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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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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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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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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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Updated: Apr 20, 2026

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

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Genome editing using Cas9 nickases.

Alexandro E Trevino1, Feng Zhang1

  • 1Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Methods in Enzymology
|November 16, 2014
PubMed
Summary
This summary is machine-generated.

Engineered Cas9n enzymes create single-strand DNA nicks for precise genome editing in mammals. This method enhances specificity and reduces off-target mutations compared to standard Cas9 tools.

Keywords:
CRISPRCas9Gene targetingGenome editingNucleases

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Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • The CRISPR-Cas9 system, derived from microbial adaptive immunity, is a powerful genome engineering tool.
  • Wild-type Cas9 can cause unintended mutations (off-target mutagenesis).
  • Cas9n enzymes induce single-strand nicks, offering a safer alternative.

Purpose of the Study:

  • To develop and validate Cas9n reagents for precision genome engineering in mammals.
  • To leverage Cas9n's high-fidelity repair mechanisms for targeted gene modification.

Main Methods:

  • Engineering of Streptococcus pyogenes Cas9 catalytic domains to create nicking enzymes (Cas9n).
  • Utilizing homology- and structure-guided mutagenesis.
  • Applying Cas9n for genome editing via nonhomologous end-joining and homology-directed repair pathways.

Main Results:

  • Cas9n enzymes successfully induce single-strand nicks in DNA.
  • These nicks are repaired with high fidelity in eukaryotic cells.
  • Demonstrated application of Cas9n reagents for precise mammalian genome engineering.

Conclusions:

  • Cas9n enzymes represent a significant advancement for precise and specific genome editing in mammals.
  • This technology minimizes off-target effects, improving the safety and reliability of genome engineering.
  • Cas9n reagents offer a valuable tool for various applications in mammalian genetics research.