CRISPR/Cas9 Construct Development for Knock-out of Root Architecture Associated 1 Gene in Rice (Oryza sativa L.)

Deeper rooting is an important trait for sustained yield under water-limited environments, as drought exhibits serious threat to rice (Oryza sativa L.) production. To mitigate the challenge, we aimed to modify the Oryza sativa ROOT ARCHITECTURE ASSOCIATED 1 (OsRAA1) gene, one of the key regulators of the cell cycle that manipulates root development, using CRISPR/Cas9 system. We used CRISPR-P v2.0 tool to select two spacer sequences targeting OsRAA1 gene. Validation of the guide RNAs (gRNAs) was conducted by analyzing their secondary structure using RNA secondary structure prediction tool. Two efficient sgRNAs were selected considering their GC content, on-target values, location on the gene, off-target sites and their location, secondary structures etc., adjacent to the Protospacer Adjacent Motif (PAM), NGG. The binary vector pRGEB32, with BsaI restriction site driven by U3 snoRNA promoter (POsU3p), was employed for cloning of sgRNAs and Cas9 by rice ubiquitin promoter (PUBIp). We successfully cloned the spacer sequences targeting OsRAA1 into a binary vector, pRGEB32. The constructs were transformed into E. coli strain DH5α and Agrobacterium tumefaciens strain EHA105. The developed gene cassettes can be used for editing of OsRAA1 gene in rice. By editing, we seek to enhance the root architecture and increase the drought-stress tolerance ability of rice. This research represents a significant step towards developing drought-tolerant rice varieties, a critical solution for ensuring global food security in the face of climate change.