Genome Editing for Crop Improvement - Applications in Clonally Propagated Polyploids With a Focus on Potato (Solanum tuberosum L.)

Abstract

Genome-editing has revolutionized biology. When coupled with a recently streamlined regulatory process by the U.S. Department of Agriculture and the potential to generate transgene-free varieties, genome-editing provides a new avenue for crop improvement. For heterozygous, polyploid and vegetatively propagated crops such as cultivated potato, Solanum tuberosum Group Tuberosum L., genome-editing presents tremendous opportunities for trait improvement. In potato, traits such as improved resistance to cold-induced sweetening, processing efficiency, herbicide tolerance, modified starch quality and self-incompatibility have been targeted utilizing CRISPR/Cas9 and TALEN reagents in diploid and tetraploid clones. However, limited progress has been made in other such crops including sweetpotato, strawberry, grapes, citrus, banana etc., In this review we summarize the developments in genome-editing platforms, delivery mechanisms applicable to plants and then discuss the recent developments in regulation of genome-edited crops in the United States and The European Union. Next, we provide insight into the challenges of genome-editing in clonally propagated polyploid crops, their current status for trait improvement with future prospects focused on potato, a global food security crop.

Keywords: Agrobacterium-mediated transformation; CRISPR/Cas system; TALENs; clonal propagation; genome-editing; polyploidy; potato (Solanum tuberosum); protoplast transformation.

FIGURE 1

Illustration of genome-editing platforms and genetic transformation procedures in potato. (A) Double stranded DNA (dsDNA) break repair in a cell occurs either by non-homologous end joining (NHEJ), where the cleaved DNA molecule is simply rejoined, often with indels in coding regions (green) that result in gene knock-out or by homologous recombination (HR), where a donor repair template (red) can be used for targeted knock-in experiments, where a single or few nucleotides alterations, insertion of an entire transgene or suites of transgenes can be made. CRISPR/Cas9 nuclease engineered to have a Cas9 protein and a guide RNA (gRNA) that is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Cas9 and gRNA complex can recognize and cleave target dsDNA that is complementary to 5′ end of target spacer sequence that is next to protospacer adjacent motif (PAM) of 5′-NGG-3′. CRISPR/Cas12a is a single CRISPR RNA guided nuclease lacking tracrRNA. Cas12a has PAM requirement of “TTTN” allowing targeting of AT rich regions and expanding the target range of RNA-guided genome-editing nucleases. Cas12a cleaves DNA at sites distal to PAM and introduces a staggered DSB with a 4–5-nt 5′ overhang, unlike blunt DSB by Cas9. Transcription activator-like effector nucleases (TALENs) bound to their target site are shown. The TALE array contains repeat variable di-residues that make sequence-specific contact with the target DNA. TALE repeats are fused to FokI, a non-specific nuclease that can cleave the dsDNA upon dimerization. Base editor constitutes fusion of nickase Cas9 (nCas9) with cytidine deaminase enabling the editing of single bases by C→T conversion of single-stranded target. (B) Agrobacterium-mediated plant transformation and regeneration in potato. 3–4-week-old in vitro propagated potato plants in a Magenta box are shown. Ex-plants are prepared from leaf and stem internodes and placed on callus induction media after Agrobacterium inoculation and co-cultivation. Callus growth observed from the ex-plants. After 6–8 weeks, shoots emerge and are grown on shoot induction media. 1–2 cm shoots are excised and transferred to root induction media. The lines that develop roots and have growth on selection media are chosen as candidates for molecular screening to confirm the gene editing events. (C) Delivery of the gene editing reagents as plasmid DNA or as preassembled Cas9 or Cas12a protein-gRNA ribonucleoproteins (RNPs) into protoplasts by polyethylene glycol (PEG) mediated transformation. The timeline from protoplast transformation to regeneration of mutagenized plants in potato is reproduced from Clasen et al. (2016) with the permission of the copyright holder (John Wiley & Sons, Inc.).