Gene editing the phytoene desaturase alleles of Cavendish banana using CRISPR/Cas9

Abstract

Bananas are a staple food source and a major export commodity worldwide. The Cavendish dessert banana is a triploid AAA genome type and accounts for around 47% of global production. Being essentially sterile, genetic modification is perhaps the only pathway available to improve this cultivar. In this study, we used the CRISPR/Cas9 gene editing system to deliver a self-cleaving polycistronic guide RNA (gRNA) designed to target exon 1 of the Phytoene desaturase (PDS) gene in the Cavendish cultivar “Williams”. Genotyping of 19 independent events showed a 100% PDS modification rate primarily in the form of insertions (1–105 nt) or deletions (1–55 nt) (indels) at the predicted cleavage site. Tri-allelic disruptive modifications were observed in 63% of plants and resulted in both albinism and dwarfing. Pale green (16%) and wildtype green (21%) phenotypes generally correlated with in-frame indels in at least one of the three PDS alleles. Editing efficiency was dependent on both target site selection and Cas9 abundance. This is the first report of a highly effective CRISPR/Cas9 modification system using a polycistronic gRNA in Cavendish banana. Such an editing platform will be of considerable utility for the development of disease resistance and novel agro-traits in this commercially important cultivar into the future.

The application of the ‘Gene-deletor’ technology in banana

“…In order to eliminate concerns about the biological safety of GM crops and food safety, several molecular strategies have been applied to GM plants in recent years (Daniell 2002;Hills et al 2007;Kausch et al 2010), including cotransformation, transposable elements, recombinase deletion and CRISPR/Cas9 techniques (Khan et al 2010;Li et al 2007;Luo et al 2007;Lutz et al 2011;Naim et al 2018). However, these existing technologies are mainly used to eliminate resistance genes and reporter genes in transgenic plants, but they cannot effectively and specifically eliminate all exogenous genes, The existence of these exogenous genes may lead to gene escape and ecological hazards (Zuo et al 2001).…”

Breeding crops to feed 10 billion

“…In both peanut and banana, resynthesis of polyploid domesticates via the use of various diploid species and speed breeding would provide access to novel traits, including disease resistance, and rapid development of new varieties. Moreover, in banana, direct editing of current elite triploid cultivars could lead to interim rapid deployment of improved lines, obviating the cost and time needed to resynthesize triploids 58,59 .…”

Application of genetic modification and genome editing for developing climate‐smart banana

“…They used a single gRNA and obtained mutation efficiency of 59%. Later, higher editing efficiency (100%) of the PDS gene was reported in “Cavendish Williams” (AAA) using polycistronic gRNAs (Naim et al., ). Similarly, high mutation efficiency was also obtained in our laboratory using multiple gRNAs targeting the PDS gene (Figure ).…”