Skip to main content
ARS Home » Northeast Area » Kearneysville, West Virginia » Appalachian Fruit Research Laboratory » Innovative Fruit Production, Improvement, and Protection » Research » Publications at this Location » Publication #408772
Research Project: Superior Fruit Tree Cultivars for Orchard Resilience, Sustainability, and Consumer Appeal

Location: Innovative Fruit Production, Improvement, and Protection

Title: Apple CRISPR-Cas9 – a recipe for successful targeting of AGAMOUS genes in domestic apple

Author
item JACOBSON, SETH - University Of Colorado
item BONDARCHUK, NATALIE - University Of Colorado
item NGUYEN, ANH - University Of Colorado
item CANADA, ALLISON - University Of Colorado
item Artlip, Timothy - Tim
item Welser, Philipp
item KLOCKO, AMY - University Of Colorado

Submitted to: Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/24/2023
Publication Date: 10/26/2023
Citation: Jacobson, S., Bondarchuk, N., Nguyen, A., Canada, A., Artlip, T.S., Welser, P.J., Klocko, A. 2023. Apple CRISPR-Cas9 – a recipe for successful targeting of AGAMOUS genes in domestic apple. Plants. 12(21):3693. https://doi.org/10.3390/plants12213693.
DOI: https://doi.org/10.3390/plants12213693

Interpretive Summary: Fruit trees may require decades of conventional breeding efforts to introduce a successful new variety. Both conventional breeding and biotechnology approaches are being utilized towards the goal of advanced crop cultivars, including fruit trees. A relatively new and promising biotechnology tool is genome editing. A few genome editing efforts in apple have been attempted, but focused on disrupting a single gene. We devised a CRISPR-Cas9 genome editing construct to target two highly-related genes involved in floral development. A main goal was to determine the effectiveness of this approach for achieving target gene changes. We analyzed the resulting stably-edited apple trees and determined that a high rate of successful gene alterations occurred in both genes. Future work will include monitoring of floral development and floral form. This work will add to the knowledge the targeting efficiency of editing two genes simultaneously in a tree fruit crop and could be applied to reduce the invasive potential of flowering ornamental fruiting species such as Pyrus calleryana (Bradford pear), a tree initially popular as an ornamental planting and source of breeding for fire-blight resistance, but now recognized as an invasive species in several states.

Technical Abstract: Fruit trees and other fruiting hardwood perennials are economically valuable and there is interest in developing improved varieties. Both conventional breeding and biotechnology approaches are being utilized towards the goal of advanced cultivars. Increased knowledge of the effec-tiveness and efficiency of biotechnology approaches can help guide use of this technology. Here, we examined CRISPR-Cas9 directed genome editing in the valuable commodity fruit tree Malus x domestica (domestic apple). We transformed two cultivars with dual CRISPR-Cas9 constructs designed to target two AGAMOUS-like genes simultaneously. A main goal was to determine the effectiveness of this approach for achieving target gene changes. We obtained 6 Cas9 and 38 independent CRISPR-Cas9 events for analysis. Of the 38 CRISPR-Cas9 events, 35 (92%) had gene edits, and 13 (34%) showed changes to all alleles of both target genes. The most common change was large deletions, which were present in 59% of all changed alleles, followed by small deletions (21%), small insertions (12%), and a combination of small insertions and deletions (7%). Overall, a high rate of successful gene alterations were found. Many of these changes are predicted to cause frameshifts and alterations to the predicted peptides. Future work will include monitoring of floral development and floral form.