Does it mandatory to segregate out the CRISPR/Cas9 cassette after generating homozygous mutant lines in plants (Arabidopsis / Tomato)?
What are the pros and cons?
Hi there,
no - but its a good idea and simple in Ath. The main reason would be to obtain a plant line that can be easily complemented by introduction of the wild-type gene. I guess in CRISPR experiments complementiation of the mutant phenotype is especially important coz of the off-target problem.
Best wishes, Henrik
Thanks Dr. Buschmann, well then it's an important step. In your crispr transgenic protocol. Do you screen them from T1 generation? how many lines you usually check?
Hi Akila Wijerathna Yapa,
The question you asked in your last answer:
CRISPR-mediated mutation can be readily found in the T0 generation in plants (or in other organisms). So, you can grow them (T0) into T1 generation to segregate away the CRISPR/Cas9 cassette.
I am not sure for the other countries. However, in the US, now that the USDA (US Department of Agriculture) has announced that they willl not regulate certain categories of CRISPR-generated products, it is important to segregate away all the CRISPR-Cas9 reagents in the final product. If the plant products still harbor those foreign genetic elements (ex. Cas9), they are still considered GMO and will be regulated. Without regulation, one can save tremondous time and money to release a product.
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