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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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Mutations01:39

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Mutations01:35

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Mutations are changes in the sequence of DNA. These changes can occur spontaneously or they can be induced by exposure to environmental factors. Mutations can be characterized in a number of different ways: whether and how they alter the amino acid sequence of the protein, whether they occur over a small or large area of DNA, and whether they occur in somatic cells or germline cells.
Chromosomal Alterations Are Large-Scale Mutations
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Genomic Imprinting and Inheritance02:30

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Diploid organisms inherit genetic material through chromosomes from both parents. Copies of the same gene are known as alleles. In most cases, both alleles are simultaneously expressed and allow various cellular processes to function optimally. If one of the alleles is missing or mutated, the expression of the other allele can compensate; however, this is not true for all genes.
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A mutation is a change in the sequence of bases of DNA or RNA in a genome. Some mutations occur during replication of the genome due to errors made by the polymerase enzymes that replicate DNA or RNA. Unlike DNA polymerase, RNA polymerase is prone to errors because it is not capable of “proofreading” its work. Viruses with RNA-based genomes, like HIV, therefore accrue mutations faster than viruses with DNA-based genomes. Because mutation and recombination provide the raw material...
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Genome Size and the Evolution of New Genes03:21

Genome Size and the Evolution of New Genes

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While every living organism has a genome of some kind (be it RNA, or DNA), there is considerable variation in the sizes of these blueprints. One major factor that impacts genome size is whether the organism is prokaryotic or eukaryotic. In prokaryotes, the genome contains little to no non-coding sequence, such that genes are tightly clustered in groups or operons sequentially along the chromosome. Conversely, the genes in eukaryotes are punctuated by long stretches of non-coding sequence.
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Related Experiment Video

Updated: Feb 13, 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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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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Precise Cas9 targeting enables genomic mutation prevention.

Alejandro Chavez1,2,3, Benjamin W Pruitt2, Marcelle Tuttle2

  • 1Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, NY 10032; ac4304@cumc.columbia.edu gchurch@genetics.med.harvard.edu.

Proceedings of the National Academy of Sciences of the United States of America
|March 21, 2018
PubMed
Summary
This summary is machine-generated.

Scientists developed a guide RNA tuning method for Cas9 gene editing to prevent specific mutations. This in vivo system successfully restricted unwanted mutations in engineered organisms, even in the mouse gut.

Keywords:
Cas9mutation preventionsynthetic biologytgRNAtuned gRNA

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR-Cas9 technology allows precise gene editing.
  • Distinguishing between similar DNA sequences, like single-nucleotide polymorphisms (SNPs), remains a challenge for Cas9.
  • Preventing specific mutations in vivo is crucial for therapeutic applications.

Purpose of the Study:

  • To develop a generalized method for tuning guide RNA specificity in Cas9.
  • To create an in vivo system for preventing undesired mutations.
  • To assess the scalability and portability of the mutation prevention system.

Main Methods:

  • Guide RNA (gRNA) engineering to enhance Cas9 specificity for single-nucleotide polymorphism (SNP) discrimination.
  • Development of an in vivo mutation prevention system using tuned Cas9.
  • Testing the system's efficacy in engineered organisms and across different Cas9 variants.
  • Evaluating system performance in the complex environment of the mouse gastrointestinal tract.

Main Results:

  • A generalized guide RNA tuning method was established, enabling Cas9 to differentiate between target sites differing by a single SNP.
  • An in vivo mutation prevention system was successfully generated, actively restricting the occurrence of specific gain-of-function mutations.
  • The system demonstrated scalability to multiple targets and portability across engineered Cas9 variants and orthologs.
  • Robust activity of the mutation prevention system was confirmed within the mouse gastrointestinal tract.

Conclusions:

  • The developed gRNA tuning method significantly enhances Cas9 specificity for SNP detection.
  • The in vivo mutation prevention system offers a powerful tool for controlling genetic mutations in engineered organisms.
  • This technology holds promise for therapeutic applications requiring precise control over gene editing and mutation prevention.