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Related Concept Videos

CRISPR01:59

CRISPR

Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced Short...
CRISPR01:59

CRISPR

Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced Short...
CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
What is Genetic Engineering?00:49

What is Genetic Engineering?

Overview
Homologous Recombination02:31

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...

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Updated: May 19, 2026

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells
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Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Published on: September 25, 2019

Genetic Engineering of Human Keratinocytes Using CRISPR/Cas9 Ribonucleoprotein Complexes or Modified Cas9-Encoding

Jaimy A Klijnhout1, Eline A W Senders1, Ellen H van den Bogaard1

  • 1Department of Dermatology, Research Institute for Medical Innovation, Radboud University Medical Center (Radboudumc), Nijmegen, The Netherlands.

Methods in Molecular Biology (Clifton, N.J.)
|May 18, 2026
PubMed
Summary
This summary is machine-generated.

This study details a CRISPR/Cas9 protocol for human keratinocytes, overcoming challenges in genome editing efficiency and cell viability. The method enables precise gene editing and selection of edited cell populations.

Keywords:
CRISPR/Cas9Cationic vectorsElectroporationKeratinocyteLaboratory protocolLipofectamineN/TERT-1N/TERT-2GRibonucleoprotein

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Area of Science:

  • Molecular Biology
  • Genetics
  • Dermatology

Background:

  • CRISPR/Cas9 is a versatile genome-editing tool used across various scientific fields.
  • Genetic manipulation of human keratinocytes using CRISPR/Cas9 presents significant challenges, including inconsistent editing efficiency, decreased cell survival, and difficulties in isolating edited cell clones.

Purpose of the Study:

  • To establish a detailed, step-by-step protocol for efficient CRISPR/Cas9-mediated genome editing in human primary keratinocytes.
  • To address and overcome the common technical hurdles associated with keratinocyte genetic modification.

Main Methods:

  • Development of a refined CRISPR/Cas9 delivery and editing system optimized for human keratinocytes.
  • Implementation of standardized methods for assessing CRISPR/Cas9 editing efficiency.
  • Establishment of robust (sub)cloning techniques for selecting heterozygous and homozygous gene-edited keratinocyte clones.
  • Inclusion of protocols for comprehensive off-target mutation analysis.

Main Results:

  • The protocol demonstrates improved and reproducible genome-editing efficiency in human keratinocytes.
  • Successful isolation and selection of specific gene-edited keratinocyte populations (heterozygous and homozygous).
  • Reliable methods for analyzing CRISPR/Cas9 efficiency and off-target effects were validated.

Conclusions:

  • This optimized protocol provides a reliable framework for CRISPR/Cas9 genome editing in human keratinocytes, facilitating research in skin biology and disease.
  • The standardized methods enhance the feasibility of genetic studies involving keratinocytes, paving the way for therapeutic applications.