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Related Concept Videos

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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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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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CRISPR Guide RNA Cloning for Mammalian Systems
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Kingdom-wide CRISPR guide design with ALLEGRO.

Amirsadra Mohseni1, Reyhane Ghorbani Nia2, Aida Tafrishi2

  • 1Computer Science and Engineering, University of California, Riverside, CA 92521, United States.

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ALLEGRO is a new algorithm for designing CRISPR single-guide RNA libraries. It enables efficient genome editing across thousands of species, advancing genetic research in diverse fungi.

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Area of Science:

  • Genetics and Genomics
  • Bioinformatics and Computational Biology
  • Molecular Biology

Background:

  • CRISPR genome editing relies on single-guide RNA (sgRNA) design.
  • Existing sgRNA design tools are species-specific and do not scale for large, diverse datasets.
  • This limits applications in comparative genomics, evolutionary studies, and biotechnology across various taxa.

Purpose of the Study:

  • To develop a scalable and effective sgRNA library design tool for diverse species.
  • To enable simultaneous targeting of multiple genes across thousands of species.
  • To facilitate universal sgRNA library development for entire taxonomic groups.

Main Methods:

  • Introduction of ALLEGRO, a combinatorial optimization algorithm using integer linear programming.
  • Development of minimal, highly effective sgRNA libraries targeting thousands of species.
  • Experimental validation of ALLEGRO-designed sgRNAs in multiple fungal species (e.g., *Kluyveromyces marxianus*, *Komagataella phaffii*, *Yarrowia lipolytica*, *Saccharomyces cerevisiae*).
  • Application of sgRNA libraries using a generalized Cas9-ribonucleoprotein delivery system.

Main Results:

  • ALLEGRO successfully designed compact sgRNA sets targeting multiple genes for over 2000 fungal species.
  • Experimental validation confirmed efficient CRISPR genome editing in tested species.
  • Successful application of ALLEGRO libraries in previously untested fungal genomes (e.g., *Rhodotorula araucariae*).

Conclusions:

  • ALLEGRO provides an efficient method for designing universal sgRNA libraries.
  • The algorithm facilitates scalable CRISPR genome editing across diverse fungal taxa.
  • This advancement supports broader genetic research and biotechnology applications in underexplored organisms.