High efficacy full allelic CRISPR/Cas9 gene editing in tetraploid potato

Abstract

CRISPR/Cas9 editing efficacies in tetraploid potato were highly improved through the use of endogenous potato U6 promoters. Highly increased editing efficiencies in the Granular Bound Starch Synthase gene at the protoplast level were obtained by replacement of the Arabidopsis U6 promotor, driving expression of the CRISPR component, with endogenous potato U6 promotors. This translated at the ex-plant level into 35% full allelic gene editing. Indel Detection Amplicon Analysis was established as an efficient tool for fast assessment of gene editing in complex genomes, such as potato. Together, this warrants significant reduction of laborious cell culturing, ex-plant regeneration and screening procedures of plants with high complexity genomes.

Figure 1

U6 promoter efficacy analysis at the protoplast level. StU6 promoters were here defined as 350 nt upstream of the transcription start site (Supplementary Fig. 1 and Supplementary Fig. 2D), and the 257 bp AtU6-1 promoter of Arabidopsis thaliana was obtained from the vector pHBT-pcoCas9. (A) Structure of the Granular Bound Starch Synthase (GBSS) gene with the guide RNA (gRNA1) targeting part of the conserved motif and proposed active site KTGGL. gRNA1 includes a diagnostic BsrI restriction enzyme site spanning the SpCas9 cleavage site, −3 bp upstream of the Photospacer Adjacent Motif (PAM) (red), which upon digestion yields the allele specific fragments: allele III (286, 140), allele I and II (289, 140) and allele IV (289, 144). The outer most primer set includes the SNPs (+SNPs) and gives rise to PCR amplicons of 426 (allele III), 429 (alleles I and II) and 433 bp (allele IV), and the innermost (-SNPs) to a PCR amplicon of 218 bp (all alleles). These length SNPs were conserved between the cultivars Desirée and Wotan. (B) Construct design and StU6-1-4 versus AtU6-1 promoter analysis at the cell pool (protoplast) level as evidenced by the presence of indel mediated destruction of the BsrI site (BsrI resistant band). (C) Indel Detection by Amplicon Analysis (IDAA) chromatograms for the + SNP and –SNP PCR amplicons of WT and AtU6-1 and StU6-1 derived indels. + SNP IDAA reveals allele complexity, while –SNP and BsrI digested + SNP amplicons permit estimation of editing efficacy. (D) Sequence analysis of 34 individual clones of the BsrI resistant band of StU6-1 (B) isolated and cloned into the pJet vector, confirmed the indel distribution of the IDAA (Fig. 1C, panel: StU6-1, -SNP) also peaking at −4 bp deletions. WT peak positions are indicated by dotted lines. PPDK and NOS designate Pyruvate phosphate dikinase promoter and nopaline synthase terminator, respectively. All experiments were done in Desirée, except for panel C (upper right side) and D, which were done in the cultivar Wotan.

Figure 2

Amylopectin only potato: Geno- and phenotypic analysis of GBSS loss of function ex-plants. (A) Regenerated ex-plants from tissue culture with resulting potato tubers.(B) In total, 23 randomly selected shoots/ex-plants were screened to identify indels using the BsrI restriction enzyme digestion. K8, K10, K18, K19, K33 & K52 appeared to have editing in all four alleles (fully BsrI resistant), K11, K12, K26, K32 & K42 displayed a mixture of WT type and edited alleles, while K1, K2, K7, K9, K13, K34, K47, K50 & K51 appeared to be un-edited. * denotes insertions. K5 and K6 appear at a first glance to be mixed, but sequence analysis revealed that the BsrI digestion bands were derived from plasmid insertions comprising the gRNA1 sequence, which reintroduced the BsrI site and that K5 and K6 in fact had full allelic editing. (C) WT and the full allelic edited ex-plants, K10 and K33, were corroborated by IDAA of the larger (+SNP) region and the inner smaller (-SNP) region without length SNPs (Fig. 1C). WT peak positions are indicated by dotted lines. Lugol staining was used to analyse tuber starch from greenhouse grown plants (Supplementary Fig. 6). The presence of amylose gives rise to the dark blue colour (WT) tubers, while amylose free/’amylopectin only’ yields the red-brownish color. Additional shoots/ex-plants selected for IDAA analysis of which a subset was propagated to set tubers were also stained with lugol (Supplementary Fig. 7). (D) Sequence analysis confirmed that both K10 and K33 were full allelic edited. K10 had a 3 bp deletion in allele III, IV and in one of the alleles I or II and a 990 bp insertion (denoted ‘*’ left to the sequence, see also arrow on gel insertion (Fig. 2C)) in the other as also evident in the IDAA analysis displaying 1:1:1 ratio of the three chromatographic peaks. K33 had 6 bp and 5 bp deletions in allele I and II, a 4 bp deletion in allele IV and a 1 bp insertion in allele III.