Optical Control of Genome Editing by Photoactivatable Cas9

Takahiro Otabe; Yuta Nihongaki; Moritoshi Sato

doi:10.1007/978-1-0716-1441-9_13

Optical Control of Genome Editing by Photoactivatable Cas9

Methods Mol Biol. 2021:2312:225-233. doi: 10.1007/978-1-0716-1441-9_13.

Authors

Takahiro Otabe¹, Yuta Nihongaki², Moritoshi Sato³

Affiliations

¹ Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo, Japan.
² Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
³ Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo, Japan. cmsato@mail.ecc.u-tokyo.ac.jp.

PMID: 34228293
DOI: 10.1007/978-1-0716-1441-9_13

Abstract

The CRISPR-Cas9 system offers targeted genome manipulation with simplicity. Combining the CRISPR-Cas9 with optogenetics technology, we have engineered photoactivatable Cas9 to precisely control the genome sequence in a spatiotemporal manner. Here we provide a detailed protocol for optogenetic genome editing experiments using photoactivatable Cas9, including that for the generation of guide RNA vectors, light-mediated Cas9 activation, and quantification of genome editing efficiency in mammalian cells.

Keywords: CRISPR-Cas9; Genome editing; Genomic PCR; Guide RNA; HDR; NHEJ; Optogenetics; T7E1 assay.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Associated Protein 9 / genetics
CRISPR-Associated Protein 9 / metabolism
CRISPR-Associated Protein 9 / radiation effects*
CRISPR-Cas Systems / radiation effects*
Cell Culture Techniques
Clustered Regularly Interspaced Short Palindromic Repeats*
DNA End-Joining Repair
Gene Editing*
Gene Expression Regulation / radiation effects*
HEK293 Cells
Humans
INDEL Mutation
Light*
Optogenetics*
Point Mutation
RNA, Guide, CRISPR-Cas Systems / genetics
RNA, Guide, CRISPR-Cas Systems / metabolism

Substances

RNA, Guide, CRISPR-Cas Systems
CRISPR-Associated Protein 9