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Mb- and FnCpf1 nucleases are active in mammalian cells

Tóth, Eszter and Czene, Bernadett C and Kulcsár, Péter István and Krausz, Sarah and Tálas, András and Nyeste, Antal and Varga, Éva and Huszár, Krisztina and Weinhardt, Nóra and Ligeti, Zoltán and Borsy, Adrienn Éva and Ayaydin-Fodor, Elfrieda and Welker, Ervin (2018) Mb- and FnCpf1 nucleases are active in mammalian cells. NUCLEIC ACIDS RESEARCH, 46 (19). pp. 10272-10285. ISSN 0305-1048

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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.

Item Type: Article
Additional Information: Funding Agency and Grant Number: National Research, Development and Innovation Office [PD 125331, K_128188, VKFI-16-1-2016-0240]; Ministry of National Economy [GINOP 1.2.2.-15-2015-01588]; GINOP [1.2.2.-15 2015-01588] Funding text: National Research, Development and Innovation Office [PD 125331 to E.T., K_128188, VKFI-16-1-2016-0240]; Ministry of National Economy [GINOP 1.2.2.-15-2015-01588]. Funding for open access charge: GINOP [1.2.2.-15 2015-01588].
Subjects: Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia > QH3011 Biochemistry / biokémia
Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia > QH3015 Molecular biology / molekuláris biológia
Q Science / természettudomány > QH Natural history / természetrajz > QH426 Genetics / genetika, örökléstan
SWORD Depositor: MTMT SWORD
Depositing User: MTMT SWORD
Date Deposited: 07 Mar 2019 08:03
Last Modified: 07 Mar 2019 08:03
URI: http://real.mtak.hu/id/eprint/91856

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