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dc.contributor.author
Bernabé-Orts, Joan Miquel
dc.contributor.author
Casas‐Rodrigo, Iván
dc.contributor.author
Minguet, Eugenio G.
dc.contributor.author
Landolfi, Viola
dc.contributor.author
Garcia-Carpintero, Victor
dc.contributor.author
Gianoglio, Silvia
dc.contributor.author
Vázquez‐Vilar, Marta
dc.contributor.author
Granell, Antonio
dc.contributor.author
Orzaez, Diego
dc.date.accessioned
2019-10-07T08:11:08Z
dc.date.available
2019-10-05T03:39:45Z
dc.date.available
2019-10-07T08:11:08Z
dc.date.issued
2019-10
dc.identifier.issn
1467-7644
dc.identifier.issn
1467-7652
dc.identifier.other
10.1111/pbi.13113
en_US
dc.identifier.uri
http://hdl.handle.net/20.500.11850/368516
dc.identifier.doi
10.3929/ethz-b-000368516
dc.description.abstract
The CRISPR/Cas12a editing system opens new possibilities for plant genome engineering. To obtain a comparative assessment of RNA‐guided endonuclease (RGEN) types in plants, we adapted the CRISPR/Cas12a system to the GoldenBraid (GB) modular cloning platform and compared the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described GB‐assembled Streptococcus pyogenes Cas9 (SpCas9) constructs in eight Nicotiana benthamiana loci using transient expression. All three nucleases showed drastic target‐dependent differences in efficiency, with LbCas12 producing higher mutagenesis rates in five of the eight loci assayed, as estimated with the T7E1 endonuclease assay. Attempts to engineer crRNA direct repeat (DR) had little effect improving on‐target efficiency for AsCas12a and resulted deleterious in the case of LbCas12a. To complete the assessment of Cas12a activity, we carried out genome editing experiments in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, we also resequenced Cas12a‐free segregating T2 lines to assess possible off‐target effects. Our results showed that the mutagenesis footprint of Cas12a is enriched in deletions of −10 to −2 nucleotides and included in some instances complex rearrangements in the surroundings of the target sites. We found no evidence of off‐target mutations neither in related sequences nor somewhere else in the genome. Collectively, this study shows that LbCas12a is a viable alternative to SpCas9 for plant genome engineering.
en_US
dc.format
application/pdf
en_US
dc.language.iso
en
en_US
dc.publisher
Wiley
en_US
dc.rights.uri
http://creativecommons.org/licenses/by-nc/4.0/
dc.subject
CRISPR
en_US
dc.subject
Cas9
en_US
dc.subject
Cas12a
en_US
dc.subject
GoldenBraid
en_US
dc.subject
Plant gene editing
en_US
dc.subject
Off-target
en_US
dc.title
Assessment of Cas12a‐mediated gene editing efficiency in plants
en_US
dc.type
Journal Article
dc.rights.license
Creative Commons Attribution-NonCommercial 4.0 International
dc.date.published
2019-04-05
ethz.journal.title
Plant biotechnology journal
ethz.journal.volume
17
en_US
ethz.journal.issue
10
en_US
ethz.journal.abbreviated
Plant biotechnol. j. (Print)
ethz.pages.start
1971
en_US
ethz.pages.end
1984
en_US
ethz.version.deposit
publishedVersion
en_US
ethz.identifier.wos
ethz.identifier.scopus
ethz.publication.place
Oxford
en_US
ethz.publication.status
published
en_US
ethz.date.deposited
2019-10-05T03:39:49Z
ethz.source
WOS
ethz.eth
yes
en_US
ethz.availability
Open access
en_US
ethz.rosetta.installDate
2019-10-07T08:11:24Z
ethz.rosetta.lastUpdated
2020-02-15T21:54:03Z
ethz.rosetta.versionExported
true
ethz.COinS
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