CRISPRi-mediated transcriptional silencing in iPSCs for the study of human brain development

Pia Annette Johansson; Anita Adami; Johan Jakobsson

doi:10.1016/j.xpro.2022.101285

CRISPRi-mediated transcriptional silencing in iPSCs for the study of human brain development

STAR Protoc. 2022 Apr 13;3(2):101285. doi: 10.1016/j.xpro.2022.101285. eCollection 2022 Jun 17.

Authors

Pia Annette Johansson^{1

2}, Anita Adami¹, Johan Jakobsson¹

Affiliations

¹ Laboratory of Molecular Neurogenetics, Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, Lund University, 221 84 Lund, Sweden.
² Cell and Gene Therapy Core, Lund Stem Cell Center, Lund University, 221 84 Lund, Sweden.

Abstract

This protocol describes the design and use of CRISPRi-mediated transcriptional silencing in human iPSCs, for loss-of-function studies in brain development research. The protocol avoids single cell selection, thereby eliminating side effects of clonal expansion and sites of viral integration. We also describe a neural progenitor differentiation protocol and discuss the challenges of target-specific lentiviral silencing, efficient silencing levels, and off-target effects. For complete details on the use and execution of this protocol, please refer to Johansson et al. (2022).

Keywords: CRISPR; Cell Differentiation; Developmental biology; Neuroscience; Stem Cells.

Publication types

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

MeSH terms

Humans
Induced Pluripotent Stem Cells*
Virus Integration