Robust CRISPR/Cas9 mediated genome editing tool for banana and plantain (Musa spp.)

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system enables precision editing of the genome of many plant species. Developing robust gene editing tools in banana and plantain would pave the way for the improvement of these crops. Here, we developed efficient CRISPR/Cas9 genome editing protocol for banana and plantain using multiple gRNAs targeting phytoene desaturase (PDS) gene. CRISPR/Cas9 construct containing two gRNAs was delivered into embryogenic cell suspension cultures of banana cultivar ‘Sukali Ndiizi’ and plantain cultivar ‘Gonja Manjaya’ by Agrobacterium-mediated transformation. The regenerated genome-edited events of ‘Sukali Ndiizi’ and ‘Gonja Manjaya’ showed albino and variegated phenotypes indicating mutations at the targeted sites disrupting the function of PDS gene. The majority of events (52/77 for ‘Sukali Ndiizi’ and 16/17 for ‘Gonja Manjaya’) were albino. Sequencing of the target sites confirmed presence of indels in all the 18 events sequenced demonstrating mutation efficiency of 100 % in both cultivars. The majority of events (6/8) of ‘Gonja Manjaya’ showed indels at both the target sites of PDS gene, however only 2/10 events of ‘Sukali Ndiizi’ showed indels at both sites, with one event (S24) having a knockout of large fragment (723 bp) indicating that both gRNAs were effective. Several of the albino events of both ‘Sukali Ndiizi’ and ‘Gonja Manjaya’ showed homozygous mutations. Further sequencing of four potential off-target sites in five events showed no mutations indicating CRISPR/Cas9 based editing in banana and plantain is targeted and precise with a very low probability of off-target sites. This study could provide a methodological framework for single or multiple knockouts in banana and plantain.