Evaluation/Investigation of Cleavage Efficiency of sgRNA Targeting TFIIAɣ5 Gene in vitro

Zulkifli Ahmad Seman, Zuraida AB. Rahman, Yun Shin Sew, Norliza Abu Bakar

Abstract


The CRISPR/cas9 becomes a popular editing system in a wide variety of organisms. Potential candidates of sgRNA targeted single genomic site can be predicted by accessible web-based tools However, designed sgRNA displays different efficiencies in term of guiding Cas9 to introduce double-stranded breaks (DSB) at a specific site of target DNA. Thus, one critical step is to test whether the designed sgRNA could successfully edit the gene of interest. Ribonucleoprotein (RNP) in CRISPR format (Cas9-crRNA-tracrRNA), has been found to be an efficient and rapid system to minimise a likely mistake of CRISPR-mediated gene editing. In this study, designed crRNA has been used via the system to cleave TFIIAɣ5 gene in vitro. The system has successfully cleaved the gene in vitro into two fragments with approximately~195bp and ~125bp, of which the total fragment size is expected to be~320bp (the original size of TFIIAɣ5 gene). The results revealed that a high efficiency of the CRISPR cleavage system to pre-validate crRNA designed for the TFIIAɣ5 gene. Overall, one step in vitro cleavage of target DNA by CRISPR/Cas9 ribonucleoprotein complex can be potentially utilised for pre-validating designed crRNAs to determine its functionality and relative efficiency prior to utilising them in gene editing study via CRISPR system

Keywords


CRISPR-Cas9, in vitro transcription, in vitro cleavage sgRNA, ribonucleoprotein (RNP)

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References


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