The Solanum tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid potato

Abstract

The StGBSSI gene was successfully and precisely edited in the tetraploid potato using gene and base editing strategies, leading to plants with impaired amylose biosynthesis. AbstractGenome editing has recently become a method of choice for basic research and functional 4 genomics, and holds great potential for molecular plant breeding applications. The powerful 5 CRISPR-Cas9 system that typically produces double-strand DNA breaks is mainly used to 6 generate knockout mutants. Recently, the development of base editors has broadened the 7 scope of genome editing, allowing precise and efficient nucleotide substitutions. In this study, 8we produced mutants in two cultivated elite cultivars of the tetraploid potato (Solanum 9 tuberosum) using stable or transient expression of the CRISPR-Cas9 components to knockout 10 the amylose-producing StGBSSI gene. We set up a rapid, highly sensitive and cost-effective 11 screening strategy based on high-resolution melting analysis followed by direct Sanger 12 sequencing and trace chromatogram analysis. Most mutations consisted of small indels, but 13 unwanted insertions of plasmid DNA were also observed. We successfully created tetra-14 allelic mutants with impaired amylose biosynthesis, confirming the loss-of-function of the 15 StGBSSI protein. The second main objective of this work was to demonstrate the proof of 16 concept of CRISPR-Cas9 base editing in the tetraploid potato by targeting two loci encoding 17 catalytic motifs of the StGBSSI enzyme. Using a cytidine base editor (CBE), we efficiently 18 and precisely induced DNA substitutions in the KTGGL-encoding locus, leading to discrete 19 variation in the amino acid sequence and generating a loss-of-function allele. The successful 20 application of base editing in the tetraploid potato opens up new avenues for genome 21 engineering in this species. 22 23 24 tuberosum. We identified single-and multi-allelic edited plants, and mutations in all four 113 alleles were observed, resulting in amylose biosynthesis impairment. To induce this 114 modification, we used protoplast transformation, which can produce non-transgenic plants. 115 Finally, we assessed the efficiency of the cytidine base editor (CBE) in two loci encoding 116 catalytic motifs of the StGBSSI enzyme, resulting in precise base conversion and amino-acid 117 substitution. Our results highlight that CRISPR-Cas9 gene and base editing can be efficiently 118 developed in a genetically complex and vegetatively propagated crop such as potato to 119 modify agronomic traits. 120 121 6 Materials and methods 122 Plant material 123 Potato cultivars Desiree (ZPC, the Netherlands) and Furia (Germicopa, France) were in vitro 124 propagated in 1X Murashige and Skoog (MS) medium (pH 5.8) including vitamins (Duchefa, 125 the Netherlands), 0.4 mg/L thiamine hydrochloride (Sigma-Aldrich, USA), 2.5% sucrose and 126 0.8% agar powder (VWR, USA). Plants were cultivated in a growth chamber at 19°C with a 127 16:8 h light:dark photoperiod. For the production of in vitro microtubers, plants w...

Potato trait development going fast-forward with genome editing

“…The introduction of the same trait was soon repeated with CRISPR-Cas9 components delivered as ribonucleoprotein (RNP) complexes, which eliminated the insertion of unintended DNA when synthetically produced RNA was used [5]. Another strategy to develop an amylopectin potato was described soon afterwards, where the use of base editing (BE) to alter the catalytic region of the GBSS enzyme was found successful [6]. Amylopectin starch is most likely the most progressed trait developed with genome editing in potato, and nontransgenic amylopectin lines are currently grown in the field for selection and seed multiplication.…”

“…The development of CRISPR-based methods that result in tailored point mutations is ongoing for potato, but they currently have a lower frequency of induced mutations. In this context, DSBs in combination with HR [3], using a template for designed repair, as well as BE [6,13] and prime editing (PE) [14] have been published. Further development of the methods will be important tasks to broaden the use of genome editing in potato from mainly providing knockouts to also being used for designed knock-ins and base substitutions.…”

Prospects for Genome Editing of Potato

“…Veillet et al used the CRISPR-Cas9 base editing, precisely in the conserved catalytic KTGGL encoding locus of the StGBSSI enzyme using a cytidine base editor (CBE). This lead to the discrete variation in the amino acid sequence and loss-offunction allele producing plants with impaired amylose biosynthesis [55].…”

Research progress in gene editing technology

“…In the laboratory, CRISPR gene editing relies on two key components: a CRISPR-associated (Cas) protein, and a trans-active single guide RNA (sgRNA). Cas9 is a nuclease that binds to sgRNA and this both activates and targets Cas9 to a specified genomic locus (termed the Protospacer Adjacent Motif or PAM site) through a 20 bp nucleotide sequence present within the sgRNA [37][38][39][40][41][42][43][44][45]. Cas9 subsequently catalyzes a DSB close to the PAM site and low-fidelity DSB repair by NHEJ will form a small insertion/deletion (indel) at the digested site thus placing a mutation within the targeted locus.…”