In Silico, Evolutionary Analysis and Designing guide RNA constructs for the Precise Modification of the Thermosensitive Genic Male Sterile (OsTMS5) Gene using the CRISPR/Cas9 System in Rice (Oryza sativaL.): A Comprehensive Study and Construct Development for Crop Improvement

Abstract The CRISPR/Cas9 system represents a state-of-the-art technology for precise genome editing in plants. In this study, we performed in silico and evolutionary analyses, as well as designed guide RNA constructs for the precise modification of the thermosensitive genic male sterile ( OsTMS5 ) gene using the CRISPR/Cas9 system in rice ( Oryza sativa L.). The OsTMS5 promoter harbors a diverse array of cis-elements, which are linked to light responsiveness, hormonal regulation, and stress-related signalling. Further, expression pattern of OsTMS5 revealed that OsTMS5 exhibited responsiveness to hormones and were activated across diverse tissues and developmental stages in rice. In addition, we meticulously designed guide RNAs (gRNAs) with a length of 20 base pairs. This design process was conducted using the CRISPR-P v2.0 online platform. The target of these gRNAs was the rice thermosensitive genic male sterile gene Os TMS5 . The selection of the top two gRNAs was made after conducting a thorough evaluation, which included assessing factors such as on-score value, minimum off-target score, GC content, potential off-target sites, and genomic location. In this study, two types of entry vectors were utilized, and the pMDC99 vector served as the destination vector for plant transformation. Following the annealing and ligation of the gRNAs through LR recombination, the resulting plasmid was named as "pMDC99-eSPCas9+OsU6- OsTMS5-target1 -gRNA+OsU6- OsTMS5-target2 -gRNA." Subsequently, this plasmid obtained from the third LR recombination was introduced into Agrobacterium EHA105 for the purpose of conducting rice transformation. Therefore, these constructs have the potential for use not only in molecular genetic analyses and molecular breeding in rice but also in a wide range of other crop species.