Generation of Cassava with Reduced Levels of Cyanogens Through CRISPR/Cas9 Targeted Mutagenesis of the Cytochrome P450 (CYP79D1) Gene

Abstract

Cassava (Manihot esculenta Crantz) is the world's most significant food root crop, providing carbohydrates to millions of subsistence farmers in Sub-Saharan Africa. Cassava leaves and roots, on the other hand, contain toxic quantities of cyanogenic glycosides. Cyanide poisoning is produced by the body's conversion of leftover cyanogens to cyanide. Inadequately processed cassava consumption, in combination with protein-deficient diets, has been associated to neurological problems. Acyanogenic cassava cultivars are essential to make cassava a consistently safe and acceptable food and commercial crop. Traditional breeding efforts have previously failed due to the crop's extended life cycle, high heterozygozity of allopolyploid plants, lack of flowering and variable seed germination in some varieties, inbreeding depression, and time-consuming and labor-intensive nature. Modern biotechnology techniques are critical for overcoming these constraints and generating more nutritional, pest-resistant, economically viable, and safe cassava varieties. In recent years, sequence-directed nucleases have emerged as the most successful strategy for crop improvement via gene-specific genome editing. The CRISPR/Cas system, which uses sequence-specific nucleases to transmit genetic modifications into crops for improvement, is the most effective and successful genome-editing tool. To establish CRISPR/Cas9 capabilities in cassava, the cytochrome P450 gene (CYP79D1) was targeted in the TMS 60444 cultivar using a construct incorporating gRNA targeting a site inside MeCYP79D1 exon 3. Agrobacterium tumefaciens strain GV3101 was utilized to infect immature cassava leaf lobes with the CRISPR/Cas9 construct. A PCR assay was utilized to confirm the incorporation of the gRNA/Cas9 into the cassava seedlings. PCR amplicons from putative transgenic cassava were sequenced to detect areas with edits. RT-PCR was used to examine the amounts of Cas9-mRNA expression in transgenic cassava lines. A picrate test and high-performance liquid chromatography (HPLC) were employed to assess the cyanide levels in transgenic cassava. To analyze the agro-morphological properties of the transgenic cassava, plant height, leaf length, leaf breadth, number of leaves per plant, and stalk length of wild type and transgenic TMS 60444 cassava plantlets were assessed in the greenhouse. The pCRISPR/Cas9-MeCYP79D1 design triggered the deletion in cotyledon-stage somatic embryos regenerating on hygromycin selection media, and the plants regenerated after CRISPR/Cas9 reagent transformation into cassava cells via Agrobacterium-mediated transformation. Mutagenesis assays were performed on eight plants (1.78 percent). The Cas9 gene was expressed by all regenerated plants. The desired gene was expressed in all of the regenerated plants. A sequencing analysis demonstrated that 8/8 (100%) of the plants studied had a mutation at the MeCYP79D1 gene, with deletions and substitutions discovered. Cyanide levels in putative transgenic cassava leaves were measured and found to be up to sevenfold lower. Cassava cyanide was not totally eliminated by deleting CYP79D1. The findings show that CRISPR/Cas9-mediated cassava genome editing is both efficient and straightforward. The new study provides the framework for CYP79D1/D2 gene targeting in Kenya's farmer-favored cassava, as well as other cassava genome-editing technologies.