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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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Mismatch Repair01:20

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Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
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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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CRISPR and crRNAs02:53

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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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Homologous Recombination02:31

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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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The Antiviral System of Bacteria and Archaea: CRISPR01:23

The Antiviral System of Bacteria and Archaea: CRISPR

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CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats is a adaptive immune system found in bacteria and archaea that protects against viral infections. This system enables prokaryotic cells to identify, remember, and neutralize foreign genetic elements, primarily bacteriophages, by storing fragments of the invader’s DNA as a genetic memory.The CRISPR immune response begins during an initial infection. Cas (CRISPR-associated) proteins play a central role in this...
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Related Experiment Video

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CIRCLE-Seq for Interrogation of Off-Target Gene Editing
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Structural basis for mismatch surveillance by CRISPR-Cas9.

Jack P K Bravo1, Mu-Sen Liu1, Grace N Hibshman1,2

  • 1Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.

Nature
|March 3, 2022
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CRISPR-Cas9 genome editing is limited by off-target DNA cleavage. This study reveals how Cas9 recognizes mismatches, enabling the design of high-fidelity variants with improved accuracy and maintained on-target cleavage efficiency.

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A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells
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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • CRISPR-Cas9 genome editing is a powerful tool but suffers from off-target DNA cleavage due to poor understanding of mismatch recognition mechanisms.
  • Existing Cas9 variants with improved mismatch discrimination often exhibit reduced on-target cleavage rates, limiting their therapeutic potential.

Purpose of the Study:

  • To elucidate the structural mechanisms underlying Cas9 mismatch recognition and cleavage.
  • To engineer next-generation high-fidelity Cas9 variants with enhanced specificity and retained activity.

Main Methods:

  • Kinetics-guided cryo-electron microscopy (cryo-EM) was employed to capture Cas9 structures at various stages of DNA mismatch cleavage.
  • Site-directed mutagenesis was used to alter specific residues involved in mismatch stabilization.

Main Results:

  • A distinct linear guide RNA-DNA duplex conformation, which inhibits Cas9 activation, was observed in the presence of mismatches.
  • Mismatches distal to the protospacer adjacent motif are stabilized by a reorganized RuvC domain loop.
  • Mutagenesis of mismatch-stabilizing residues successfully reduced off-target cleavage while preserving rapid on-target cleavage.

Conclusions:

  • Targeting Cas9 regions involved in mismatch tolerance offers a viable strategy for developing high-fidelity genome editing tools.
  • This research provides a structural basis for designing improved CRISPR-Cas9 systems with enhanced specificity for therapeutic applications.