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

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.

Recent advances and challenges in potato improvement using CRISPR/Cas genome editing

“…2 , Model plant). The second path has employed CRISPR/Cas techniques to create or improve phenotypic features and has often generated genome-edited lines with the potential for commercialization (Andersson et al 2017 ; Kusano et al 2018 ; Carlsen et al 2022 ) (Fig. 2 , Food crop).…”

“…Among the numerous transformation methods proposed to generate transgenic potato, the effective delivery of CRISPR/Cas components has been confirmed so far only for Agrobacterium -mediated transformation and for protoplast transfection (Tiwari et al 2022 ). In the case of protoplasts, efficient target mutagenesis can be achieved by both transient expression of DNA constructs containing CRISPR/Cas genes and a direct application of active Cas/sgRNA nucleoprotein complexes (RNPs) (Andersson et al 2017 , 2018 ; Zong et al 2018 ; Veillet et al 2019a ; González et al 2021 ; Carlsen et al 2022 ).…”

Invited Mini-Review Research Topic: Utilization of Protoplasts to Facilitate Gene Editing in Plants: Schemes for In Vitro Shoot Regeneration From Tissues and Protoplasts of Potato and Rapeseed: Implications of Bioengineering Such as Gene Editing of Broad-Leaved Plants

“…Lately, we mapped the editing efficiency in relation to gene structure and placement of CRISPR/gRNA-targeting, i.e. start versus end, of a gene, and the effect of applying several RNPs (multiplexing) targeting the same gene versus targeting genes residing on different chromosomes ( Carlsen et al, 2022 ). Although CRISPR derived off-target events have attained some focus, unintentional small genetic changes (somaclonal variation) associated with cell and tissue culture e.g.…”

“…Fresh sterile 15 ml centrifuge tubes are prepared for each transfection or control. Ca 10 µg of plasmid DNA (10–20 µl) or ribonucleoprotein (RNP) (for amount and incubation see manufacturer instructions and Carlsen et al, 2022 ) are pipetted in each tube followed by 100 µl of protoplasts in transformation buffer 2that equals approximately 160.000 protoplasts. 9.…”

“…Plant multiplication through clonal vegetative propagation is in many crops imperative as it preserves existing elite or superior traits ( Nadakuduti et al, 2018 ). For example, the complex genome of tetraploid potato contains an extreme prevalence of single-nucleotide and even-length polymorphisms between alleles ( Potato Genome Sequencing Consortium et al, 2011 ; D’Hoop et al, 2014 ) underlying the genetic basis for the important traits of elite cultivars ( Johansen et al, 2019 , Carlsen et al, 2022 ).…”