CRISPR/Cas9 based genome editing of Phytoene desaturase (PDS) gene in chilli pepper (Capsicum annuum L.)

Abstract

An effective CRISPR/Cas9 reagent delivery system has been developed in a commercially significant crop, the chilli pepper using a construct harboring two distinct gRNAs targeting exons 14 and 15 of the Phytoene desaturase (CaPDS) gene, whose loss-of-function mutation causes a photo-bleaching phenotype and impairs the biosynthesis of carotenoids. The construct carrying two sgRNAs was observed to create visible albino phenotypes in cotyledons regenerating on a medium containing 80 mg/L kanamycin, and plants regenerated therefrom after biolistic-mediated transfer of CRISPR/Cas9 reagents into chilli pepper cells. Analysis of CRISPR/Cas9 genome-editing events, including kanamycin screening of mutants and assessing homozygosity using the T7 endonuclease assay (T7E1), revealed 62.5 % of transformed plants exhibited successful editing at the target region and displayed both albino and mosaic phenotypes. Interestingly, the sequence analysis showed that insertions and substitutions were present in all the plant lines in the targeted CaPDS region. The detected mutations were mostly 12- to 24-bp deletions that disrupted the exon-intron junction, along with base substitutions and the insertion of 1-bp at the protospacer adjacent motif (PAM) region of the target site. The reduction in essential photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoid) in knockout chilli pepper lines provided further evidence that the CaPDS gene had been functionally disrupted. In this present study, we report that the biolistic delivery of CRISPR/Cas9 reagents into chilli peppers is very effective and produces multiple mutation events in a short span of time.

Keywords: Biolistic delivery; CRISPR/Cas9; Chilli pepper; Phytoene desaturase; T7E1 assay.

Fig.1

CRISPR/Cas9-mediated modification of CaPDS in Chilli pepper (Capsicum annuum L). A. Schematic representations of the targeted Chilli pepper PDS gene. The number of exons has been mentioned below each box representing an exons and red lines represent the introns. The two different gRNAs (gRNA1 and gRNA2) were designed from the exon 14 and 15 respectively. The PAM sequence was highlighted in green color. B. The CRISPR/Cas9 binary vector containing 2XCaMV 35S promoter driven Cas9 protein employed for the stable biolistic transformation of chilli pepper. C. CRISPR/Cas9-mediated mutations of CaPDS in in vitro regenerated shoots of chilli pepper. Control Wild-type plants with completely green shoots. (i), edited albino shoots displaying a mosaic green and white shoot (ii &iii), and fully albino shoots, (iv) cultured on selection media (MS salts + B5 vitamins + 0.5 mg/L TDZ + 0.2 mg/L IAA + 80 mg/L kanamycin). Phenotype of CRISPR/Cas9 based CaPDS mutations in 5 w old in vitro chilli pepper plantlets (v) and leaf from mosaic and albino plants (vi). WT represents non-edited wild-type control plants with fully green tissues (leaf and shoots), 2,5, and 6 represents Capds edited lines showing full albino phenotype. 11 and 15 represents heterozygous Capds edited lines showing a mixture of green and white patches in leaves. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Assessment of CaPDS gene edited chilli pepper plants. A. T7 endonuclease assay for confirmation of mutation in transformed chilli pepper lines. PCR amplified target from control (WT) and transformed lines (2, 5, 6, 11, and 15) were subjected to T7E1 assay. The edited lines showed fragmentation of target into two bands and corresponding bands were absent in the WT plants. M, 1 kb plus DNA ladder; WT, genomic DNA from wild type chilli pepper cultivar G4; 2, 5, 6, 11, and 15 represents Capds edited lines. B. Summarization of biolistic‐delivered CRISPR/Cas9 based mutagenesis of the CaPDS gene in Chilli Pepper. Mutation events were confirmed after T7E1 assay. HM, Homozygous; and HE, Heterozygous. C. Sequence analysis of mutant chilli pepper plants. The target sequences of CaPDS are denoted in boldface and the PAM is indicated in the reference sequence for the wild type (WT). Below the WT sequence, sequences from each mutant line (Capds-2, and Capds-5, etc.) are shown. Substitutions are marked in red; insertions are highlighted in yellow, and deletions are represented by blue dashes. On the left side of the panel, the size and mutation type (insertion (+), deletion (−), or substitution (S)) are indicated. HM, Homozygous; and HE, Heterozygous Capds edited lines. D. Quantification of chlorophyll (Chl a), E. chlorophyll (Chl b), F. Total chlorophyll, and G. Carotenoid concentration in leaf tissue of Capds edited lines (Heterozygous lines, capds-11, and capds-15; Homozygous lines, capds-2, capds-5, and capds-6; Control, wild type chilli pepper cultivar G4). Pigment Concentration was calculated in mg/g of fresh weight. The student’s t-test was used to assess the data in relation to the WT control to determine statistical significance. Statistically significant differences are indicated by an asterisk (*p < 0.05, **p < 0.01). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)