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Abstract Key messageTransgene-free genome editing of the gene of interest in citrus and poplar has been achieved by co-editing theALSgene via transient transgene expression of an efficient cytosine base editor. AbstractCRISPR-Cas genome editing systems have been widely used in plants. However, such genome-edited plants are nearly always transgenic in the first generation whenAgrobacterium-mediated transformation is used. Transgene-free genome-edited plants are valuable for genetic analysis and breeding as well as simplifying regulatory approval. It can be challenging to generate transgene-free genome-edited plants in vegetatively propagated or perennial plants. To advance transgene-free genome editing in citrus and poplar, we investigated a co-editing strategy using an efficient cytosine base editor (CBE) to edit theALSgene to confer herbicide resistance combined with transient transgene expression and potential mobile RNA-based movement of CBE transcripts to neighboring, non-transgenic cells. An FCY-UPP based cytotoxin system was used to select non-transgenic plants that survive after culturing on 5-FC containing medium. While the editing efficiency is higher in poplar than in citrus, our results show that the CBE-based co-editing strategy works in both citrus and poplar, albeit with low efficiency for biallelic edits. Unexpectedly, the addition of the TLS mobile RNA sequence reduced genome editing efficiency in both transgenic and non-transgenic plants. Although a small fraction of escaping plants is detected in both positive and negative selection processes, our data demonstrate a promising approach for generating transgene-free base-edited plants. 

Abstract BackgroundCas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants. ResultsTo improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis inE. coliand identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency inT₀plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites. ConclusionsOur results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential of these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms. 

Abstract Fusarium head blight (FHB; caused byFusarium graminearum) is a destructive disease of wheat (Triticumspp.), barley (Hordeum vulgare), rye (Secale cerealeL.), and triticale (×TriticosecaleWittmack) not only reducing their yield but also contaminating the grain with mycotoxins such as deoxynivalenol (DON). Developing varieties with genetic resistance is integral to successfully manage FHB. Triticale acreage worldwide is steadily increasing. However, the genetic diversity of triticale for FHB resistance is not well characterized. In the present study, a sequential screening of a set of winter triticale accessions from a global collection was done for their type‐2 FHB resistance and DON accumulation. In the first‐year screening, 298 triticale accessions were tested for FHB in an artificially inoculated, misted‐field nursery with high inoculum density. Most of the triticale accessions were susceptible to FHB, and only 8% of the accessions showed resistance in the field nursery screening. Next, the 24 resistant accessions identified in the nursery screening were tested for 2 years in greenhouse and 17 accessions showed significantly lower FHB severity in Year 2 and/or Year 3. These 17 resistant accessions were further tested for their FHB severity and DON accumulation in Year 4 in greenhouse and for DON accumulation in Year 5 in the field FHB nursery. Eight accessions showed significantly lower FHB severity and nine accessions showed DON accumulation of less than 1 mg/kg in Year 4 greenhouse testing. Eleven accessions had significantly lower DON concentration than the susceptible check in the Year 5 field screening. The resistant accessions common across all years identified in the study can be used for enhancing FHB resistance and reducing DON accumulation in triticale breeding programs.