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

Iana Fedorova, Anatolii Arseniev, Polina Selkova, Georgii Pobegalov, Ignatiy Goryanin, Aleksandra Vasileva, Olga Musharova, Marina Abramova, Maksim Kazalov, Tatyana Zyubko, Tatyana Artamonova, Daria Artamonova, Sergey Shmakov, Mikhail Khodorkovskii, Konstantin Severinov

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)2026-2034
Number of pages9
JournalNucleic acids research
Issue number4
StatePublished - Feb 28 2020
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Genetics


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