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Related Experiment Video

Updated: Dec 26, 2025

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation
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Efficient SNP editing in haploid human pluripotent stem cells.

Lauren Zakarin Safier1,2, Michael V Zuccaro3, Dietrich Egli4,5

  • 1Department of Obstetrics and Gynecology and Columbia University Fertility Center, Columbia University, College of Physicians & Surgeons, New York, NY, 10032, USA.

Journal of Assisted Reproduction and Genetics
|March 13, 2020
PubMed
Summary
This summary is machine-generated.

CRISPR gene editing successfully corrected a mutation in haploid human embryonic stem cells. This advance enables new strategies for studying genetic diseases using haploid cells and recessive alleles.

Keywords:
CRISPR/Cas-9Gene editingHaploid stem cells

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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

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Area of Science:

  • Stem cell biology
  • Gene editing technologies
  • Human genetics

Background:

  • Haploid human embryonic stem cells (hESCs) offer a unique model for genetic studies due to their single set of chromosomes.
  • Correcting mutations in hESCs is crucial for understanding gene function and developing therapeutic strategies.
  • Existing gene editing methods face challenges in efficiency and off-target effects, particularly in sensitive cell types.

Purpose of the Study:

  • To demonstrate the efficacy of CRISPR/Cas9 gene editing for correcting a specific mutation in haploid human embryonic stem cells.
  • To establish and maintain a culture of haploid cells suitable for gene editing experiments.
  • To investigate the potential of haploid cell cultures for studying recessive alleles.

Main Methods:

  • Exome sequencing identified an SLC10A2 gene mutation in a haploid embryonic stem cell line (pES12).
  • CRISPR/Cas9 components (guide RNA and correction template) were designed to target the mutation.
  • Haploid enrichment was achieved through serial fluorescence-activated cell sorting (FACS), followed by nucleofection and Cas9-expressing cell sorting.

Main Results:

  • CRISPR/Cas9 editing achieved a 77.1% efficiency, with 14.6% of clones showing successful correction of the SLC10A2 mutation.
  • Maintenance of haploid cell populations was confirmed via FACS and centromere quantification.
  • Karyotype and off-target effect analyses revealed normal karyotypes and no detectable off-target mutations.

Conclusions:

  • CRISPR/Cas9 is an effective tool for gene editing in haploid human embryonic stem cells.
  • The establishment and maintenance of haploid cell cultures provide a novel platform for CRISPR/Cas9 applications.
  • This approach is particularly valuable for the study of recessive gene variants and their associated conditions.