Workshop: Practical Considerations for Plant Genome Editing
Practical Considerations for Plant Genome Editing
Virtual event held March 6th, 2024
11:00AM-1:30PM ET/17:00-19:30 CET
ABOUT THE WORKSHOP
Recent advances in genome editing have greatly increased the accessibility of editing tools, but it can be difficult to successfully utilize CRISPR systems in plants. The aim of this workshop is to provide practical information and best practices for a successful CRISPR experiment. This event will be accessible to beginners and will help more experienced CRISPR users improve their workflow.
CERTIFICATE OF PARTICIPATION
https://docs.google.com/forms/d/e/1FAIpQLSfmrVDpfE4bIq7m1rwsc4eQGGheHglynKN3LIS8tEQNRltvpQ/viewform?usp=sf_link
VIDEOS OF THE EVENT
Introduction Christian Rogers & Bill Gordon Kamm
Getting Started With CRISPR/Cas9 Editing in Plants - Dr. Colby Starker, University of Minnesota
Download the slides:
CRISPR/Cas9 in Crops: Where to Start - Dr. Laurens Pauwels, VIB-UGent
Download the slides:
QUESTIONS AND ANSWERS FROM THE ZOOM CHAT
Below are conversations between workshop attendees. PlantGENE does not endorse or fact-check any comments made by attendees.
AC: Has there been any effective way to rapidly test sgRNA-Cas9 targeting efficiency in vivo? ZK: if you have a protoplast system going, this can be a quick check for gRNA efficiency in real plant chromosomes. FS: I prefer Hairy root for transient exp. |
AC: Question on RUBY, can you expand on why you are not using it? We are currently using RUBY as a visual marker and we have yet to generate mutants. JVE: We’ve seen the same with RUBY as Colby mentioned. Our high expressing tomato, don’t set fruit. Lower expressing ones do. RS: RUBY is a physiologically linked marker. As such, it depends on the plant species in which you are working, and in what tissues you would like to paint with the red color. Tyrosine is not the most abundant product of the Shikimate pathway, thus user beware, there is an inherent risk. MW: Anyone add tyrosine to their media to account for RUBY’s use of it? RS: Perfusion studies can work, but it is important to remember to use the free base and NOT the sodium salt. JVE: We have also added tyrosine to our medium for tomato. 250 mg/l. Phytotech- T873 https://phytotechlab.com/l-tyrosine.html |
LV: Can someone please recommend a publication or site to look at for specifics when designing genome editing project? Would be really appreciated A: Addgene has good short and quick info pages: https://www.addgene.org/guides/crispr/ Gordon-Kamm, William. “Strategies for CRISPR/Cas9-Mediated Genome Editing: From Delivery to Production of Modified Plants,” 2021 |
AB: In our experience with wheat transformation using Agrobacterium we get a larger proportion of plants with multiple copy integration events (>4). What is your experience in maize or other monocots and how transgene copy number can be reduced in T0 plants? JVE: Our experience with direct regeneration is that we do not see good editing. We had to modify our method in golden berry to produce a bit of callus before regeneration. VV: This depend on many factors, what is the agrobacterium type and origin of replication of your binary vector? EW: We generally get a good number of wheat plants edited with single T-DNA insertions AB: We use AGL1 with pVS1 StaA, pVS1 RepA and pVS1 EW: We also use Agl1 and EHA105 AB: This is our experience with JD633 plasmid having GRF-GIF. |
HELPFUL LINKS FROM THE ZOOM CHAT
Addgene has a discounted plasmid program: https://help.addgene.org/hc/en-us/articles/206133475-My-lab-cannot-afford-the-standard-prices-do-you-offer-discounts
If you are not familiar with the Society for In Vitro Biology, I highly recommend you checkout their site and attend the conference in June in St. Louis, Missouri. It’s a great group of plant tissue culture, transformation, and genome editing researchers from academia, non-profit research institutes and industry. https://sivb.org/