Prime editing is achievable in the tetraploid potato, but needs improvement

Veillet, Florian; Kermarrec, Marie-Paule; Chauvin, Laura; Guyon‐Debast, Anouchka; Chauvin, Jean-Éric; Gallois, Jean‐Luc; Nogué, Fabien

doi:10.1101/2020.06.18.159111

Cited by 35 publications

(34 citation statements)

References 17 publications

(20 reference statements)

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“…PE activity was shown to be locus-specific, and several parameters, such as PBS and RT template lengths, may also affect PE efficiency in plants 6 . During our study, several PE data were reported that showed comparable PE efficiencies among the PE2, PE3, and PE3b constructs 6–8, 10 , and in some cases, the PE2 approach performed better than the PE3 approach 12, 13 . We then extended the PE experiment to 7 more loci using the PE2 approach and single replicon-based vectors (Fig.…”

Section: Resultssupporting

confidence: 56%

“…However, the majority of the data showed successful PE at the acetolactate synthase (ALS) locus. Very limited data regarding the applications of PE in dicots were released, with relatively low efficacy in potato 13 and tomato 14 , and this process would need to be improved for further applications. Therefore, we sought to investigate the activity of PE complexes in plants using a transient assay with a synthetic substrate in tobacco and stable transformation for editing ten loci in tomato.…”

Section: Introductionmentioning

confidence: 99%

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The obstacles and potential clues of prime editing applications in tomato, a dicot plant

Kim

Das

et al. 2021

Preprint

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Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a priming extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. However, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. However, our data obtained by targeted deep sequencing also revealed inefficient PE activity in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. These data could also help explain the recent successes of some prime editors in plants using improved expression systems. Our work provides an important clue for the application of the PE approach in crop improvement.

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Section: Resultssupporting

confidence: 56%

Section: Introductionmentioning

confidence: 99%

The obstacles and potential clues of prime editing applications in tomato, a dicot plant

Kim

Das

et al. 2021

Preprint

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“…The highest efficiency overall, 53.2%, was in maize, obtained by optimization of pegRNA expression (Jiang et al, 2020). In contrast to results in mammalian cells (Anzalone et al, 2019), PE3 did not increase editing efficiency in plants relative to PE2 (Butt et al, 2020;Lin et al, 2020;Veillet et al, 2020;Xu et al, 2020a). In some studies, in fact, PE2 yielded a much higher editing efficiency than PE3 (Jiang et al, 2020;Tang et al, 2020).…”

Section: Introductionmentioning

confidence: 82%

“…While the editing efficiencies ranged from 1.55 to 31.3% in rice (Butt et al, 2020;Hua et al, 2020;Li et al, 2020;Lin et al, 2020;Tang et al, 2020;Xu et al, 2020a,b), the highest efficiency observed in tomato was 1.66% (Lu et al, 2020). Potato was similar to tomato (Veillet et al, 2020). The highest efficiency overall, 53.2%, was in maize, obtained by optimization of pegRNA expression (Jiang et al, 2020).…”

Section: Introductionmentioning

confidence: 98%

Spelling Changes and Fluorescent Tagging With Prime Editing Vectors for Plants

et al. 2021

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Prime editing is an adaptation of the CRISPR-Cas system that uses a Cas9(H840A)-reverse transcriptase fusion and a guide RNA amended with template and primer binding site sequences to achieve RNA-templated conversion of the target DNA, allowing specified substitutions, insertions, and deletions. In the first report of prime editing in plants, a variety of edits in rice and wheat were described, including insertions up to 15 bp. Several studies in rice quickly followed, but none reported a larger insertion. Here, we report easy-to-use vectors for prime editing in dicots as well as monocots, their validation in Nicotiana benthamiana, rice, and Arabidopsis, and an insertion of 66 bp that enabled split-GFP fluorescent tagging.

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“…In their latest work they showed a proof of concept of application of a CRISPR/Cas9-mediated prime editor, which allows targeted 'rewriting' of short stretches genomic DNA sequences, in potato. Preliminary results show the expected substitution of three nucleotides at the StALS1 target locus, which would provide herbicide tolerance (Veillet et al 2020c).…”

Section: Genome Editing and Polyploid Cropsmentioning

confidence: 99%

Genome editing of polyploid crops: prospects, achievements and bottlenecks

2021

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Plant breeding aims to develop improved crop varieties. Many crops have a polyploid and often highly heterozygous genome, which may make breeding of polyploid crops a real challenge. The efficiency of traditional breeding based on crossing and selection has been improved by using marker-assisted selection (MAS), and MAS is also being applied in polyploid crops, which helps e.g. for introgression breeding. However, methods such as random mutation breeding are difficult to apply in polyploid crops because there are multiple homoeologous copies (alleles) of each gene. Genome editing technology has revolutionized mutagenesis as it enables precisely selecting targets. The genome editing tool CRISPR/Cas is especially valuable for targeted mutagenesis in polyploids, as all alleles and/or copies of a gene can be targeted at once. Even multiple genes, each with multiple alleles, may be targeted simultaneously. In addition to targeted mutagenesis, targeted replacement of undesirable alleles by desired ones may become a promising application of genome editing for the improvement of polyploid crops, in the near future. Several examples of the application of genome editing for targeted mutagenesis are described here for a range of polyploid crops, and achievements and bottlenecks are highlighted.

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Prime editing is achievable in the tetraploid potato, but needs improvement

Abstract: Since its discovery and first applications for genome editing in plants, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology has revolutionized plant research and precision crop breeding.

Cited by 35 publications

References 17 publications

The obstacles and potential clues of prime editing applications in tomato, a dicot plant

The obstacles and potential clues of prime editing applications in tomato, a dicot plant

Spelling Changes and Fluorescent Tagging With Prime Editing Vectors for Plants

Genome editing of polyploid crops: prospects, achievements and bottlenecks

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