Generation of Efficient Knock-in Mouse and Human Pluripotent Stem Cells Using CRISPR-Cas9

Tatsuya Anzai; Hiromasa Hara; Nawin Chanthra; Taketaro Sadahiro; Masaki Ieda; Yutaka Hanazono; Hideki Uosaki

doi:10.1007/978-1-0716-1484-6_22

Generation of Efficient Knock-in Mouse and Human Pluripotent Stem Cells Using CRISPR-Cas9

Methods Mol Biol. 2021:2320:247-259. doi: 10.1007/978-1-0716-1484-6_22.

Authors

Tatsuya Anzai^#^{1

2}, Hiromasa Hara^#^{1

3}, Nawin Chanthra¹, Taketaro Sadahiro⁴, Masaki Ieda⁴, Yutaka Hanazono^{1

5}, Hideki Uosaki^{6

7}

Affiliations

¹ Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan.
² Department of Pediatrics, Jichi Medical University, Tochigi, Japan.
³ Animal Resource Laboratory, Center for Development of Advanced Medical Technology, Jichi Medical University, Tochigi, Japan.
⁴ Department of Cardiology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
⁵ Translational Research Laboratory, Center for Development of Advanced Medical Technology, Jichi Medical University, Tochigi, Japan.
⁶ Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan. [email protected].
⁷ Translational Research Laboratory, Center for Development of Advanced Medical Technology, Jichi Medical University, Tochigi, Japan. [email protected].

^# Contributed equally.

PMID: 34302663
DOI: 10.1007/978-1-0716-1484-6_22

Abstract

A knock-in can generate fluorescent or Cre-reporter under the control of an endogenous promoter. It also generates knock-out or tagged-protein with fluorescent protein and short tags for tracking and purification. Recent advances in genome editing with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) significantly increased the efficiencies of making knock-in cells. Here we describe the detailed protocols of generating knock-in mouse and human pluripotent stem cells (PSCs) by electroporation and lipofection, respectively.

Keywords: CRISPR-Cas9; Genome editing; Homology-directed repair; Knock-in; Pluripotent stem cells.

Publication types

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

MeSH terms

Animals
CRISPR-Cas Systems*
Cells, Cultured
Clone Cells
Culture Media
DNA Primers
Drug Resistance / genetics
Electroporation
Embryonic Stem Cells / cytology
Gene Editing / methods
Gene Knock-In Techniques / methods*
Genes, Reporter
Humans
Induced Pluripotent Stem Cells / cytology*
Induced Pluripotent Stem Cells / metabolism
Mice
Mice, Inbred C57BL
Puromycin / pharmacology
RNA, Guide, CRISPR-Cas Systems / genetics
Recombinational DNA Repair / genetics

Substances

Culture Media
DNA Primers
RNA, Guide, CRISPR-Cas Systems
Puromycin