Genome editing in potato via CRISPR‐Cas9 ribonucleoprotein delivery

Mariette Andersson; Helle Turesson; Niklas Olsson; Ann‐Sofie Fält; Pia Ohlsson; Matías N. Gonzalez; Mathias Samuelsson; Per Hofvander

doi:10.1111/ppl.12731

journal article Open Access Apr 27, 2018

Genome editing in potato via CRISPR‐Cas9 ribonucleoprotein delivery

Mariette Andersson

Helle Turesson Niklas Olsson Ann‐Sofie Fält Pia Ohlsson Matías N. Gonzalez Mathias Samuelsson Per Hofvander

Physiologia Plantarum Vol. 164 No. 4 pp. 378-384 · Wiley

View at Publisher Save 10.1111/ppl.12731

Abstract

Clustered regularly interspaced short palindromic repeats and CRISPR‐associated protein‐9 (CRISPR‐Cas9) can be used as an efficient tool for genome editing in potato (Solanum tuberosum). From both a scientific and a regulatory perspective, it is beneficial if integration of DNA in the potato genome is avoided. We have implemented a DNA‐free genome editing method, using delivery of CRISPR‐Cas9 ribonucleoproteins (RNPs) to potato protoplasts, by targeting the gene encoding a granule bound starch synthase (GBSS, EC 2.4.1.242). The RNP method was directly implemented using previously developed protoplast isolation, transfection and regeneration protocols without further adjustments. Cas9 protein was preassembled with RNA produced either synthetically or by in vitro transcription. RNP with synthetically produced RNA (cr‐RNP) induced mutations, i.e. indels, at a frequency of up to 9%, with all mutated lines being transgene‐free. A mutagenesis frequency of 25% of all regenerated shoots was found when using RNP with in vitro transcriptionally produced RNA (IVT–RNP). However, more than 80% of the shoots with confirmed mutations had unintended inserts in the cut site, which was in the same range as when using DNA delivery. The inserts originated both from DNA template remnants from the in vitro transcription, and from chromosomal potato DNA. In 2–3% of the regenerated shoots from the RNP‐experiments, mutations were induced in all four alleles resulting in a complete knockout of the GBSS enzyme function.

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382

Citations

23

References

Details

Published: Apr 27, 2018
Vol/Issue: 164(4)
Pages: 378-384
License: View

Authors

M

Mariette Andersson

Department of Plant Breeding Swedish University of Agricultural Sciences P.O. Box 101, SE 23053 Alnarp Sweden

H

Helle Turesson

Department of Plant Breeding Swedish University of Agricultural Sciences P.O. Box 101, SE 23053 Alnarp Sweden

N

Niklas Olsson

Department of Plant Breeding Swedish University of Agricultural Sciences P.O. Box 101, SE 23053 Alnarp Sweden

A

Ann‐Sofie Fält

Department of Plant Breeding Swedish University of Agricultural Sciences P.O. Box 101, SE 23053 Alnarp Sweden

P

Pia Ohlsson

Department of Plant Breeding Swedish University of Agricultural Sciences P.O. Box 101, SE 23053 Alnarp Sweden

M

Matías N. Gonzalez

Concejo Nacional de Investigaciones Científicas y Técnicas (CONICET) Buenos Aires Argentina; Laboratorio de Agrobiotecnología, INTA–EEA Balcarce, B7620CNQ Argentina

M

Mathias Samuelsson

Lyckeby Starch AB Degebergavägen 60‐20, SE 291 91 Kristianstad Sweden

P

Per Hofvander

Department of Plant Breeding Swedish University of Agricultural Sciences P.O. Box 101, SE 23053 Alnarp Sweden

Funding

Einar and Inga Nilssons Stiftelse

Lyckeby Research Foundation (Stiftelsen Stärkelsen Forskning Utveckling)

Cite This Article

Mariette Andersson, Helle Turesson, Niklas Olsson, et al. (2018). Genome editing in potato via CRISPR‐Cas9 ribonucleoprotein delivery. Physiologia Plantarum, 164(4), 378-384. https://doi.org/10.1111/ppl.12731

Genome editing in potato via CRISPR‐Cas9 ribonucleoprotein delivery

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