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Updated: Sep 10, 2025
06:48
CRISPR Guide RNA Cloning for Mammalian Systems
Published on: October 2, 2018
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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.
Nucleic Acids Research
|August 19, 2025
Summary
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.
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.
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