Identification of early flowering mutant gene in Phalaenopsis amabilis (L.) Blume for sgRNA construction in CRISPR/Cas9 genome editing system; Identificação do gene mutante de floração precoce em Phalaenopsis amabilis (L.) Blume para construção de sgRNA no sistema de edição de genoma CRISPR/Cas9

Phalaenopsis amabilis (L.) Blume commonly called Moth Orchid (Orchidaceae) is a natural orchid species designated
as the National Flower of Indonesia for its beautiful flower shape and long-lasting flowering period. Basically,
P. amabilis has a long vegetative phase that cause late flowering, about 2 to 3 years for flowering, hence a method
to shorten vegetative period is desired. The latest technological approach that can be used to accelerate flowering
of P. amabilis is the CRISPR/Cas9 genome editing method to inactivate the GAI (Gibberellic Acid Insensitive) gene as
a mutant gene that can accelerate the regulation of FLOWERING TIME (FT) genes flowering biosynthesis pathway.
The approach that needs to be taken is to silence the GAI gene with a knockout system which begins with identifying
and characterizing the GAI target gene in the P. amabilis which will be used as a single guide RNA. CRISPR/Cas9
mediated knockout efficiency is highly dependent on the properties of the sgRNA used. SgRNA consists of a target
sequence, determining its specificity performance. We executed phylogenetic clustering for the PaGAI protein
with closely related orchid species such as Dendrobium capra, Dendrobium cultivars and Cymbidium sinensis.
SWISS-Model as tool webserver for protein structure homology modeling. Results show that P. amabilis has a
specific domain with the occurrence of point mutations in the two conservative domains. Therefore, a single guide
RNA reconstruction needs to be implemented