CRISPR/Cas9-mediated efficient editing in phytoene desaturase (PDS) demonstrates precise manipulation in banana cv. Rasthali genome

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been reported for precise genome modification in many plants. In the current study, we demonstrate a successful mutation in phytoene desaturase (RAS-PDS) of banana cv. Rasthali using the CRISPR/Cas9 system. Two PDS genes were isolated from Rasthali (RAS-PDS1 and RAS-PDS2), and their protein sequence analysis confirmed that both PDS comprises conserved motifs for enzyme activity. Phylogenetic analysis of RAS-PDS1 and RAS-PDS2 revealed a close evolutionary relationship with other monocot species. The tissue-specific expression profile of RAS-PDS1 and RAS-PDS2 in Rasthali suggested differential regulation of the genes. A single 19-bp guide RNA (gRNA) was designed to target the conserved region of these two RAS-PDS and transformed with Cas9 in embryogenic cell suspension (ECS) cultures of cv. Rasthali. Complete albino and variegated phenotype were observed among regenerated plantlets. DNA sequencing of 13 plants confirmed the indels with 59% mutation frequency in RAS-PDS, suggesting activation of the non-homologous end-joining (NHEJ) pathway. The majority of mutations were either insertion (1-5) or deletion (1-4) of nucleotides near to protospacer adjacent motif (PAM). These mutations have created stop codons in RAS-PDS sequences which suggest premature termination of RAS-PDS protein synthesis. The decreased chlorophyll and total carotenoid contents were detected in mutant lines that revealed the functional disruption of both RAS-PDS genes. Our results demonstrate that genome editing through CRISPR/Cas9 can be applied as an efficient tool for banana genome modification.