CRISPR/Cas9 Editing of Human Keratinocytes for Organotypic Modeling of Genetic Epidermal Disorders.

Anthony Coon, Jessica L Ayers, Christopher Cole, Afua Tiwaa, Arti Parihar, Thomas Vincent, Johann E Gudjonsson, Mrinal K Sarkar, Cory L Simpson

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Article in Methods in molecular biology (Clifton, N.J.), 2027. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.

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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

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4 · The record

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5 · Who and what money

Authors and funding

9 authors.

Anthony CoonDepartment of Dermatology, University of Michigan, Ann Arbor, MI, USA.

Jessica L AyersDepartment of Dermatology, University of Washington, Seattle, WA, USA.

Christopher ColeDepartment of Dermatology, University of Michigan, Ann Arbor, MI, USA.

Afua TiwaaDepartment of Dermatology, University of Washington, Seattle, WA, USA.

Arti PariharDepartment of Dermatology, University of Washington, Seattle, WA, USA.

Thomas VincentDepartment of Dermatology, University of Washington, Seattle, WA, USA.

Johann E GudjonssonDepartment of Dermatology, University of Michigan, Ann Arbor, MI, USA.

Mrinal K SarkarDepartment of Dermatology, University of Michigan, Ann Arbor, MI, USA. mksarkar@med.umich.edu.

Cory L SimpsonDepartment of Dermatology, University of Washington, Seattle, WA, USA. csimp99@uw.edu.

Funding

No grant is acknowledged in the PubMed record.

6 · The paper itself

Abstract

CRISPR/Cas9-based DNA editing has revolutionized biomedical research by enabling investigators to precisely and permanently inactivate genes in human cells to provide greater insight into the mechanisms that support normal physiology and drive disease pathology. To leverage this technology for cutaneous biology research, we optimized a strategy for efficient gene editing in human keratinocytes, which can be grown into a differentiated organotypic epidermis to extend studies of gene function to a mature 3D tissue model. Here, we describe step-by-step methods for deploying CRISPR/Cas9 to engineer a TERT-immortalized human epidermal keratinocyte line (N/TERT-2G) to generate monoclonal heterozygous and homozygous knockout cell lines. Additionally, we provide a detailed protocol for growing gene-edited keratinocytes into organotypic epidermis, which we have used to model the pathology of monogenic dermatologic disorders that were not replicated in traditional knockout mice.

Indexed as

CRISPR-Cas SystemsEpidermisGene EditingKeratinocytesSkin Diseases, GeneticAnimalsCell LineGene Knockout TechniquesHumansMiceCRISPR/Cas9DNA editingEpidermisGenetic diseaseKeratinocytes

Identifiers

PMID42668338

DOI10.1016/j.jid.2022.12.002

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