A Simple gRNA in vitro Screening Method

doi:10.3969/j.issn.1674-5817.2018.02.009

Abstract

Abstract: Objective To explore a simple guide ribonucleic acid (gRNA) in vitro screening method.Methods gRNAs target rabbits torsin family 2 member A (TOR2A) gene were used as an example and screened out two efficiency gRNAs.First,design the gRNAs by online software.Three or more gRNA sequences were choose and inserted behind T7 promoter.After in vitro transcription,gRNAs were purified and then quantified by NanoDrop.Secondly,a pair of primers were designed to amplify about 300~1000 bp genomic sequence which included the gRNAs target sequences.The last step,PCR products,gRNA candidate and Cas9 nuclease were incubated together and then were checked by agarose gel electrophoresis.Results In this study,three gRNAs that target rabbits TOR2A gene were screened.The gRNA1 was failed to guide Cas9 to cleave the PCR products that contain the target sequences,while gRNA2 and gRNA3 were identified to successfully guide the Cas9 cleavage.Conclusion The gRNA in vitro screening method that used in this experiment was simple and efficient.

Key words: Guide ribonucleic acid (gRNA), In vitro screening, Gene editing

CLC Number:

Q95-33

CHENG Da-xin, XU Li-ran, YU Qing-qing, GAO Shou-cui, WANG Xiao-jing, LIU Yi, LIU En-qi, ZHAO Si-hai. A Simple gRNA in vitro Screening Method[J]. Laboratory Animal and Comparative Medicine, 2018, 38(2): 126-129.

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