Tracking Transgenes with Color: RUBY as a Visual Marker in CRISPR-Edited Mutant Plants in Two Triticum Species
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Abstract
CRISPR-Cas9 is a powerful tool for precise genome editing in plants, but the presence of foreign DNA, such as T-DNA, raises regulatory concerns and complicates mutant screening and field studies of edited material. Detecting plants with good transgene expression and later removing the T-DNA from edited plants is both time-consuming and costly. To address this, we developed a system that uses the non-destructive RUBY reporter, linked to the CRISPR-Cas9 cassette, and expressed under the ZmUbi1 promoter. To assess the applicability of the system, it was tested on two Triticum species, targeting three genes in either tetraploid or hexaploid wheat. Strong correlations were observed in both T0 and T1 plants between betalain content and Cas9 expression, allowing for the quick identification of plants likely to be edited. Furthermore, the RUBY reporter could be used to select against the transgenic CRISPR-Cas9 cassette in subsequent generations at both the seed and seedling stages, thereby reducing the number of plants that need to be screened to identify edited lines without a T-DNA. This approach, using a nondestructive reporter, enabled rapid distinction between transgene expression in primary transgenics and served as a negative selection in the T1 generation, streamlining selection towards edited and T-DNA-free progeny.
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This complicates the ability to quickly clear the T-DNAs when multiple T-DNA fragments lacking a reporter are present in the transformant
Earlier in the paragraph you suggest that T-DNA molecules are undergoing concatemerization prior to chromosomal integration, rather than multiple independent partial T-DNA insertions. However, if this was the case, then the mutlple transgene copies would be linked and segregation would occur as if there was a single insertion event.
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This demonstrates that seed-coat RUBY intensity reflects transgene dosage in the T1 generation and can be used to select against it.
The seed-coat is T0 maternal tissue, so how would this be helpful to determine segregation of the transgene in the T1 seeds?
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The strongest ruby coloration was on the ventral side of the grain around the crease and on the dorsal side containing the embryo. Whereas coloration on the dorsal side of the grain distal to the embryo, was generally weaker
Based on your segregation data, is it possible to determine if the RUBY observed in the seeds is being expressed in the T1 seeds, or if it is being expressed in T0 maternal floral tissue and being transferred/leaked into the seed?
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Sequencing analysis of T1 plants indicated the presence of new mutations not observed in T0 indicate functional activity of the ribonucleoprotein (RNP) complex at later developmental stages of T0 or during the initial phase of T1 seedling development
Since editing continues through T0 generation development, which developmental stage did you use for checking T0 CRISPR editing efficiency? How soon is too soon, and is there a recommended tissue and/or developmental time point to perform the T0 screening?
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TaGRF4-GIF1
It's exciting to see that Cas9 can be linked to RUBY using P2A, and I can see multiple benefits. Not just for selection, but it also helps reduce the size of the binary vector for plant transformation. With that in mind, since ZmUbi1 promoter is used for GIF1 and Cas9-RUBY, is there a reason for the current design over ZmUbi1::GRF-GIF1-P2A-CAS9-P2A-RUBY?
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