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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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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...
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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...
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Conservative Site-specific Recombination and Phase Variation02:53

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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Genome Editing in Mammalian Cell Lines using CRISPR-Cas
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Genome Editing in Mammalian Cell Lines using CRISPR-Cas

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CRISPR-Mediated Genome Engineering in Cell Lines.

Ya-Ju Chang1,2, Joseph Ryu1,3, Xuan Cui1,2

  • 1Edward S. Harkness Eye Institute, Department of Ophthalmology, Columbia University Irving Medical Center, New York, NY, USA.

Methods in Molecular Biology (Clifton, N.J.)
|December 9, 2022
PubMed
Summary
This summary is machine-generated.

This chapter details CRISPR-Cas9 genome editing tools for precise gene modification. It provides guidelines for designing targets, assessing editing efficiency, and analyzing off-target effects in cell lines.

Keywords:
CRISPRGene editingGene modificationGenome engineeringPrecise gene editingSNP

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • The CRISPR-Cas9 system is a microbial adaptive immune mechanism enabling precise genome engineering.
  • Cas9 nuclease, guided by RNA, facilitates targeted DNA modifications in various cell types.

Purpose of the Study:

  • To present a comprehensive protocol for Cas9-mediated genome editing.
  • To guide the generation of modified cell lines for functional studies using non-homologous end joining (NHEJ) or homology-directed repair (HDR).

Main Methods:

  • Utilizing a 20-nucleotide targeting sequence for Cas9-RNA complex.
  • Implementing NHEJ or HDR pathways for gene modification.
  • Developing experimental guidelines for target site selection and cleavage efficiency evaluation.

Main Results:

  • Established a protocol for generating modified cell lines.
  • Provided methods for assessing Cas9 editing efficiency.
  • Included strategies for analyzing off-target mutations.

Conclusions:

  • Cas9-mediated genome editing offers a precise method for altering cellular genomes.
  • The described tools and guidelines facilitate the creation of modified cell lines for downstream research.