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Cpf1 nucleases demonstrate robust activity to induce DNA modification by exploiting homology directed repair pathways in mammalian cells

Tóth, Eszter and Weinhardt, Nóra and Bencsura, Petra and Huszár, Krisztina and Kulcsár, Péter István and Ayaydin-Fodor, Elfrieda and Welker, Ervin (2016) Cpf1 nucleases demonstrate robust activity to induce DNA modification by exploiting homology directed repair pathways in mammalian cells. Biology Direct, 11. pp. 1-14. ISSN 1745-6150

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Abstract

Background: Cpf1 nucleases have recently been repurposed for site-specific genome modification. Two members of the Cpf1 family, the AsCpf1 from Acidaminococcus sp. and the LbCpf1 from Lachnospiraceae bacterium were shown to induce higher indel frequencies than SpCas9 when examining four randomly-selected target sequences for each type of nuclease. Whether they are a real match for Cas9 nucleases, however, remains to be verified. Results: Here, we used AsCpf1 and LbCpf1 to induce homology directed repair, either single strand annealing (SSA) or homologous recombination (HR), in N2a mouse neuroblastoma cells. Exploiting a plasmid that contains two GFP halves with overlapping sequences and exploring 20 targets, on all but one both nucleases consistently performed with above 10 % efficiency. Several Cas9 nucleases have been previously characterised in order to find an orthogonal counterpart for the most widely used promiscuous SpCas9. Here, we found that AsCpf1 and LbCpf1 might be better candidates than three of the best such counterparts: Cas9 from Staphylococcus aureus, from Streptococcus thermophilus and from Neisseria meningitidis, when assessed for inducing efficient SSA mediated repair in N2a cells. When tested on genomic targets exploiting HR, both nucleases were able to induce the integration of a donor cassette with 1000 bp-long homologous arms. We also generated plasmids that express these Cpf1 nucleases together with their cognate crRNAs and that are equipped with type IIS restriction enzyme sites to facilitate spacer cloning. Conclusions: Our results suggest that employing As-or LbCpf1 nuclease to induce homology directed repair in N2a cells, although is less effective at present than employing SpCas9, it is an equally or more effective tool than the most frequently used orthogonal Cas9 counterparts of SpCas9. These findings support the position of Cpf1 nucleases on the side of SpCas9 on the palette of effective genome engineering tools.

Item Type: Article
Uncontrolled Keywords: PROTEINS; EVOLUTION; MECHANISM; RECOGNITION; SPECIFICITIES; endonuclease; GENE-REGULATION; ADAPTIVE IMMUNITY; CRISPR-CAS SYSTEMS; DNA; RNA; genome engineering; StCas9; NmCas9; SaCas9; SpCas9; GFxFP assay; Cas9 nuclease; CRISPR; LbCpf1; AsCpf1; Cpf1 nuclease
Subjects: Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia > QH3011 Biochemistry / biokémia
Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia > QH3020 Biophysics / biofizika
SWORD Depositor: MTMT SWORD
Depositing User: MTMT SWORD
Date Deposited: 17 Oct 2016 09:52
Last Modified: 17 Oct 2016 09:52
URI: http://real.mtak.hu/id/eprint/41866

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