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Related Concept Videos

CRISPR01:59

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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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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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Related Experiment Video

Updated: Feb 2, 2026

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
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High Efficiency Targeting of Non-coding Sequences Using CRISPR/Cas9 System in Tilapia.

Minghui Li1, Xingyong Liu2, Shengfei Dai2

  • 1Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, 400715, China limh@163.com wdeshou@swu.edu.cn.

G3 (Bethesda, Md.)
|November 29, 2018
PubMed
Summary

This study optimized CRISPR/Cas9 to delete non-coding DNA, including microRNA and 3' UTRs, in tilapia. This method efficiently generates germline-transmissible mutations for functional studies in non-model fish.

Keywords:
CRISPR/Cas9germline transmissionnon-coding sequencessDNA

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Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9
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Area of Science:

  • Genetics
  • Molecular Biology
  • Aquaculture

Background:

  • The majority of animal genomes consist of non-coding sequences that regulate biological processes.
  • Understanding the function of these non-coding elements is crucial for biological research.
  • CRISPR/Cas9 is effective for protein-coding gene disruption but less explored for non-coding sequences.

Purpose of the Study:

  • To develop and optimize CRISPR/Cas9 system for targeted deletion of non-coding sequences in tilapia.
  • To investigate the functional significance of microRNA and 3' UTRs in tilapia.
  • To establish a platform for generating non-coding sequence mutants in non-model fish.

Main Methods:

  • Utilized single and dual guide RNA (gRNA) guided Cas9 nuclease for targeted DNA deletion.
  • Co-injected fertilized tilapia eggs with gRNAs, Cas9 mRNA, and short single-stranded DNA (ssDNA) donors.
  • Employed Cas9-vasa 3'-UTR construct to enhance germline transmission efficiency.

Main Results:

  • Achieved efficient deletion of non-coding sequences, including microRNA and 3' UTRs, with maximum efficiency up to 19% using ssDNA donors.
  • Demonstrated germline transmission of deletions with an average efficiency of 8.7%, improved to 14.9% using the Cas9-vasa 3'-UTR.
  • Successfully deleted the vasa gene 3'-UTR, leading to reduced vasa mRNA expression in gonads.

Conclusions:

  • The optimized CRISPR/Cas9 system is a powerful tool for generating non-coding sequence mutants in tilapia.
  • This approach facilitates the functional study of non-coding elements in non-model fish species.
  • The study provides a valuable platform for genetic research in aquaculture and evolutionary biology.