PCR-mediated One-day Synthesis of Guide RNA for the CRISPR/Cas9 System

Naim Hassan; Farhana Easmin; Keisuke Ekino; Satoshi Harashima

doi:10.21769/BioProtoc.4082

PCR-mediated One-day Synthesis of Guide RNA for the CRISPR/Cas9 System

Bio Protoc. 2021 Jul 5;11(13):e4082. doi: 10.21769/BioProtoc.4082.

Authors

Naim Hassan¹, Farhana Easmin¹, Keisuke Ekino¹, Satoshi Harashima¹

Affiliation

¹ Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto, Japan.

Abstract

Nowadays, CRISPR (clustered regularly interspaced short palindromic repeats) and the CRISPR-associated protein (Cas9) system play a major role in genome editing. To target the desired sequence of the genome successfully, guide RNA (gRNA) is indispensable for the CRISPR/Cas9 system. To express gRNA, a plasmid expressing the gRNA sequence is typically constructed; however, construction of plasmids involves much time and labor. In this study, we propose a novel procedure to express gRNA via a much simpler method that we call gRNA-TES (gRNA-transient expression system). This method employs only PCR, and all the steps including PCR and yeast transformation can be completed within 1 day. In comparison with the plasmid-based gRNA delivery system, the performance of gRNA-TES is more effective, and its total time and cost are significantly reduced.

Keywords: CRISPR/Cas9; Genome editing; Guide RNA; PCR-based; Yeast.