Using the dCas9-KRAB system to repress gene expression in hiPSC-derived NGN2 neurons

Aiqun Li; Samuel Cartwright; Alex Yu; Seok-Man Ho; Nadine Schrode; P J Michael Deans; Marliette R Matos; Meilin Fernandez Garcia; Kayla G Townsley; Bin Zhang; Kristen J Brennand

doi:10.1016/j.xpro.2021.100580

Using the dCas9-KRAB system to repress gene expression in hiPSC-derived NGN2 neurons

STAR Protoc. 2021 Jun 3;2(2):100580. doi: 10.1016/j.xpro.2021.100580. eCollection 2021 Jun 18.

Authors

Aiqun Li^{1

2

3

4

5}, Samuel Cartwright^{1

2

3}, Alex Yu^{1

2

3}, Seok-Man Ho^{1

3

4

5

6}, Nadine Schrode^{1

3

4

5

6}, P J Michael Deans^{1

3

4

5

6}, Marliette R Matos^{1

3

4

5

6}, Meilin Fernandez Garcia^{1

3

4

5

6}, Kayla G Townsley^{1

3

4

5

6}, Bin Zhang^{1

2

3}, Kristen J Brennand^{1

3

4

5}

Affiliations

¹ Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.
² Mount Sinai Center for Transformative Disease Modeling, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.
³ Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.
⁴ Nash Family Department of Neuroscience, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, New York, NY 10029, USA.
⁵ Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, New York, NY 10029, USA.
⁶ Department of Psychiatry, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, New York, NY 10029, USA.

Abstract

We describe a CRISPR inhibition (CRISPRi) protocol to repress endogenous gene expression (e.g., ATP6V1A) in human induced pluripotent stem cell-derived NGN2-induced glutamatergic neurons. CRISPRi enables efficient and precise gene repression of one or multiple target genes via delivering gRNA(s) to direct a dCas9-KRAB fusion protein to the gene(s) of interest. This protocol can also be adapted for gene activation and high-throughput gene manipulation, allowing assessment of the transcriptomic and phenotypic impact of candidate gene(s) associated with neurodevelopment or brain disease. For complete details on the use and execution of this protocol, please refer to Ho et al. (2017) and Wang et al. (2021).

Keywords: CRISPR; Cell Differentiation; Molecular Biology; Neuroscience; Stem Cells.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems*
Gene Expression Regulation*
Humans
Induced Pluripotent Stem Cells / metabolism*
Nerve Tissue Proteins / genetics*
Neurons / cytology
Neurons / metabolism*
Transcriptome

Substances

Nerve Tissue Proteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding