Strategies for Efficient Gene Editing in Protoplasts of Solanum tuberosum Theme: Determining gRNA Efficiency Design by Utilizing Protoplast (Research)

Frida Meijer Carlsen; Ida Elisabeth Johansen; Zhang Yang; Ying Liu; Ida Nøhr Westberg; Nam Phuong Kieu; Bodil Jørgensen; Marit Lenman; Erik Andreasson; Kåre Lehmann Nielsen; Andreas Blennow; Bent Larsen Petersen

doi:10.3389/fgeed.2021.795644

Strategies for Efficient Gene Editing in Protoplasts of Solanum tuberosum Theme: Determining gRNA Efficiency Design by Utilizing Protoplast (Research)

Front Genome Ed. 2022 Jan 20:3:795644. doi: 10.3389/fgeed.2021.795644. eCollection 2021.

Authors

Affiliations

¹ Department of Plant and Environmental Sciences, Faculty of Science, The University of Copenhagen, Copenhagen, Denmark.
² Kartoffel Mel Centralen Amba, Brande, Denmark.
³ Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
⁴ Department of Plant Breeding, Swedish University of Agricultural Sciences, Alnarp, Sweden.
⁵ Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Alnarp, Sweden.
⁶ Bioscience, Aalborg University, Aalborg, Denmark.

Abstract

Potato, Solanum tuberosum is a highly diverse tetraploid crop. Elite cultivars are extremely heterozygous with a high prevalence of small length polymorphisms (indels) and single nucleotide polymorphisms (SNPs) within and between cultivars, which must be considered in CRISPR/Cas gene editing strategies and designs to obtain successful gene editing. In the present study, in-depth sequencing of the gene encoding glucan water dikinase (GWD) 1 and the downy mildew resistant 6 (DMR6-1) genes in the potato cultivars Saturna and Wotan, respectively, revealed both indels and a 1.3-2.8 higher SNP prevalence when compared to the heterozygous diploid RH genome sequence as expected for a tetraploid compared to a diploid. This complicates guide RNA (gRNA) and diagnostic PCR designs. At the same time, high editing efficiencies at the cell pool (protoplast) level are pivotal for achieving full allelic knock-out in tetraploids. Furthermore, high editing efficiencies reduce the downstream cumbersome and delicate ex-plant regeneration. Here, CRISPR/Cas ribonucleoprotein particles (RNPs) were delivered transiently to protoplasts by polyethylene glycol (PEG) mediated transformation. For each of GWD1 and the DMR6-1, 6-10 gRNAs were designed to target regions comprising the 5' and the 3' end of the two genes. Similar to other studies including several organisms, editing efficiency of the individual RNPs varied significantly, and some generated specific indel patterns. RNP's targeting the 5' end of GWD1 yielded significantly higher editing efficiency as compared to targeting the 3' end. For DMR6-1, such an effect was not seen. Simultaneously targeting each of the two target regions with two RNPs (multiplexing) yielded a clear positive synergistic effect on the total editing when targeting the 3' end of the GWD1 gene only. Multiplexing of the two genes, residing on different chromosomes, yielded no or a slightly negative effect on editing from the single or combined gRNA/RNPs. These initial findings may instigate much larger studies needed for facilitating and optimizing precision breeding in plants.

Keywords: CRISPR/Cas; complex genome; gene editing; protoplast; ribonucleoprotein/gRNA design.