CRISPR/Cas9-mediated editing of PHYTOENE DESATURASE gene in onion (Allium cepa L.)

Abstract

Introduction: Clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) is a precise genome editing tool used to introduce genetic modifications in a wide range of crop species. Thus far, there is no report of CRISPR/Cas9-mediated genome editing in onions (Allium cepa L.).

Methods: In the present study, we targeted two exons of the gene coding for Phytoene desaturase (AcPDS) in onion cv. Bhima Super. The sgRNA-carrying constructs were co-cultivated with 8-week-old embryogenic calli using an Agrobacterium-mediated transformation protocol and incubated on the media without hygromycin B selection.

Results and discussion: Out of the total 617 co-cultivated calli, 21 (3.4%) regenerated shoots exhibited three distinct phenotypes: albino, chimeric, and pale green; in comparison to the wild-type non-transformed regenerated shoots. Total chlorophyll content was drastically reduced in albino shoots and significantly decreased in chimeric shoots. Out of the six Cas9 gene PCR-confirmed regenerated shoots, two exhibited the albino phenotype due to insertions/deletions (InDels) and substitution-based mutations in and around the AcPDS target sites. Deep amplicon sequencing revealed a significantly variable InDel frequency between two sgRNAs, ranging from 1.2% to 63.4%, along with a 53.4% substitution frequency. The mutation of the AcPDS gene generated a visually detectable albino phenotype, thus confirming the successful editing of the AcPDS gene. This is the first time a CRISPR/Cas9-mediated genome editing protocol has been successfully established in onion, with the AcPDS gene serving as an example. This study will provide the necessary momentum for researchers to further basic and applied research on onions.

Keywords: CRISPR/Cas9; PDS; chlorophyll; genome editing; onion transformation.

Figure 1

In silico analysis of AcPDS gene, (A) Schematic representation of conserved amino oxidase domain in AcPDS protein. (B) Phylogenetic relationship of AcPDS protein of Allium cepa L. cv. B. Super with PDS protein sequences from 11 dicot and seven monocot species was constructed using the MEGA 11 tool.

Figure 2

CRISPR/Cas9-mediated genome-editing of onion by targeting phytoene desaturase (AcPDS) gene. (A) Schematic presentation of binary vector pRGEB31-AcPDS E3 target. (B) Schematic presentation of binary vector pRGEB31-AcPDS E4 target. (C) Protospacer sequences for exon 3 target (cyan highlighted) and for exon 4 target (yellow highlighted); PAM sequences in pink highlighted. (D) Schematic representation of gene structure of AcPDS isolated from onion cv. B. Super, showing the gRNA targets. Blue blocks indicate exons of the gene. (E) Co-cultivated regenerated shoots showing three distinct phenotypes, viz. albino (regenerated shoots #3 and #41), chimeric (regenerated shoots #1 and #2), and pale green (regenerated shoots #78 and #83). (F) Nontransformed control regenerated shoots showing dark green shoots. (G) Regenerated plantlets showing albino and normal green wild-type phenotypes. (H) Chlorophyll content estimation in the three phenotypically distinct regenerated shoots. Bars represent the mean ± SD of the three independent replicates (n = 3). (I) Representative PCR analysis of phenotypically distinct shoots to confirm the presence of Cas9 gene. (J) PCR analysis of albino and chimeric shoots showing varying sizes of amplicons in the target regions. (L, 1 kb plus ladder in (H) and 50 bp ladder in (I); P, pRGEB31-plasmid (positive control); BS: onion cv. B. Super (negative control); 1, 2, 3, 8, 41, 48, 78, and 83: phenotypically distinct co-cultivated regenerated shoots). (K, L) Schematic chromatogram presentation of site-specific mutations of AcPDS induced by exon 3 and exon 4 sgRNAs in albino shoots regenerated from callus #3 and #41, respectively. Dot lines specify the type of mutation, and parentheses indicates large (4 nt and 10 nt) deletions. PAM is in red dots, and protospacers are underlined.