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.

CRISPR-Cas9 has proven to be highly valuable for genome editing in plants, including the model plant Physcomitrium patens. However, the fact that most of the editing events produced using the native Cas9 nuclease correspond to small insertions and deletions is a limitation. CRISPR-Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic genomes and therefore overcome this limitation. Here, we report two programmable baseediting systems to induce precise cytosine or adenine conversions in P. patens. Using cytosine or adenine base editors, site-specific single-base mutations can be achieved with an efficiency up to 55%, without off-target mutations. Using the APT gene as a reporter of editing, we could show that both base editors can be used in simplex or multiplex, allowing for the production of protein variants with multiple amino-acid changes. Finally, we set up a co-editing selection system, named selecting modification of APRT to report gene targeting (SMART), allowing up to 90% efficiency site-specific base editing in P. patens. These two base editors will facilitate gene functional analysis in P. patens, allowing for sitespecific editing of a given base through single sgRNA base editing or for in planta evolution of a given gene through the production of randomly mutagenised variants using multiple sgRNA base editing.

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...