Sequence Variability and in Vitro Cleavage of Chitinase Homologs Toward Crispr/Cas9-Based Genome-Editing in Philippine Bananas

Abstract

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 for elucidation of the functional role of chit6 and chac in host resistance response to TR4 in bananas.