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Mb- and FnCpf1 nucleases are active in mammalian cells: activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants.
Tóth, Eszter; Czene, Bernadett C; Kulcsár, Péter I; Krausz, Sarah L; Tálas, András; Nyeste, Antal; Varga, Éva; Huszár, Krisztina; Weinhardt, Nóra; Ligeti, Zoltán; Borsy, Adrienn É; Fodor, Elfrieda; Welker, Ervin.
Afiliación
  • Tóth E; Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, H-1117, Hungary.
  • Czene BC; Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, H-1117, Hungary.
  • Kulcsár PI; Gene Design Kft., Szeged, H-6726, Hungary.
  • Krausz SL; Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, H-1117, Hungary.
  • Tálas A; Doctoral School of Multidisciplinary Medical Science, University of Szeged, Szeged, H-6726, Hungary.
  • Nyeste A; Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, H-1117, Hungary.
  • Varga É; School of Ph.D. Studies, Semmelweis University, Budapest, H-1085, Hungary.
  • Huszár K; Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, H-1117, Hungary.
  • Weinhardt N; School of Ph.D. Studies, Semmelweis University, Budapest, H-1085, Hungary.
  • Ligeti Z; Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, H-1117, Hungary.
  • Borsy AÉ; Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, H-1117, Hungary.
  • Fodor E; Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, H-1117, Hungary.
  • Welker E; Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, H-1117, Hungary.
Nucleic Acids Res ; 46(19): 10272-10285, 2018 11 02.
Article en En | MEDLINE | ID: mdl-30239882
ABSTRACT
Cpf1s, the RNA-guided nucleases of the class II clustered regularly interspaced short palindromic repeats system require a short motive called protospacer adjacent motif (PAM) to be present next to the targeted sequence for their activity. The TTTV PAM sequence of As- and LbCpf1 nucleases is relatively rare in the genome of higher eukaryotic organisms. Here, we show that two other Cpf1 nucleases, Fn- and MbCpf1, which have been reported to utilize a shorter, more frequently occurring PAM sequence (TTN) when tested in vitro, carry out efficient genome modification in mammalian cells. We found that all four Cpf1 nucleases showed similar activities and TTTV PAM preferences. Our approach also revealed that besides their activities their PAM preferences are also target dependent. To increase the number of the available targets for Fn- and MbCpf1 we generated their RVR and RR mutants with altered PAM specificity and compared them to the wild-type and analogous As- and LbCpf1 variants. The mutants gained new PAM specificities but retained their activity on targets with TTTV PAMs, redefining RR-Cpf1's PAM-specificities as TTYV/TCCV, respectively. These variants may become versatile substitutes for wild-type Cpf1s by providing an expanded range of targets for genome engineering applications.
Asunto(s)

Texto completo: 1 Banco de datos: MEDLINE Asunto principal: Endonucleasas / Proteínas Asociadas a CRISPR / Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas / Francisella / Moraxella Límite: Animals / Humans Idioma: En Revista: Nucleic Acids Res Año: 2018 Tipo del documento: Article País de afiliación: Hungria

Texto completo: 1 Banco de datos: MEDLINE Asunto principal: Endonucleasas / Proteínas Asociadas a CRISPR / Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas / Francisella / Moraxella Límite: Animals / Humans Idioma: En Revista: Nucleic Acids Res Año: 2018 Tipo del documento: Article País de afiliación: Hungria