Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been used for genome editing in various fruit trees, including apple (Malus x domestica). In previous studies, transfer DNA (T-DNA) expressing genome editing tools, Streptococcus pyogenes Cas9 (SpCas9) and single guide RNA (sgRNA), was stably integrated into the apple genome via Agrobacterium-mediated transformation. However, due to self-incompatibility, long generation period, and the high heterozygosity of apple, removing only the integrated T-DNA from the apple genome by crossbreeding while maintaining the introduced varietal trait is difficult. Therefore, an efficient SpCas9-sgRNA delivery system without transgene insertion is required for genome editing of apple. In this study, we used geminivirus-derived replicons (GVRs) for the transient expression of genome editing tools. Small DNA vectors were deconstructed by splitting the elements necessary for the production of GVRs from bean yellow dwarf virus into two vectors. Production of GVRs using these vectors was demonstrated in Arabidopsis and apple cells. Genome editing was improved by using the GVR-producing vectors with genome editing tools in Arabidopsis protoplasts. The use of the GVR-producing vectors for SpCas9 and sgRNA delivery into apple leaves improved the expression levels of SpCas9 and sgRNA, enabling the detection of targeted mutations introduced in the endogenous apple genome. These findings demonstrate the utility of GVRs in genome editing via transient gene expression in apple. It can be expected that our GVR-based genome editing technology has potential utility for transgene-free genome editing in apple.
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Claims 4
Claim · model-written summary
Geminivirus-derived replicons were produced from the split vectors in apple cells.
- Taxon
- Malus x domestica
Malus domesticae0 - Outcome
- Production of GVRse2
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“apple cultivar”
e0“including apple (Malus x domestica)”
e2“Production of GVRs using these vectors was demonstrated in Arabidopsis and apple cells”
Claim · model-written summary
GVR-producing vectors improved genome editing in Arabidopsis protoplasts.
- Taxon
- Arabidopsis
Arabidopsise3 - Organ / stage
- protoplastse3
- Treatment
- GVR-producing vectors with genome editing toolse3
- Outcome
- Genome editinge3
- Direction
- improvede3
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“apple cultivar”
e3“Genome editing was improved by using the GVR-producing vectors with genome editing tools in Arabidopsis protoplasts”
Claim · model-written summary
GVR-producing vectors for SpCas9 and sgRNA delivery improved SpCas9 and sgRNA expression levels in apple leaves.
- Taxon
- Malus x domesticaMalus domesticae0
- Organ / stage
- leavese4
- Treatment
- GVR-producing vectors for SpCas9 and sgRNA deliverye4
- Gene
- SpCas9e4
- Outcome
- expression levels of SpCas9 and sgRNAe4
- Direction
- improvede4
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“apple cultivar”
e0“including apple (Malus x domestica)”
e4“The use of the GVR-producing vectors for SpCas9 and sgRNA delivery into apple leaves improved the expression levels of SpCas9 and sgRNA”
Claim · model-written summary
The GVR-based delivery enabled detection of targeted mutations in the endogenous apple genome.
- Taxon
- Malus x domesticaMalus domesticae0
- Outcome
- detection of targeted mutationse5
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“apple cultivar”
e0“including apple (Malus x domestica)”
e5“enabling the detection of targeted mutations introduced in the endogenous apple genome”
The same questions in other papers
loss of function · molecular: expression · Malus domestica (apple)Positive and negative results
3 papers in the same direction as this paper · 11 in the opposite direction
- negative
- negative
- negative
11 papers more in this group
See all 15 papers →Claims are grouped by organism, treatment and what was measured or targeted. A paper counts as positive when any of its claims in the group is positive.