nucleic Acids Research 48(4). p.2026-2034
Abstract

Type II CRISPR-Cas9 RNA-guided nucleases are widely used for genome engineering. Type II-A SpCas9 protein from Streptococcus pyogenes is the most investigated and highly used enzyme of its class. Nevertheless, it has some drawbacks, including a relatively big size, imperfect specificity and restriction to DNA targets flanked by an NGG PAM sequence. Cas9 orthologs from other bacterial species may provide a rich and largely untapped source of biochemical diversity, which can help to overcome the limitations of SpCas9. Here, we characterize CcCas9, a Type II-C CRISPR nuclease from Clostridium cellulolyticum H10. We show that CcCas9 is an active endonuclease of comparatively small size that recognizes a novel two-nucleotide PAM sequence.... (More)

Type II CRISPR-Cas9 RNA-guided nucleases are widely used for genome engineering. Type II-A SpCas9 protein from Streptococcus pyogenes is the most investigated and highly used enzyme of its class. Nevertheless, it has some drawbacks, including a relatively big size, imperfect specificity and restriction to DNA targets flanked by an NGG PAM sequence. Cas9 orthologs from other bacterial species may provide a rich and largely untapped source of biochemical diversity, which can help to overcome the limitations of SpCas9. Here, we characterize CcCas9, a Type II-C CRISPR nuclease from Clostridium cellulolyticum H10. We show that CcCas9 is an active endonuclease of comparatively small size that recognizes a novel two-nucleotide PAM sequence. The CcCas9 can potentially broaden the existing scope of biotechnological applications of Cas9 nucleases and may be particularly advantageous for genome editing of C. cellulolyticum H10, a bacterium considered to be a promising biofuel producer.

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publishing date
type
Contribution to journal
publication status
published
keywords
CRISPR-Associated Protein 9/chemistry, CRISPR-Cas Systems/genetics, Clostridium cellulolyticum/enzymology, Crystallography, X-Ray, DNA/chemistry, Gene Editing, Mutation, Nucleotide Motifs/genetics, RNA, Guide, CRISPR-Cas Systems/genetics, Streptococcus pyogenes/enzymology, Substrate Specificity
in
Nucleic Acids Research
volume
48
issue
4
pages
2026 - 2034
publisher
Oxford University Press
external identifiers
  • pmid:31943070
  • scopus:85081102381
ISSN
1362-4962
DOI
10.1093/nar/gkz1225
language
English
LU publication?
no
additional info
© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.
id
b397b040-4295-4f84-961f-3ffb45de868f
date added to LUP
2026-01-29 09:52:56
date last changed
2026-01-30 04:01:46
@article{b397b040-4295-4f84-961f-3ffb45de868f,
  abstract     = {{<p>Type II CRISPR-Cas9 RNA-guided nucleases are widely used for genome engineering. Type II-A SpCas9 protein from Streptococcus pyogenes is the most investigated and highly used enzyme of its class. Nevertheless, it has some drawbacks, including a relatively big size, imperfect specificity and restriction to DNA targets flanked by an NGG PAM sequence. Cas9 orthologs from other bacterial species may provide a rich and largely untapped source of biochemical diversity, which can help to overcome the limitations of SpCas9. Here, we characterize CcCas9, a Type II-C CRISPR nuclease from Clostridium cellulolyticum H10. We show that CcCas9 is an active endonuclease of comparatively small size that recognizes a novel two-nucleotide PAM sequence. The CcCas9 can potentially broaden the existing scope of biotechnological applications of Cas9 nucleases and may be particularly advantageous for genome editing of C. cellulolyticum H10, a bacterium considered to be a promising biofuel producer.</p>}},
  author       = {{Fedorova, Iana and Arseniev, Anatolii and Selkova, Polina and Pobegalov, Georgii and Goryanin, Ignatiy and Vasileva, Aleksandra and Musharova, Olga and Abramova, Marina and Kazalov, Maksim and Zyubko, Tatyana and Artamonova, Tatyana and Artamonova, Daria and Shmakov, Sergey and Khodorkovskii, Mikhail and Severinov, Konstantin}},
  issn         = {{1362-4962}},
  keywords     = {{CRISPR-Associated Protein 9/chemistry; CRISPR-Cas Systems/genetics; Clostridium cellulolyticum/enzymology; Crystallography, X-Ray; DNA/chemistry; Gene Editing; Mutation; Nucleotide Motifs/genetics; RNA, Guide, CRISPR-Cas Systems/genetics; Streptococcus pyogenes/enzymology; Substrate Specificity}},
  language     = {{eng}},
  month        = {{02}},
  number       = {{4}},
  pages        = {{2026--2034}},
  publisher    = {{Oxford University Press}},
  series       = {{Nucleic Acids Research}},
  title        = {{DNA targeting by Clostridium cellulolyticum CRISPR-Cas9 Type II-C system}},
  url          = {{http://dx.doi.org/10.1093/nar/gkz1225}},
  doi          = {{10.1093/nar/gkz1225}},
  volume       = {{48}},
  year         = {{2020}},
}