An optimized FusX assembly-based technique to introduce mitochondrial TC-to-TT variations in human cell lines

Bibekananda Kar; Ankit Sabharwal; Santiago Restrepo-Castillo; Brandon W Simone; Karl J Clark; Stephen C Ekker

doi:10.1016/j.xpro.2022.101288

An optimized FusX assembly-based technique to introduce mitochondrial TC-to-TT variations in human cell lines

STAR Protoc. 2022 Apr 14;3(2):101288. doi: 10.1016/j.xpro.2022.101288. eCollection 2022 Jun 17.

Authors

Bibekananda Kar¹, Ankit Sabharwal¹, Santiago Restrepo-Castillo², Brandon W Simone¹, Karl J Clark¹, Stephen C Ekker¹

Affiliations

¹ Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA.
² Mayo Clinic Graduate School of Biomedical Sciences, Virology and Gene Therapy Track, Mayo Clinic, Rochester, MN, USA.

Abstract

The FusX TALE Based Editor (FusXTBE) is a programmable base editing platform that can introduce specific TC-to-TT variations in the mitochondrial DNA (mtDNA). Here, we provide a protocol describing the synthesis and testing of the FusXTBE plasmids in cultured human cell lines. This tool is designed to be easily modified to work in diverse applications where editing of mitochondrial DNA is desired. For complete details on the use and execution of this protocol, please refer to Sabharwal et al. (2021) and Ma et al. (2016).

Keywords: CRISPR; Cell Biology; Genetics; Molecular Biology; Sequencing.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Cell Line
DNA, Mitochondrial* / genetics
Humans
Mitochondria* / genetics

Substances

DNA, Mitochondrial

Grants and funding

U01 AI142773/AI/NIAID NIH HHS/United States