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

Eszter Tóth, Nóra Weinhardt, Petra Bencsura, Krisztina Huszár, Péter I. Kulcsár, András Tálas, E. Fodor, E. Welker

Research output: Contribution to journalArticle

25 Citations (Scopus)

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. Reviewers: This article was reviewed by Eugene Koonin, Haruhiko Siomi and Jean-Yves Masson.

Original languageEnglish
Article number46
JournalBiology Direct
Volume11
Issue number1
DOIs
Publication statusPublished - Sep 14 2016

Fingerprint

nucleases
homology
repair
Repair
Homology
Pathway
DNA
Homologous Recombination
Cells
annealing
plasmid
Annealing
Recombination
Acidaminococcus
recombination
Target
Cell
Genome
Plasmids
genome

Keywords

  • AsCpf1
  • Cas9 nuclease
  • Cpf1 nuclease
  • CRISPR
  • DNA
  • Genome engineering
  • GFxFP assay
  • LbCpf1
  • NmCas9
  • RNA
  • SaCas9
  • SpCas9
  • StCas9

ASJC Scopus subject areas

  • Immunology
  • Ecology, Evolution, Behavior and Systematics
  • Modelling and Simulation
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • Applied Mathematics

Cite this

Cpf1 nucleases demonstrate robust activity to induce DNA modification by exploiting homology directed repair pathways in mammalian cells. / Tóth, Eszter; Weinhardt, Nóra; Bencsura, Petra; Huszár, Krisztina; Kulcsár, Péter I.; Tálas, András; Fodor, E.; Welker, E.

In: Biology Direct, Vol. 11, No. 1, 46, 14.09.2016.

Research output: Contribution to journalArticle

Tóth, Eszter ; Weinhardt, Nóra ; Bencsura, Petra ; Huszár, Krisztina ; Kulcsár, Péter I. ; Tálas, András ; Fodor, E. ; Welker, E. / Cpf1 nucleases demonstrate robust activity to induce DNA modification by exploiting homology directed repair pathways in mammalian cells. In: Biology Direct. 2016 ; Vol. 11, No. 1.
@article{3a714831c83045a6be15cce97fa9a21f,
title = "Cpf1 nucleases demonstrate robust activity to induce DNA modification by exploiting homology directed repair pathways in mammalian cells",
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. Reviewers: This article was reviewed by Eugene Koonin, Haruhiko Siomi and Jean-Yves Masson.",
keywords = "AsCpf1, Cas9 nuclease, Cpf1 nuclease, CRISPR, DNA, Genome engineering, GFxFP assay, LbCpf1, NmCas9, RNA, SaCas9, SpCas9, StCas9",
author = "Eszter T{\'o}th and N{\'o}ra Weinhardt and Petra Bencsura and Krisztina Husz{\'a}r and Kulcs{\'a}r, {P{\'e}ter I.} and Andr{\'a}s T{\'a}las and E. Fodor and E. Welker",
year = "2016",
month = "9",
day = "14",
doi = "10.1186/s13062-016-0147-0",
language = "English",
volume = "11",
journal = "Biology Direct",
issn = "1745-6150",
publisher = "BioMed Central",
number = "1",

}

TY - JOUR

T1 - Cpf1 nucleases demonstrate robust activity to induce DNA modification by exploiting homology directed repair pathways in mammalian cells

AU - Tóth, Eszter

AU - Weinhardt, Nóra

AU - Bencsura, Petra

AU - Huszár, Krisztina

AU - Kulcsár, Péter I.

AU - Tálas, András

AU - Fodor, E.

AU - Welker, E.

PY - 2016/9/14

Y1 - 2016/9/14

N2 - 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. Reviewers: This article was reviewed by Eugene Koonin, Haruhiko Siomi and Jean-Yves Masson.

AB - 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. Reviewers: This article was reviewed by Eugene Koonin, Haruhiko Siomi and Jean-Yves Masson.

KW - AsCpf1

KW - Cas9 nuclease

KW - Cpf1 nuclease

KW - CRISPR

KW - DNA

KW - Genome engineering

KW - GFxFP assay

KW - LbCpf1

KW - NmCas9

KW - RNA

KW - SaCas9

KW - SpCas9

KW - StCas9

UR - http://www.scopus.com/inward/record.url?scp=85043549846&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85043549846&partnerID=8YFLogxK

U2 - 10.1186/s13062-016-0147-0

DO - 10.1186/s13062-016-0147-0

M3 - Article

VL - 11

JO - Biology Direct

JF - Biology Direct

SN - 1745-6150

IS - 1

M1 - 46

ER -