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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

79
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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CRISPR01:59

CRISPR

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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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Related Experiment Video

Updated: Jul 28, 2025

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.
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Efficient CRISPR/Cas9 mediated large insertions using long single-stranded oligonucleotide donors in C. elegans.

Matthew Eroglu1,2, Bin Yu1, W Brent Derry1,2

  • 1Developmental and Stem Cell Biology Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, Canada.

The FEBS Journal
|May 31, 2023
PubMed
Summary

This study simplifies creating long single-stranded DNA (ssDNA) for CRISPR/Cas9 genome editing in Caenorhabditis elegans. The new method significantly boosts the efficiency of inserting large DNA sequences, overcoming previous limitations.

Keywords:
CRISPR/Cas9Caenorhabditis eleganslambda exonucleaselarge insertionsssDNA

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR/Cas9 enables precise genome editing in Caenorhabditis elegans.
  • Inserting large DNA sequences (e.g., fluorescent markers) using single-stranded oligonucleotide donors is challenging.
  • Current methods for preparing long ssDNA donors are inefficient and labor-intensive.

Purpose of the Study:

  • To develop a simplified and efficient method for generating long single-stranded DNA (ssDNA) donors for CRISPR/Cas9 genome editing.
  • To improve the efficiency of inserting large DNA sequences into the C. elegans genome.
  • To provide a standardized and labor-minimal protocol for generating long ssDNA donors.

Main Methods:

  • Utilized a standard Polymerase Chain Reaction (PCR) followed by lambda exonuclease digestion to generate long ssDNA.
  • Compared the efficiency of long ssDNA donors with double-stranded DNA (dsDNA) donors for large sequence insertion.
  • Demonstrated the ability to perform multiple large insertions simultaneously.

Main Results:

  • Achieved high yields of long ssDNA donors rapidly and efficiently.
  • ssDNA donors demonstrated orders of magnitude higher insertion frequency compared to dsDNA donors.
  • Successfully generated large insertions without the need for selection or co-CRISPR markers.

Conclusions:

  • The developed method simplifies the generation of long ssDNA donors for CRISPR/Cas9 in C. elegans.
  • This approach significantly enhances the efficiency of inserting large DNA sequences, enabling complex genome modifications.
  • The protocol is standardized, labor-minimal, and expands the CRISPR/Cas9 toolkit for C. elegans research.