Methods for in vitro CRISPR/CasRx-Mediated RNA Editing

Yu-Fan Chuang; Peng-Yuan Wang; Satheesh Kumar; Suraj Lama; Fan-Li Lin; Guei-Sheung Liu

doi:10.3389/fcell.2021.667879

Methods for in vitro CRISPR/CasRx-Mediated RNA Editing

Front Cell Dev Biol. 2021 Jun 11:9:667879. doi: 10.3389/fcell.2021.667879. eCollection 2021.

Authors

Yu-Fan Chuang^{1

2}, Peng-Yuan Wang¹, Satheesh Kumar², Suraj Lama², Fan-Li Lin^{1

2}, Guei-Sheung Liu^{2

3

4}

Affiliations

¹ Shenzhen Key Laboratory of Biomimetic Materials and Cellular Immunomodulation, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.
² Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS, Australia.
³ Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, VIC, Australia.
⁴ Aier Eye Institute, Changsha, China.

Abstract

Specific changes in the genome have been accomplished by the revolutionary gene-editing tool known as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system. The advent of programmable RNA editing CRISPR/Cas nucleases has made this gene-editing tool safer and more precise. Specifically, CasRx, a family member of the Cas13d family, has shown great therapeutic potential. Here, we describe the in vitro methods of utilizing this powerful RNA editing platform and determine the RNA editing efficiencies for CasRx with different forms of guide RNAs (also known as gRNA or sgRNA).

Keywords: AAV (Adeno-associated virus); CRISPR/Cas13d; CasRx; RNA editing; VEGF (Vascular endothelial growth factor).