Multiplex CRISPR/Cas9-mediated knockout of the phytoene desaturase gene in Coffea canephora

Abstract

Coffea canephora (2n = 2x = 22 chromosomes) is a species with extensive genetic diversity and desirable agronomic traits for coffee breeding programs. However, obtaining a new coffee cultivar through conventional breeding techniques may require more than 30 years of crossing cycles and selection, which hampers the effort of keeping up with market demands and rapidly proposing more resilient to climate change varieties. Although, the application of modern biotechnology tools such as precision genetic engineering technologies may enable a faster cultivar development process. Therefore, we aimed to validate the CRISPR/Cas9 system to generate mutations on a selected genotype of C. canephora, the clone 14. Embryogenic calli and a multiplex binary vector containing two sgRNAs targeting different exons of the CcPDS gene were used. The sgRNAs were under the C. canephora U6 promoter regulation. The target gene encodes phytoene desaturase, an enzyme essential for photosynthesis involved in β-carotene biosynthesis. Somatic seedlings and embryos with albino, variegated and green phenotypes regenerated after Agrobacterium tumefaciens-mediated genetic transformation were analyzed by verifying the insertion of the Cas9 gene and later by sequencing the sgRNAs target regions in the genome of Robusta modified seedlings. Among them, 77% had the expected mutations, and of which, 50% of them had at least one target with a homozygous mutation. The genotype, temperature of co-cultivation with the bacteria, and light intensity used for subsequent embryo regeneration appeared to strongly influence the successful regeneration of plants with a mutated CcPDS gene in the Coffea genus.

Figure 1

Coffee plant material regenerated after CRISPR/Cas9-mediated knockout of the phytoene desaturase gene using Agrobacterium tumefaciens-mediated transformation. Images representing the development of embryogenic calli, transformed somatic embryos and somatic seedlings and the different embryo phenotypes obtained. (A) Hygromycin-resistant yellowish embryogenic calli growing from oxidized primary calli formed on the explant. (B) Albino, green, and variegated cotyledonary transformed embryos. (C) WT somatic seedling. (D) One of the albino somatic seedlings obtained after CRISPR/Cas9 transformation; (E,F) Aspect of transformed embryos with abnormal development of albino (E) and variegated (F) cotyledons.

Figure 2

Schematic representation of CcPDS gene structure. The alignment results for the reference gene (Cc04_g00540—chr4:412113..419155, Coffee Genome Hub, http://coffee-genome.org/), control (WT), and transformed seedlings CcPDS sequences. CcPDS: reference gene; a: comparisons of sequencing results for target 1, at the exon 3; b: comparisons of sequencing results for target 2, at exon 5. Abbreviations refer to the following samples: CcPDS: reference gene sequence from Coffee Genome Hub; WT: control, untransformed; P: albino seedlings (transformed); G: green seedlings (transformed); and S: variegated seedlings (transformed). At the sequence results, each color represents the following: Blue: sgRNA target sequence; Yellow: PAM sequence; Black: base substitutions; Pink: base insertions; Green: base deletions; Black scissor with vertical dashed lines: Cas9 cleavage site. Each number in parentheses represents the number of sequenced colonies for a given sample in which the same sequencing result was found. In the case of WT and green seedlings, the number of sequenced seedlings is also represented.

Figure 3

Schematic representation of the amino acid sequences translated from the edited CcPDS gene sequences after coffee genetic transformation using the CRISPR/Cas9 methodology. Altered amino acids are represented in orange, the bar represents an amino acid deletion, and the asterisk represents a stop codon. The underlined regions in the control sequence (WT) represent the amino acids corresponding to the regions of the two sgRNAs.