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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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CRISPR and crRNAs02:53

CRISPR and crRNAs

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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

Homologous Recombination

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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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Conservative Site-specific Recombination and Phase Variation02:53

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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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Related Experiment Video

Updated: May 15, 2025

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

Published on: May 25, 2018

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Directed evolution expands CRISPR-Cas12a genome editing capacity.

Enbo Ma, Kai Chen, Honglue Shi

    Biorxiv : the Preprint Server for Biology
    |April 8, 2025
    PubMed
    Summary
    This summary is machine-generated.

    Researchers engineered CRISPR-Cas12a to recognize new DNA sequences, expanding genome editing capabilities. This breakthrough broadens access to previously unreachable genetic targets for therapeutic and agricultural applications.

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    Area of Science:

    • Molecular Biology
    • Biotechnology
    • Genomics

    Background:

    • CRISPR-Cas12a is a powerful genome editing tool.
    • Its utility is limited by a narrow protospacer-adjacent motif (PAM) recognition (5'-TTTV-3'), restricting targeting to ~1% of the genome.

    Purpose of the Study:

    • To overcome the PAM limitation of CRISPR-Cas12a.
    • To engineer variants with expanded PAM recognition for broader genomic targeting.

    Main Methods:

    • Directed evolution using a bacterial assay.
    • Rational engineering of *Lachnospiraceae bacterium* Cas12a (LbCas12a).
    • Biochemical and cell-based assays to characterize variants.

    Main Results:

    • Identified LbCas12a variants with expanded PAM recognition, including non-canonical motifs.
    • Developed Flex-Cas12a, which recognizes 5'-NYHV-3' PAMs.
    • Expanded DNA recognition to ~25% of the human genome.

    Conclusions:

    • Engineered Cas12a variants significantly enhance genome targeting versatility.
    • Flex-Cas12a provides access to previously inaccessible genomic loci.
    • Offers new opportunities for therapeutic and agricultural genome engineering.