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Related Concept Videos

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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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 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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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Updated: Dec 30, 2025

CRISPR Guide RNA Cloning for Mammalian Systems
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Versatile 3' Functionalization of CRISPR Single Guide RNA.

Cody M Palumbo1, Jeton M Gutierrez-Bujari1, Henriette O'Geen2

  • 1Department of Chemistry, University of California Davis, One Shields Ave., Davis, CA, 95616, USA.

Chembiochem : a European Journal of Chemical Biology
|January 17, 2020
PubMed
Summary
This summary is machine-generated.

Chemically modifying the 3' end of guide RNAs (sgRNAs) is compatible with CRISPR/Cas systems. This strategy enables the development of novel tools for CRISPR applications and the identification of sgRNA-binding proteins.

Keywords:
RNAclick chemistrynucleic acidsprotein engineeringproteomics

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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetic Engineering

Background:

  • CRISPR/Cas genome editing systems offer powerful tools for genetic manipulation.
  • Chemical modifications of guide RNAs (sgRNAs) can enhance the utility of CRISPR/Cas applications.
  • Functionalizing the 3'-end of sgRNAs presents an opportunity for novel applications.

Purpose of the Study:

  • To develop a versatile and efficient strategy for chemical functionalization of the 3'-end of sgRNAs.
  • To assess the compatibility of modified sgRNAs with the Streptococcus pyogenes Cas9 enzyme.
  • To demonstrate the utility of modified sgRNAs as affinity reagents for identifying interacting proteins.

Main Methods:

  • Synthesis of six chemically modified sgRNAs, including those with crosslinkers, a fluorophore, and biotin.
  • In vitro DNA cleavage assays using Streptococcus pyogenes Cas9 and modified sgRNAs.
  • Affinity purification using 3'-biotinylated sgRNA followed by mass spectrometry to identify binding proteins in HEK293T cell extracts.

Main Results:

  • The chemical modification of the sgRNA 3'-end was well-tolerated by Streptococcus pyogenes Cas9 in vitro.
  • A library of six modified sgRNAs was successfully prepared.
  • 3'-biotinylated sgRNAs were used to identify IGF2BP1, YB1, and hnRNP K as sgRNA-binding proteins in HEK293T cells.

Conclusions:

  • A robust strategy for 3'-end functionalization of sgRNAs was established.
  • Modified sgRNAs are compatible with Cas9 activity and can be used for protein identification.
  • This approach expands the toolkit for CRISPR/Cas system applications, including target identification and molecular probing.