۱- Introduction

۲- Methodology

۳- Results

۴- Discussion

۵- Conclusion

References

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.

Introduction

Genome editing technologies using specific nucleases have been developed as effective genetic engineering methods for targeted mutations at specific sites in the genome of an organism [1]. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated protein 9) based genome editing is the emerging most powerful tool with ability to create targeted mutations in the genome of crops and to understand the functional biology. These nucleases respond in a target-specific manner and induce a double-strand break (DSB) in the gene sequence [2]. The DSB is repaired either by homologous recombination (HR) or errorprone non-homologous end-joining (NHEJ) mechanisms, which may lead to disruption of gene function [3]. CRISPR/Cas9 system has emerged as a potent genome editing tool due to its simplicity, design flexibility, and high efficiency, and has been applied successfully in several plant species [4]. It is based on the type II CRISPR/Cas immune system of bacteria that protects against invading DNA viruses and/or plasmids. The CRISPR/Cas9 molecular immunity system is made up of the Cas9 endonuclease from Streptococcus pyogenes and a single guide RNA (sgRNA). The sgRNA directs the Cas9 endonuclease to a target sequence complementary to the 20 nucleotides preceding the protospacer-adjacent motif (PAM) NGG or NAG, which is required for Cas9 activity [5-6]. Multiplexing of CRISPR/Cas9 system by integrating two or more gRNAs, which are distance apart, is useful in generating large deletions in plants [7].