Efficient bi-allelic tagging in human induced pluripotent stem cells using CRISPR

Xingjie Ren; Maya Asami Takagi; Yin Shen

doi:10.1016/j.xpro.2023.102084

Efficient bi-allelic tagging in human induced pluripotent stem cells using CRISPR

STAR Protoc. 2023 Mar 17;4(1):102084. doi: 10.1016/j.xpro.2023.102084. Epub 2023 Feb 1.

Authors

Xingjie Ren¹, Maya Asami Takagi², Yin Shen³

Affiliations

¹ Institute for Human Genetics, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: xingjie.ren@ucsf.edu.
² Institute for Human Genetics, University of California, San Francisco, San Francisco, CA 94143, USA.
³ Institute for Human Genetics, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Neurology, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: yin.shen@ucsf.edu.

Abstract

Allelic tagging of endogenous genes enables studying gene function and transcriptional control in the native genomic context. Here, we present an efficient protocol for bi-allelic tagging of protein-coding genes with fluorescent reporters in human iPSCs using the CRISPR-Cas9-mediated homology-directed repair. We detail steps for design, cloning, electroporation, and single-cell clone isolation and validation. The tagging strategy described in this protocol is readily applicable for knockin of other reporters in diverse cell types for biomedical research.

Keywords: CRISPR; Cell Culture; Genetics; Molecular Biology; Stem Cells.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

CRISPR-Cas Systems / genetics
Clone Cells
Gene Editing* / methods
Humans
Induced Pluripotent Stem Cells* / metabolism
Recombinational DNA Repair

Abstract

Publication types

MeSH terms

Grants and funding