Targeting silencing of Root architecture associated 1 gene in rice using CRISPR/Cas9 system

Abstract

Rice (Oryza sativa L.) is the second most cultivated crop, feeding around two-thirds of the population after wheat worldwide (Pirdashti et al., 2009). The semi-aquatic nature of rice makes it more susceptible to water stress (Lafitte et al., 2006). Drought stress is the major obstacle in rice production, affecting around 45% of agricultural areas worldwide (Ambavaram et al., 2014; Todaka et al., 2015; Heinemann et al., 2015). Climate change has resulted in unpredictable, more frequent, and distressing weather patterns, which are likely to continue with increasing global warming (IPCC, 2023). The root structure of a plant plays a significant role in scavenging limited resources and coping with stressed conditions, remains unexplored. Researchers have identified genotypes with deep rooting habits that had a better edge towards growth and survival in stressful conditions before the outbreak of stress period exhibit better productivity (Venuprasad et al., 2002). The ROOT ARCHITECTURE ASSOCIATED 1 (OsRAA1) gene belongs to a new small protein family having GTP binding activity that negatively regulates a wide range of cellular processes and can be controlled by phytohormone signaling, particularly auxin (Ge et al., 2004; Xu et al., 2010). According to Ge et al. (2004), constitutive overexpression of the gene led to an increased number of adventitious roots, reduced gravitropic response, and reduced growth of primary roots in rice. The presence of two auxin response elements (AuRE) suggests that the OsRAA1 gene is probably regulated by auxin (Ge et al., 2004). Xu et al. (2010) found that degradation of RAA1 protein is associated with Anaphase Promoting Complex (APC), ascertaining a functional association between RAA1 and APC/C complex. RAA1 was established as a cell cycle candidate and an APC/C substrate for proteolysis. Degradation of RAA1 by the ubiquitin-proteosome structure is necessary for the transition of the cell cycle to anaphase during root growth in rice (Xu et al., 2010). Although a lot of research conveys the biochemical and regulatory role of OsRAA1, several gaps exist in comprehending the function of the OsRAA1 gene under various stress conditions. Thus, the main goal of this study is to knock out the OsRAA1 gene using CRISPR/Cas9 system to produce mutant rice lines with deeper rooting traits. In the current study, Oryza sativa ssp. japonica cv. Nipponbare was used as plant material. The sequence of OsRAA1 was retrieved from the Rice Annotation Project Database (RAP-DB) and Rice Genome Annotation Project (RGAP). The Locus ID of OsRAA1 was identified from RAP DB. The spacer sequence or guide sequences for the sgRNA that target potential protospacers were designed using the CRISPR-P v2.0 portal. Based on the GC content (60-80%), on-score value, off-target sites, and location in the genome, two 20 bp length gRNAs were selected. The gRNAs located towards the 5’ end of the coding sequence of the gene with fewer off target sites and located mostly on the first or initial exons, were preferred. The secondary structures of the gRNAs were also validated using RNA secondary structure prediction tool. In this study, guide RNA constructs were developed using the pRGEB32 vector. The gRNA scaffold is the vector flanked by BsaI restriction sites, enabling easy insertion of designed spacer sequences. The CRISPR/Cas9 construct for cloning was developed by annealing and ligating the gRNAs to the pRGEB32 vector followed by cloning in E. coli strain DH5α. The putative positive clones were identified by colony and plasmid PCR and further confirmed by Sanger sequencing with M13 reverse primer. The sequences of the clones were confirmed by analyzing the results using BioEdit v7.2 software. The multiple sequence alignment in the software confirmed the presence of both the gRNAs (OsRAA1#R1 and OsRAA1#R2). The vector-gRNA constructs, pRGEB32:OsRAA1#R1 and pRGEB32:OsRAA1#R2 were confirmed positive for cloning after sequence analysis and were further mobilized into Agrobacterium strain EHA105 by freeze-thaw method (Holsters et al., 1978). Randomly selected colonies were screened for inserts by colony PCR with gRNA specific and M13 reverse primer. Expected bands of size ~450 bp were observed on 1% agarose gel. Positive colonies of OsRAA1#R1(4) and OsRAA1#R2(6) constructs in EHA105 were then used for rice genetic transformation. The calli induced from the seeds of rice cv. Nipponbare were inoculated into MS medium supplemented with 2,4-D (2.5 mg/L) for callus induction. After 2 weeks of inoculation, seeds and shoots were removed and only the callus cultures were transferred to fresh callus induction media. The established calli were transformed by co-cultivation with positive EHA colonies. To remove excess Agrobacterium load, calli were washed with a solution of sterile water containing cefotaxime and timentin. The selection of transformed calli was carried out in three steps through selection media I, II, and III for hygromycin resistance. From selection media III, only the micro-calli of the proliferating calli were transferred to regeneration media. In the future, regenerated shoots will be analyzed for mutation and the expected mutant rice lines may confer drought tolerance.