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CRISPR/Cas9 is a technique in molecular biology yielding effective DNA cleavage. For effective DNA cutting, CRISPR/Cas9 system requires appropriate single guide RNA (sgRNA) and a functional sgRNA binding site on Cas9 enzyme. The goal of this project is to design a functional CRISPR/Cas9 test tube model system for further research in the lab. The costs and inaccuracy associated with RNA synthesis constitute a limitation for CRISPR technology use. To this end, a truncated functional sgRNA is desired. In this study, we will also design a shortened sgRNA that is missing the trans-activating CRISPR RNA (tracrRNA) portion. The cleaving potential of sgRNA system composed entirely of shortened sgRNA is evaluated thermodynamically using isothermal titration calorimetry (ITC) and in vitro gel cleavage assays. The findings indicate cleavage ineffectiveness of the designed RNA, while full sgRNA is functioning normally. The ITC data suggests no noticeable difference in the thermodynamic interactions between the modified sgRNA cas9 system and the full sgRNA cas9 system.
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Ratnikov, Danil, "Design and Characterization of an In Vitro CRISPR/Cas9 Model System" (2021). Senior Projects Spring 2021. 241.
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