Widespread genome instability in<i>Solanum tuberosum</i>plants regenerated from protoplasts

Fossi, Michelle; Comai, Luca

doi:10.1101/382861

Cited by 2 publications

(2 citation statements)

References 42 publications

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“…Although some alternative delivery methods such as particle bombardment or protoplast transfection using plasmid DNA or ribonucleoprotein (RNP) complexes have been demonstrated to result in non-transgenic mutants, these approaches are often highly sensitive and limited to some species due to bottlenecks in the regeneration process. Regeneration from protoplasts has been recently shown to result in a substantial genome instability in the tetraploid potato, affecting the prospects for protoplast utilization in genome editing for this species [13]. Whether this syndrome may be reduced using regeneration from Agrobacterium -transformed explants is a question of upmost importance.…”

Section: Introductionmentioning

confidence: 99%

Transgene-Free Genome Editing in Tomato and Potato Plants Using Agrobacterium-Mediated Delivery of a CRISPR/Cas9 Cytidine Base Editor

Veillet

Perrot

Chauvin

et al. 2019

IJMS

272

173

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Genome editing tools have rapidly been adopted by plant scientists for gene function discovery and crop improvement. The current technical challenge is to efficiently induce precise and predictable targeted point mutations valuable for crop breeding purposes. Cytidine base editors (CBEs) are CRISPR/Cas9 derived tools recently developed to direct a C-to-T base conversion. Stable genomic integration of CRISPR/Cas9 components through Agrobacterium-mediated transformation is the most widely used approach in dicotyledonous plants. However, elimination of foreign DNA may be difficult to achieve, especially in vegetatively propagated plants. In this study, we targeted the acetolactate synthase (ALS) gene in tomato and potato by a CBE using Agrobacterium-mediated transformation. We successfully and efficiently edited the targeted cytidine bases, leading to chlorsulfuron-resistant plants with precise base edition efficiency up to 71% in tomato. More importantly, we produced 12.9% and 10% edited but transgene-free plants in the first generation in tomato and potato, respectively. Such an approach is expected to decrease deleterious effects due to the random integration of transgene(s) into the host genome. Our successful approach opens up new perspectives for genome engineering by the co-edition of the ALS with other gene(s), leading to transgene-free plants harboring new traits of interest.

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Section: Introductionmentioning

confidence: 99%

Transgene-Free Genome Editing in Tomato and Potato Plants Using Agrobacterium-Mediated Delivery of a CRISPR/Cas9 Cytidine Base Editor

Veillet

Perrot

Chauvin

et al. 2019

IJMS

272

173

View full text Add to dashboard Cite

show abstract

“…In potato, CRISPR/Cas9 technology was mostly used in combination with Agrobacterium -mediated stable transformation [ 10 , 57 , 64 ]. The alternative option is protoplast transfection [ 42 ], which is in potato problematic as regeneration of plants from protoplast is long lasting and difficult to establish for a specific genotype ([ 2 , 3 , 21 , 55 ]).…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9-mediated fine-tuning of miRNA expression in tetraploid potato

Lukan

Veillet

Križnik

et al. 2022

Horticulture Research

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MicroRNAs (miRNAs) are small noncoding RNAs, which modulate the abundance and spatiotemporal accumulation of target mRNAs at transcriptional and post-transcriptional levels and through that play important roles in several biological processes in plants. Here we show that in polyploid species, CRISPR/Cas9 system can be used for fine-tuning of miRNA expression, which can have broader range of applications compared to knock-out mutants. We established the complete pipeline for CRISPR-Cas9-mediated modulation of miRNA expression in potato. It consists of (1) design and assembly of dual sgRNA CRISPR/Cas9 constructs, (2) transient transfection of protoplasts following fast and efficient screening by high resolution melting analysis to select functional sgRNAs, and (3) stable transformation of potato explants with functional sgRNAs and selection of regenerated transgenic lines with desired mutations and desired miRNA abundance based on sequencing and RT-qPCR. We show that miRNA-editing using dual sgRNA approach results in different types of mutations among transgenic lines but also in different alleles of the same plant, which are target site-dependent. The most frequent were short deletions, but we also detected 1-nt insertions (T or G), deletions between two sgRNAs and larger deletions. miRNA abundance correlates with the frequency and type of introduced mutations, as more extensive mutations in more alleles result in lower miRNA abundance. Interestingly, some mutated loci can generate alternative miRNAs, now novel targets were however predicted for those. In all transgenic lines with Cas9 expression, we detected mutations, suggesting high efficiency of Cas9-editing. We confirmed the miRNA-editing efficiency of our optimised approach in two different potato genotypes and three different loci.

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Widespread genome instability inSolanum tuberosumplants regenerated from protoplasts

Cited by 2 publications

References 42 publications

Transgene-Free Genome Editing in Tomato and Potato Plants Using Agrobacterium-Mediated Delivery of a CRISPR/Cas9 Cytidine Base Editor

Transgene-Free Genome Editing in Tomato and Potato Plants Using Agrobacterium-Mediated Delivery of a CRISPR/Cas9 Cytidine Base Editor

CRISPR/Cas9-mediated fine-tuning of miRNA expression in tetraploid potato

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