CRISPR-Cas9 has been reported for precise genome modification in many
plants. However, it has not been reported in banana. To prove this concept,
researchers from the National Agri-Food Biotechnology Institute in India
aims to perform a successful mutation in the phytoene desaturase gene
(RAS-PDS) of banana cv. Rasthali using the CRISPR-Cas9 system.
Two PDS genes, RAS-PDS1 and RAS-PDS2, were isolated from Rasthali and
analysis confirmed that both PDS comprises conserved motifs for enzyme
activity. A single guide RNA (sgRNA) was designed to target the conserved
region of these two RAS-PDS genes and transformed with Cas9 in embryogenic
cell suspension (ECS) cultures.
Complete albino and variegated phenotype were observed among the generated
plantlets. DNA sequencing of 13 plants confirmed indels with a 59% mutation
frequency in RAS-PDS. The decreased chlorophyll and total carotenoid
contents were observed in mutant lines, revealing the functional disruption
of both RAS-PDS genes.
These results demonstrate that genome editing through CRISPR-Cas9 can be
applied for banana genome modification.