The technology based on CRISPR/Cas9, one of the potential solutions recognized for addressing the worsening situation of fusarium wilt caused by Fusarium oxysporum f. sp. cubense ‘Tropical Race 4’ (TR4) plaguing the Philippine banana industry, still lacks information on its use to target host resistance in Philippine bananas. This study reports the elucidation of the gene sequence of chitinase homologs chit6 and chac in the leading Philippine dessert banana varieties, ‘Lakatan’ and ‘Latundan,’ and the design, selection, and pre-validation of sgRNAs using in vitro cleavage assay. Multiple sequence alignment revealed the conserved sites, SNPs, and indels. Evolutionary analysis disclosed that the pattern of nucleotide substitution resulted in an overall bias in favor of adenine and thymine changing to cytosine and guanine. It said transitions outnumber transversions typical of clonally propagated crops. The in silico prediction initially identified a total of 58 sgRNAs for chit6 and 68 sgRNAs for chac, with 58.62% and 58.97% found across ‘Latundan’ and ‘Lakatan’/’Mapilak’ backgrounds, respectively. A design criteria imposition for plants and consideration of the predicted efficiency and localization of sgRNAs along the targets narrowed down the sgRNAs. Four selected efficient sgRNAs were demonstrated to cleave all targets under in vitro assay with Cas9, showing its potential for gene editing by the SDN-1 or SDN-2 mechanism. With unavailable protocols for embryogenic cell suspension and corresponding delivery systems for these varieties, this in vitro approach provides a strategy to identify potential sgRNAs to streamline resources for the gene-editing pipeline and a guide to employing CRISPR/Cas9

Keywords: Banana, chitinase, CRISPR/Cas9, in vitro cleavage assay, Foc TR4, sequence variation

Key findings: This reports the elucidation of the full-length sequence of the chitinase gene homologs chit6 and chac in the leading Philippine dessert banana varieties ‘Lakatan,’ its mutant derivative ‘Mapilak,’ and ‘Latundan,’ revealing conserved sites, point mutations, and their classification. The selection criteria employing in silico prediction resulted in designing and selecting efficient sgRNAs that exhibited cleavage of designated targets in all genomic backgrounds evaluated under in vitro cleavage assay, offering valuable information for prospective research harnessing CRISPR/Cas9 to manipulate chit6 and chac for breeding TR4 resistance or functional studies in ‘Lakatan’/’Mapilak’ and ‘Latundan.’