Executive Summary : | Soybean is an annual legume crop having high economic importance worldwide. It has been a preferred crop for attempting genetic modification and even with the reoriented focus towards CRISPR-based genome editing soybean still remains relevant. The success of genome editing largely depends on simultaneous-, elevated- and preferably equimolar- expression of, the Cas9 nuclease and the single guide RNA (sgRNA). At present, there are four different strategies which have been demonstrated to express Cas9 and sgRNA in plants. Mixed Dual Promoter (MDP) system is the most common expression strategy as of now which utilizes a RNA polymerase II (Poll II) promoter to drive Cas9 gene while a RNA Polymerase III (Pol III) promoter to drive sgRNA. But the strength Pol III promoters is much lesser than Pol II promoters. Hence, the expression level of Cas9 and sgRNA in this case can never be matched which ultimately limits the overall editing efficiency. Besides, RNA Polymerase III has naturally evolved to transcribe short RNA transcripts making it a less attractive option to transcribe polycistronic sgRNA during CRISPR-multiplexing. Using a Duel Pol II Promoter (DPIIP) system, where the Pol III promoter of MDP is replaced by a Pol II promoter, can circumvent these problems but it makes the overall size of the plant transformation vector too big to handle. Another available option in this regard is known as Single Transcription Unit (STU) system, where both Cas9 and sgRNA is driven by a strong Pol II promoter. But it has found to possess reduced editing efficiency than expected due to the non-synchrony between the molar ration of produced sgRNA and Cas9 enzyme. However, the most recent development in this area is the use of Bidirectional Promoter (BiP) system which utilize a single- but bidirectional- Pol II promoter. BiP system combines the ‘Pros’ of STU and DPIIP leaving the ‘cons’ apart. Like STU system, BiP system can also simultaneously drive the expression of two different expression units (sgRNA and Cas9 in this case) when the units flank the BiP and are placed in opposite orientations to each other. But, like DPIIP system, it also provide the flexibility to fine tune expression level individual units independently by using different 3′-UTR and/or terminators. Besides, the use of a single promoter also economize on the overall size of the plant transformation vector. To the best of our knowledge, there is no report of identification and cloning of any native BiP from soybean which might further boost the overall efficiency of CRISPR-system in soybean. Objectives: • Identification and isolation of bidirectional promoter from soybean capable of driving gene expression in two opposite directions. • Construction of binary plant transformation vector containing a strategically placed bidirectional promoter • Investigating the efficiency of Bidirectional Promoter System in generating CRISPR-Cas9 based knockouts. |