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Related Experiment Video

Updated: Aug 30, 2025

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells
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Exploiting CRISPR/Cas9 to engineer precise segmental deletions in mouse embryonic stem cells.

Rajula Elango1, Arvind Panday1, Nicholas A Willis1

  • 1Department of Medicine, Division of Hematology-Oncology and Cancer Research Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA.

STAR Protocols
|August 31, 2022
PubMed
Summary

This study details a CRISPR/Cas9 protocol for creating gene deletions in mouse stem cells. It enables efficient screening of guide RNAs to generate precise in-frame or frameshift mutations for protein studies.

Keywords:
CRISPRGeneticsMolecular BiologyStem Cells

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Studying multi-domain proteins requires precise genetic modifications.
  • CRISPR/Cas9 technology offers powerful tools for genome engineering.

Purpose of the Study:

  • To present an optimized CRISPR/Cas9 protocol for generating large and precise gene deletions in mouse embryonic stem cells.
  • To facilitate the study of multi-domain proteins by enabling systematic engineering of in-frame deletions.

Main Methods:

  • Utilized CRISPR/Cas9 to create large deletions of gene coding regions, establishing hemizygous cell lines.
  • Engineered precise in-frame deletions within the wild-type allele.
  • Developed a method for rapid screening of effective sgRNA pairs.

Main Results:

  • Achieved high frequencies of in-frame deletions and frameshift mutations in mouse embryonic stem cells.
  • The protocol allows for efficient generation of modified cell lines for protein research.

Conclusions:

  • The described protocol provides an efficient and rapid method for CRISPR/Cas9-mediated gene editing.
  • This technique is valuable for studying the function of multi-domain proteins through precise genetic manipulation.