PMID- 30950179
OWN - NLM
STAT- MEDLINE
DCOM- 20200304
LR  - 20231213
IS  - 1467-7652 (Electronic)
IS  - 1467-7644 (Print)
IS  - 1467-7644 (Linking)
VI  - 17
IP  - 10
DP  - 2019 Oct
TI  - Assessment of Cas12a-mediated gene editing efficiency in plants.
PG  - 1971-1984
LID - 10.1111/pbi.13113 [doi]
AB  - 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.
CI  - (c) 2019 The Authors. Plant Biotechnology Journal published by Society for 
      Experimental Biology and The Association of Applied Biologists and John Wiley & 
      Sons Ltd.
FAU - Bernabe-Orts, Joan Miquel
AU  - Bernabe-Orts JM
AUID- ORCID: 0000-0001-9407-2678
AD  - Instituto de Biologia Molecular y Celular de Plantas (IBMCP), Consejo Superior de 
      Investigaciones Cientificas, Universidad Politecnica de Valencia, Valencia, 
      Spain.
FAU - Casas-Rodrigo, Ivan
AU  - Casas-Rodrigo I
AD  - Instituto de Biologia Molecular y Celular de Plantas (IBMCP), Consejo Superior de 
      Investigaciones Cientificas, Universidad Politecnica de Valencia, Valencia, 
      Spain.
FAU - Minguet, Eugenio G
AU  - Minguet EG
AUID- ORCID: 0000-0003-0568-4299
AD  - Instituto de Biologia Molecular y Celular de Plantas (IBMCP), Consejo Superior de 
      Investigaciones Cientificas, Universidad Politecnica de Valencia, Valencia, 
      Spain.
FAU - Landolfi, Viola
AU  - Landolfi V
AD  - Department of Agricultural, Forest and Food Sciences, University of Torino, 
      Grugliasco, Italy.
FAU - Garcia-Carpintero, Victor
AU  - Garcia-Carpintero V
AD  - Institute for the Conservation and Breeding of Agricultural Biodiversity 
      (COMAV-UPV), Universitat Politecnica de Valencia, Valencia, Spain.
FAU - Gianoglio, Silvia
AU  - Gianoglio S
AD  - Department of Agricultural, Forest and Food Sciences, University of Torino, 
      Grugliasco, Italy.
FAU - Vazquez-Vilar, Marta
AU  - Vazquez-Vilar M
AD  - Instituto de Biologia Molecular y Celular de Plantas (IBMCP), Consejo Superior de 
      Investigaciones Cientificas, Universidad Politecnica de Valencia, Valencia, 
      Spain.
FAU - Granell, Antonio
AU  - Granell A
AD  - Instituto de Biologia Molecular y Celular de Plantas (IBMCP), Consejo Superior de 
      Investigaciones Cientificas, Universidad Politecnica de Valencia, Valencia, 
      Spain.
FAU - Orzaez, Diego
AU  - Orzaez D
AD  - Instituto de Biologia Molecular y Celular de Plantas (IBMCP), Consejo Superior de 
      Investigaciones Cientificas, Universidad Politecnica de Valencia, Valencia, 
      Spain.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20190419
PL  - England
TA  - Plant Biotechnol J
JT  - Plant biotechnology journal
JID - 101201889
RN  - EC 3.1.- (Endonucleases)
SB  - IM
MH  - Arabidopsis/genetics
MH  - *CRISPR-Cas Systems
MH  - Endonucleases
MH  - *Gene Editing
MH  - *Genome, Plant
MH  - Solanum lycopersicum/genetics
MH  - Mutagenesis
MH  - Sequence Deletion
MH  - Nicotiana/genetics
PMC - PMC6737022
OTO - NOTNLM
OT  - CRISPR/Cas12a
OT  - CRISPR/Cas9
OT  - GoldenBraid
OT  - off-target
OT  - plant gene editing
COIS- The authors declare no conflict of interest.
EDAT- 2019/04/06 06:00
MHDA- 2020/03/05 06:00
PMCR- 2019/09/11
CRDT- 2019/04/06 06:00
PHST- 2018/02/06 00:00 [received]
PHST- 2019/02/19 00:00 [revised]
PHST- 2019/03/20 00:00 [accepted]
PHST- 2019/04/06 06:00 [pubmed]
PHST- 2020/03/05 06:00 [medline]
PHST- 2019/04/06 06:00 [entrez]
PHST- 2019/09/11 00:00 [pmc-release]
AID - PBI13113 [pii]
AID - 10.1111/pbi.13113 [doi]
PST - ppublish
SO  - Plant Biotechnol J. 2019 Oct;17(10):1971-1984. doi: 10.1111/pbi.13113. Epub 2019 
      Apr 19.