Genome editing in potato via CRISPR‐Cas9 ribonucleoprotein delivery

Andersson, Mariette; Turesson, Helle; Olsson, Niklas; Fält, Ann Sofie; Ohlsson, Pia; Gonzalez, Matías Nicolás; Samuelsson, Mathias; Hofvander, Per

doi:10.1111/ppl.12731

Cited by 346 publications

(218 citation statements)

References 22 publications

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“…It is entirely feasible that mutants will be found with nutritionally relevant increases in resistant starch and no other deleterious changes. Recent developments in CRISPR‐derived technology for potatoes promise to improve the efficiency of generation of transgene‐free mutants (Andersson et al ., , ; Kusano et al ., ), thus allowing large‐scale screens for nutritionally desirable phenotypes.…”

Section: Discussionmentioning

confidence: 99%

Cas9‐mediated mutagenesis of potato starch‐branching enzymes generates a range of tuber starch phenotypes

Tuncel

Corbin

Ahn‐Jarvis

et al. 2019

Plant Biotechnology Journal

123

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Summary We investigated whether Cas9‐mediated mutagenesis of starch‐branching enzymes (SBEs) in tetraploid potatoes could generate tuber starches with a range of distinct properties. Constructs containing the Cas9 gene and sgRNAs targeting SBE1, SBE2 or both genes were introduced by Agrobacterium‐mediated transformation or by PEG‐mediated delivery into protoplasts. Outcomes included lines with mutations in all or only some of the homoeoalleles of SBE genes and lines in which homoeoalleles carried several different mutations. DNA delivery into protoplasts resulted in mutants with no detectable Cas9 gene, suggesting the absence of foreign DNA. Selected mutants with starch granule abnormalities had reductions in tuber SBE1 and/or SBE2 protein that were broadly in line with expectations from genotype analysis. Strong reduction in both SBE isoforms created an extreme starch phenotype, as reported previously for low‐SBE potato tubers. HPLC‐SEC and 1H NMR revealed a decrease in short amylopectin chains, an increase in long chains and a large reduction in branching frequency relative to wild‐type starch. Mutants with strong reductions in SBE2 protein alone had near‐normal amylopectin chain‐length distributions and only small reductions in branching frequency. However, starch granule initiation was enormously increased: cells contained many granules of <4 μm and granules with multiple hila. Thus, large reductions in both SBEs reduce amylopectin branching during granule growth, whereas reduction in SBE2 alone primarily affects numbers of starch granule initiations. Our results demonstrate that Cas9‐mediated mutagenesis of SBE genes has the potential to generate new, potentially valuable starch properties without integration of foreign DNA into the genome.

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Section: Discussionmentioning

confidence: 99%

Cas9‐mediated mutagenesis of potato starch‐branching enzymes generates a range of tuber starch phenotypes

Tuncel

Corbin

Ahn‐Jarvis

et al. 2019

Plant Biotechnology Journal

123

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“…transgenes) were on a par in terms of mutagenesis efficiency (Svitashev et al ). A particularly valuable application of using gRNA/Cas9 RNP complexes was demonstrated by Andersson et al (), who generated an amylopectin potato via knock‐out of GBSS . In addition, the utilization of Cas12a has been exemplified along with gRNA in the format of RNPs that were transfected into protoplasts to induce targeted mutations in the two FAD2‐1 gene variants of the palaeopolyploid soybean (Kim et al ).…”

Section: Selection Of Target Motifsmentioning

confidence: 99%

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

Kumlehn

Pietralla

Hensel

et al. 2018

JIPB

102

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Since the discovery that nucleases of the bacterial CRISPR (clustered regularly interspaced palindromic repeat)‐associated (Cas) system can be used as easily programmable tools for genome engineering, their application massively transformed different areas of plant biology. In this review, we assess the current state of their use for crop breeding to incorporate attractive new agronomical traits into specific cultivars of various crop plants. This can be achieved by the use of Cas9/12 nucleases for double‐strand break induction, resulting in mutations by non‐homologous recombination. Strategies for performing such experiments − from the design of guide RNA to the use of different transformation technologies − are evaluated. Furthermore, we sum up recent developments regarding the use of nuclease‐deficient Cas9/12 proteins, as DNA‐binding moieties for targeting different kinds of enzyme activities to specific sites within the genome. Progress in base deamination, transcriptional induction and transcriptional repression, as well as in imaging in plants, is also discussed. As different Cas9/12 enzymes are at hand, the simultaneous application of various enzyme activities, to multiple genomic sites, is now in reach to redirect plant metabolism in a multifunctional manner and pave the way for a new level of plant synthetic biology.

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“…Protoplast modification via nucleoprotein complexes results in high efficiency editing and transgene-free genomes (Woo et al, 2015;Andersson et al, 2018). When regeneration is possible, at least some of the resulting plants should display only the targeted changes.…”

mentioning

confidence: 99%

Regeneration of Solanum tuberosum Plants from Protoplasts Induces Widespread Genome Instability

et al. 2019

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Nontransgenic genome editing in regenerable protoplasts, plant cells free of their cell wall, could revolutionize crop improvement because it reduces regulatory and technical complexity. However, plant tissue culture is known to engender frequent unwanted variation, termed somaclonal variation. To evaluate the contribution of large-scale genome instability to this phenomenon, we analyzed potatoes (Solanum tuberosum) regenerated from either protoplasts or stem explants for copy number changes by comparison of Illumina read depth. Whereas a control set of eight plants that had been propagated by cuttings displayed no changes, all 15 protoplast regenerants tested were affected by aneuploidy or structural chromosomal changes. Certain chromosomes displayed segmental deletions and duplications ranging from one to many. Resampling different leaves of the same plant found differences in three regenerants, indicating frequent persistence of instability. By comparison, 33 regenerants from stem explants used for Agrobacterium-mediated transformation displayed less frequent but still considerable (18%) large-scale copy number changes. Repetition of certain instability patterns suggested greater susceptibility in specific genomic sites. These results indicate that tissue culture, depending on the protocol used, can induce genomic instability resulting in large-scale changes likely to compromise final plant phenotype. 78

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Genome editing in potato via CRISPR‐Cas9 ribonucleoprotein delivery

Cited by 346 publications

References 22 publications

Cas9‐mediated mutagenesis of potato starch‐branching enzymes generates a range of tuber starch phenotypes

Cas9‐mediated mutagenesis of potato starch‐branching enzymes generates a range of tuber starch phenotypes

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

Regeneration of Solanum tuberosum Plants from Protoplasts Induces Widespread Genome Instability

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