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Related Concept Videos

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...
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...

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Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing
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Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Published on: May 10, 2020

Optimization of scarless human stem cell genome editing.

Luhan Yang1, Marc Guell, Susan Byrne

  • 1Department of Genetics, Harvard Medical School, Boston, 02115 MA, USA, Biological and Biomedical Sciences Program, Harvard Medical School, Boston, 02115 MA, USA, Children's Hospital, Boston, 02115 MA, USA, Chemistry and Chemical Biology program, Harvard, 02138 Cambridge, MA, USA and Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, 02138 MA, USA.

Nucleic Acids Research
|August 3, 2013
PubMed
Summary

Cas9-gRNA demonstrated superior genome editing efficiency in human-induced pluripotent cells (hiPSCs) compared to re-coded TALENs (reTALENs). This study optimized gene editing tools for faster, seamless genome correction in hiPSCs.

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Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells
09:04

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

Published on: September 25, 2019

Area of Science:

  • Molecular Biology
  • Genetics
  • Stem Cell Biology

Background:

  • Precise genome editing in human-induced pluripotent cells (hiPSCs) is crucial for research and clinical applications.
  • Programmable nucleases like Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA have advanced targeted genetic modifications.
  • Optimization of existing tools and expansion of their applications remain important research areas.

Purpose of the Study:

  • To develop improved genome editing tools for hiPSCs.
  • To compare the efficiency of re-coded TALENs (reTALEs) and Cas9-gRNA systems in hiPSCs.
  • To optimize single-stranded oligodeoxynucleotide (ssODN) design for precise mutation introduction.

Main Methods:

  • Development of functional re-coded TALEs (reTALEs) for one-pot synthesis and lentiviral vector applications.
  • Comparison of genome editing efficiencies using 15 pairs of reTALENs and Cas9-gRNA targeting the CCR5 gene in hiPSCs.
  • Optimization of ssODN donor design for both editing systems to introduce specific mutations.

Main Results:

  • Cas9-gRNA exhibited 7-8 times higher non-homologous end joining (NHEJ) efficiency (3%) compared to reTALENs (0.4%) in hiPSCs.
  • Cas9-gRNA showed moderately superior homology-directed repair (HDR) efficiency (1.0%) versus reTALENs (0.6%) when combined with ssODN donors.
  • A streamlined process for seamless genome-corrected hiPSCs was achieved within 3 weeks using optimized methods.

Conclusions:

  • Cas9-gRNA is a more efficient genome editing tool than reTALENs in hiPSCs for both NHEJ and HDR-mediated modifications.
  • Optimized ssODN design enhances the efficiency of genome editing in hiPSCs.
  • The developed methods enable rapid and seamless genome correction of hiPSCs, facilitating future research and therapeutic applications.