PMID- 29492697
OWN - NLM
STAT- MEDLINE
DCOM- 20180829
LR  - 20240104
IS  - 1432-2048 (Electronic)
IS  - 0032-0935 (Linking)
VI  - 247
IP  - 4
DP  - 2018 Apr
TI  - Gene editing by CRISPR/Cas9 in the obligatory outcrossing Medicago sativa.
PG  - 1043-1050
LID - 10.1007/s00425-018-2866-1 [doi]
AB  - The CRISPR/Cas9 technique was successfully used to edit the genome of the 
      obligatory outcrossing plant species Medicago sativa L. (alfalfa). RNA-guided 
      genome engineering using Clustered Regularly Interspersed Short Palindromic 
      Repeats (CRISPR)/Cas9 technology enables a variety of applications in plants. 
      Successful application and validation of the CRISPR technique in a multiplex 
      genome, such as that of M. sativa (alfalfa) will ultimately lead to major 
      advances in the improvement of this crop. We used CRISPR/Cas9 technique to mutate 
      squamosa promoter binding protein like 9 (SPL9) gene in alfalfa. Because of the 
      complex features of the alfalfa genome, we first used droplet digital PCR (ddPCR) 
      for high-throughput screening of large populations of CRISPR-modified plants. 
      Based on the results of genome editing rates obtained from the ddPCR screening, 
      plants with relatively high rates were subjected to further analysis by 
      restriction enzyme digestion/PCR amplification analyses. PCR products 
      encompassing the respective small guided RNA target locus were then sub-cloned 
      and sequenced to verify genome editing. In summary, we successfully applied the 
      CRISPR/Cas9 technique to edit the SPL9 gene in a multiplex genome, providing some 
      insights into opportunities to apply this technology in future alfalfa breeding. 
      The overall efficiency in the polyploid alfalfa genome was lower compared to 
      other less-complex plant genomes. Further refinement of the CRISPR technology 
      system will thus be required for more efficient genome editing in this plant.
FAU - Gao, Ruimin
AU  - Gao R
AD  - Agriculture and Agri-Food Canada, London, ON, Canada.
FAU - Feyissa, Biruk A
AU  - Feyissa BA
AD  - Agriculture and Agri-Food Canada, London, ON, Canada.
AD  - Department of Biology, University of Western Ontario, London, ON, Canada.
FAU - Croft, Mana
AU  - Croft M
AD  - Agriculture and Agri-Food Canada, London, ON, Canada.
FAU - Hannoufa, Abdelali
AU  - Hannoufa A
AUID- ORCID: 0000-0003-0064-620X
AD  - Agriculture and Agri-Food Canada, London, ON, Canada. 
      Abdelali.Hannoufa@agr.gc.ca.
AD  - Department of Biology, University of Western Ontario, London, ON, Canada. 
      Abdelali.Hannoufa@agr.gc.ca.
LA  - eng
GR  - J-000260/Agriculture and Agri-Food Canada/
PT  - Journal Article
DEP - 20180228
PL  - Germany
TA  - Planta
JT  - Planta
JID - 1250576
SB  - IM
MH  - *CRISPR-Cas Systems
MH  - Gene Editing/*methods
MH  - Genes, Plant/genetics
MH  - High-Throughput Screening Assays
MH  - Medicago sativa/*genetics
MH  - Plants, Genetically Modified/genetics
MH  - Polymerase Chain Reaction/methods
OTO - NOTNLM
OT  - Alfalfa
OT  - CRISPR/Cas9
OT  - Droplet digital PCR (ddPCR)
OT  - Gene editing
OT  - Multiplex mutagenesis
EDAT- 2018/03/02 06:00
MHDA- 2018/08/30 06:00
CRDT- 2018/03/02 06:00
PHST- 2017/11/17 00:00 [received]
PHST- 2018/02/21 00:00 [accepted]
PHST- 2018/03/02 06:00 [pubmed]
PHST- 2018/08/30 06:00 [medline]
PHST- 2018/03/02 06:00 [entrez]
AID - 10.1007/s00425-018-2866-1 [pii]
AID - 10.1007/s00425-018-2866-1 [doi]
PST - ppublish
SO  - Planta. 2018 Apr;247(4):1043-1050. doi: 10.1007/s00425-018-2866-1. Epub 2018 Feb 
      28.