PJB-2018-1780
An efficient transient assay for CRISPR CAS9 system delivering targeted mutation using synthetic oligo SgRNA in soybean (Glycine max)
Noor Al Amin, Naveed Ahmad, Wu Nan, Fu Xiuming, Wang Nan Bo Xiaoxue, Ma Tong and Wang Piwu
Abstract
In recent years CRISPR Cas9 system has been proved as a versatile genetic tool for efficient target mutation in a variety of plants. (CRISPR)-associated Cas9 Nuclease system can efficiently incorporate high frequency targeted mutation wherein Cas9 act as endonuclease coupled with an oligosgRNA sequence. This RNA guided nuclease complex causes a Cas9-directed nick production in nuclear DNA in the form of double-strand breaks (DSBs). Importantly, our findings revealed various factors affecting the specificity and activity of small guide RNAs (sgRNAs), casting a new light on flexibility and efficiency of sgRNA encountering with targeted mutagenesis in various agronomical crops. We demonstrated an efficient protocol and online resources used for the optimization of best possible sgRNA designing and it’s (PAM) Protospacer Adjacent Motif to achieve targeted mutation in FAD2-1A. Collectively our findings confirmed an efficient transient expression assay of FAD2-1A gene in isolated protoplasts via PEG mediated transformation. High throughput protein modeling revealed the successful integration of targeted mutation induced by Small guide RNA induced CRISPR Cas9 system. Preferably, these findings can be replicated in designing multiple novel genome models to facilitate efficient target mutagenesis and gene expression approaches in several other higher plants.
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