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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

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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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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
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Multiple genome modifications by the CRISPR/Cas9 system in zebrafish.

Satoshi Ota1, Yu Hisano, Yoshiya Ikawa

  • 1Laboratory for Cardiovascular Molecular Dynamics, RIKEN Quantitative Biology Center (QBiC), Furuedai 6-2-3, Suita, Osaka, 565-0874, Japan; Laboratory for Developmental Biology, Center for Medical Education and Sciences, Graduate School of Medical Science, University of Yamanashi, Shimogatou 1110, Chuo, Yamanashi, 409-3898, Japan.

Genes to Cells : Devoted to Molecular & Cellular Mechanisms
|May 23, 2014
PubMed
Summary

The CRISPR/Cas9 system enables multiple genome edits in zebrafish, causing pigment and heart development defects. These edits, including indels and deletions, are heritable and detectable with an improved heteroduplex mobility assay.

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Area of Science:

  • Genetics and Genomics
  • Developmental Biology
  • Molecular Biology

Background:

  • The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is a bacterial adaptive immune mechanism repurposed for genome editing.
  • CRISPR/Cas9 technology offers precise gene modification capabilities across diverse model organisms.
  • Zebrafish (Danio rerio) are a valuable vertebrate model for studying gene function and developmental processes.

Purpose of the Study:

  • To demonstrate the efficacy of the CRISPR/Cas9 system for multiplex genome editing in zebrafish.
  • To investigate the effects of disrupting genes involved in pigment formation and cardiac development.
  • To assess the heritability of CRISPR/Cas9-induced mutations and develop efficient screening methods.

Main Methods:

  • Co-injection of multiple guide RNAs (gRNAs) and Cas9 mRNA into zebrafish embryos.
  • Targeting genes such as golden (gol), tyrosinase (tyr), s1pr2, and spns2.
  • Phenotypic analysis of F0 embryos for pigment and cardiac abnormalities.
  • Detection of indels, deletions, and inversions using molecular assays.
  • Improvement of heteroduplex mobility assay (HMA) for multi-locus mutation screening.

Main Results:

  • Simultaneous disruption of multiple genes led to observed phenotypes including hypopigmentation and a two-hearts condition.
  • CRISPR/Cas9 induced indel mutations and a specific 7.1-kb deletion between s1pr2 target sites.
  • Chromosomal translocations were not detected among the five targeted loci.
  • Indel mutations and locus-specific deletions were confirmed to be heritable in F1 generation zebrafish.
  • An improved multi-locus HMA effectively screened for multiple genome modifications.

Conclusions:

  • CRISPR/Cas9 is a versatile tool for multiplex genome editing in zebrafish, facilitating the study of gene function.
  • The system can induce various mutations, including indels and deletions, which are heritable across generations.
  • The developed multi-locus HMA is an efficient method for identifying multiple CRISPR/Cas9-induced modifications in founder screening.