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Related Concept Videos

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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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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Each human somatic cell contains 6 billion base-pairs of DNA. Each base-pair is 0.34 nm long, which means that each diploid cell contains a staggering 2 meters of DNA. How is such a long DNA strand packed inside a nucleus measuring only 10 - 20 microns in diameter? 
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Genome-wide association studies or GWAS are used to identify whether common SNPs are associated with certain diseases. Suppose specific SNPs are more frequently observed in individuals with a particular disease than those without the disease. In that case, those SNPs are said to be associated with the disease. Chi-square analysis is performed to check the probability of the allele likely to be associated with the disease.
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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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The genome of most prokaryotic organisms consists of double-stranded DNA organized into one circular chromosome in a region of cytoplasm called the nucleoid. The chromosome is tightly wound, or supercoiled, for efficient storage. Prokaryotes also contain other circular pieces of DNA called plasmids. These plasmids are smaller than the chromosome and often carry genes that confer adaptive functions, such as antibiotic resistance.
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Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq
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DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA.

Daesik Kim1, Jin-Soo Kim1,2

  • 1Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea.

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Summary
This summary is machine-generated.

Chromatin structure impacts CRISPR-Cas9 gene editing. Mismatched guide RNAs showed sensitivity to chromatin, suggesting it hinders off-target DNA cleavage but not on-target activity.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR-Cas9 is a powerful gene editing tool.
  • Understanding its activity within the complex cellular environment is crucial.
  • Chromatin structure is known to influence DNA accessibility and protein binding.

Purpose of the Study:

  • To investigate the effect of chromatin on CRISPR-Cas9 on-target and off-target activities.
  • To determine if chromatin influences DNA cleavage by Cas9 and guide RNAs.

Main Methods:

  • Identified identical endogenous DNA sequences in open and closed chromatin regions.
  • Measured mutation frequencies using Cas9 with matched and mismatched sgRNAs in human cells.
  • Performed Digenome-seq (DIG-seq) on cell-free chromatin DNA and histone-free genomic DNA.

Main Results:

  • Mismatched sgRNAs, unlike matched ones, were sensitive to chromatin states, indicating chromatin hinders off-target DNA cleavage.
  • Only a subset of sites cleaved in histone-free DNA were cut in chromatin DNA.
  • Chromatin appears to inhibit Cas9 off-target effects.

Conclusions:

  • Chromatin structure plays a role in modulating CRISPR-Cas9 activity.
  • Chromatin can enhance the specificity of CRISPR-Cas9 by inhibiting off-target DNA cleavage.
  • These findings are important for optimizing CRISPR-Cas9 applications in eukaryotic cells.