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Optimized guide RNA design and evaluation of CRISPR/Cas9 cleavage efficiency in LEP gene knockout

Nguyen Le Tram Anh, Au Duong Tuyet Mai, Nguyen Thi Thuong Huyen, Thai Ke Quan, Nguyen Dang Quan

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Int. J. Biosci.19( 4), 193-206, October 2021

DOI: http://dx.doi.org/10.12692/ijb/19.4.193-206


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Streptococcus pyogenes (SpCas9)-derived CRISPR/Cas9 system (SpCas9) is now a valuable tool for gene-editing technology, although their widespread applications is obstructed by a backwardness of knowledge about the activity of guide RNA (gRNA). A specific gRNA must maximize on-target (effective Cas protein guidance) while minimizing off-target sites. Balancing these two requirements is a challenging task. Therefore, the optimization of the sgRNA design is essential for the application of CRISPR/Cas9 towards knockout gene modeling, significantly lowering prices and timeoptimizing to generate genetically modified animals. In this study, sgRNAs had been screened and validated in silico before inserted into the pX330 vectors in vitro validation. We randomly screened 468 single-stranded RNA sequences in the protein-coding regions of the lep gene. The sgRNAs were evaluated for their pairing in the target sequence region and validated for genomic random pairing activity. Next, we determined their activity on the pX330 plasmid by assessing the fluorescence expression in the HEK293 cell line. Of 468 screened sgRNAs, two sgRNA 28 and sgRNA 95 showed the most specific shear potential in vitro model. These sgRNAs will be checked further for production of CRISPR/Cas-generated lep KO mice as animal model for studying obesity and diabetes in the future.


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Optimized guide RNA design and evaluation of CRISPR/Cas9 cleavage efficiency in LEP gene knockout

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