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.

CRISPR technology towards genome editing of the perennial and semi-perennial crops citrus, coffee and sugarcane

“…Among them, we may cite soybean ( Glycine max ) ( Jacobs et al., 2015 ), cassava ( Manihot esculenta ) ( Odipio et al., 2017 ), banana ( Musa spp.) ( Kaur et al., 2018 ), coffee ( Coffea canephora ) ( Casarin et al., 2022 ), grapevine ( Vitis vinifera ) ( Malnoy et al., 2016 ), rice ( Oryza sativa ) ( Lowder et al., 2015 ), wheat ( Triticum aestivum ) ( Upadhyay et al., 2013 ) and sweet orange ( Citrus sinensis ) ( Wang et al., 2014 ). And the list grows quickly, reaching species once intractable by conventional genetic methodologies.…”

“…Breeding perennial plants is time-consuming, and the possibility to modify specific traits by editing target genes with no other potential change in the genetic background is very appealing. In coffee, gene editing studies available so far ( Table 3 ) are merely proof of concepts in C. canephora plants ( Breitler et al., 2018 ; Casarin et al., 2022 ). In both studies, PDS was used as a target gene and the CRISPR machinery was delivered to embryogenic callus by A. tumefaciens .…”

“…In both studies, PDS was used as a target gene and the CRISPR machinery was delivered to embryogenic callus by A. tumefaciens . The overall editing efficiencies were 30.4% ( Breitler et al., 2018 ) and 76.9% ( Casarin et al., 2022 ), with 7.6% and 54% of homozygous mutations, respectively. Besides albine and variegated seedlings, Casarin et al.…”

“…The overall editing efficiencies were 30.4% ( Breitler et al., 2018 ) and 76.9% ( Casarin et al., 2022 ), with 7.6% and 54% of homozygous mutations, respectively. Besides albine and variegated seedlings, Casarin et al. (2022) also observed seedlings with abnormal cotyledon and root development, and limited growth.…”

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

“… 26 It also allows for simple screening of mutant lines. 27 , 28 Recent studies have shown the establishment of efficient genome editing using the PDS gene through the CRISPR/Cas9 system in several crops, including potato, 24 pea, 29 papaya, 30 coffee, 31 chickpea, 32 hop, 23 onion 25 and squash. 33 Nevertheless, the development of a biolistic-mediated CRISPR/Cas9-reagent delivery system using the PDS gene in chilli pepper has not yet been reported.…”