Kingdom-wide CRISPR guide design with ALLEGRO

Abstract Designing CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) single guide RNA (sgRNA) libraries targeting entire kingdoms of life will significantly advance genetic research in diverse and underexplored taxa. Current sgRNA design tools are often species-specific and fail to...

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Published inNucleic acids research Vol. 53; no. 15
Main Authors Mohseni, Amirsadra, Nia, Reyhane Ghorbani, Tafrishi, Aida, López, Mario León, Liu, Xin-Zhan, Stajich, Jason E, Lonardi, Stefano, Wheeldon, Ian
Format Journal Article
LanguageEnglish
Published England Oxford University Press 11.08.2025
Subjects
Algorithms
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems
Gene Editing - methods
Gene Library
Genome, Fungal
Kluyveromyces - genetics
Methods
Rhodotorula - genetics
RNA, Guide, CRISPR-Cas Systems - genetics
Saccharomyces cerevisiae - genetics
Saccharomycetales
Yarrowia - genetics
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ISSN0305-1048
1362-4962
1362-4962
DOI10.1093/nar/gkaf783

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Summary:Abstract Designing CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) single guide RNA (sgRNA) libraries targeting entire kingdoms of life will significantly advance genetic research in diverse and underexplored taxa. Current sgRNA design tools are often species-specific and fail to scale to large, phylogenetically diverse datasets, limiting their applicability to comparative genomics, evolutionary studies, and biotechnology. Here, we introduce ALLEGRO, a combinatorial optimization algorithm designed to compose minimal, yet highly effective sgRNA libraries targeting thousands of species at the same time. Leveraging integer linear programming, ALLEGRO identified compact sgRNA sets simultaneously targeting multiple genes of interest for over 2000 species across the fungal kingdom. We experimentally validated sgRNAs designed by ALLEGRO in Kluyveromyces marxianus, Komagataella phaffii, Yarrowia lipolytica, and Saccharomyces cerevisiae, confirming successful genome edits. Additionally, we employed a generalized Cas9–ribonucleoprotein delivery system to apply ALLEGRO’s sgRNA libraries to untested fungal genomes, such as Rhodotorula araucariae. Our experimental findings, together with cross-validation, demonstrate that ALLEGRO facilitates efficient CRISPR genome editing, enabling the development of universal sgRNA libraries applicable to entire taxonomic groups. Graphical Abstract Graphical Abstract
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Amirsadra Mohseni and Reyhane Ghorbani Nia Contributed equally to this work.
ISSN:0305-1048
1362-4962
1362-4962
DOI:10.1093/nar/gkaf783