Cas9/gRNA-mediated genome editing of yeast mitochondria and
CRISPR
Cas9/gRNA
Chlamydomonas
Chloroplast
DNA replacement
Edit Plasmid
Genome editing
Mitochondria
Organelle
Yeast
Journal
PeerJ
ISSN: 2167-8359
Titre abrégé: PeerJ
Pays: United States
ID NLM: 101603425
Informations de publication
Date de publication:
2020
2020
Historique:
received:
16
10
2018
accepted:
05
12
2019
entrez:
15
1
2020
pubmed:
15
1
2020
medline:
15
1
2020
Statut:
epublish
Résumé
We present a new approach to edit both mitochondrial and chloroplast genomes. Organelles have been considered off-limits to CRISPR due to their impermeability to most RNA and DNA. This has prevented applications of Cas9/gRNA-mediated genome editing in organelles while the tool has been widely used for engineering of nuclear DNA in a number of organisms in the last several years. To overcome the hurdle, we designed a new approach to enable organelle genome editing. The plasmids, designated "Edit Plasmids," were constructed with two expression cassettes, one for the expression of Cas9, codon-optimized for each organelle, under promoters specific to each organelle, and the other cassette for the expression of guide RNAs under another set of promoters specific to each organelle. In addition, Edit Plasmids were designed to carry the donor DNA for integration between two double-strand break sites induced by Cas9/gRNAs. Each donor DNA was flanked by the regions homologous to both ends of the integration site that were short enough to minimize spontaneous recombination events. Furthermore, the donor DNA was so modified that it did not carry functional gRNA target sites, allowing the stability of the integrated DNA without being excised by further Cas9/gRNAs activity. Edit Plasmids were introduced into organelles through microprojectile transformation. We confirmed donor DNA insertion at the target sites facilitated by homologous recombination only in the presence of Cas9/gRNA activity in yeast mitochondria and
Identifiants
pubmed: 31934513
doi: 10.7717/peerj.8362
pii: 8362
pmc: PMC6951285
doi:
Types de publication
Journal Article
Langues
eng
Pagination
e8362Informations de copyright
© 2020 Yoo et al.
Déclaration de conflit d'intérêts
Hajime Sakai is CEO at Napigen. Byung-Chun Yoo, Narendra Yadav and Emil Orozco, Jr are employees at Napigen. All authors are co-inventors on patent application US2019/0136249 A1 “organelle genome modification using polynucleotide guided endonuclease,” submitted by Napigen. The authors declare that they have no other competing interests.
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