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

Eszter Tóth, Bernadett C. Czene, Péter I. Kulcsár, Sarah L. Krausz, András Tálas, Antal Nyeste, Éva Varga, Krisztina Huszár, Nóra Weinhardt, Zoltán Ligeti, Adrienn Borsy, Elfrieda Fodor, Ervin Welker

Research output: Contribution to journalArticle

8 Citations (Scopus)

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. Themutants 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 wildtype Cpf1s by providing an expanded range of targets for genome engineering applications.

Original languageEnglish
Pages (from-to)10272-10285
Number of pages14
JournalNucleic acids research
Volume46
Issue number19
DOIs
Publication statusPublished - Jan 1 2018

    Fingerprint

ASJC Scopus subject areas

  • Genetics

Cite this

Tóth, E., Czene, B. C., Kulcsár, P. I., Krausz, S. L., Tálas, A., Nyeste, A., Varga, É., Huszár, K., Weinhardt, N., Ligeti, Z., Borsy, A., Fodor, E., & Welker, E. (2018). 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. Nucleic acids research, 46(19), 10272-10285. https://doi.org/10.1093/nar/gky815