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1. Kingdom-wide CRISPR guide design with ALLEGRO

   https://doi.org/10.1093/nar/gkaf783  

   Mohseni, Amirsadra; Nia, Reyhane Ghorbani; Tafrishi, Aida; López, Mario León; Liu, Xin-Zhan; Stajich, Jason E; Lonardi, Stefano; Wheeldon, Ian (August 2025, Nucleic Acids Research)

   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. 

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2. Conditional Expression of Cas9 and dCas9 in Lucilia cuprina Reveals dCas9-Associated Lethality

   https://doi.org/10.1177/25731599261436373  

   Kriete, Alexis; Basika, Tatiana; Novas, Rossina; Mathieson, Olivia L; Belikoff, Esther J; Kleiner, Manuel; Scott, Maxwell J (April 2026, The CRISPR Journal)

   Conditional sex transformation systems could improve genetic control strategies against insect pests. Here, we developed and tested CRISPR-based, tetracycline-repressible sex transformation strains in the Australian sheep blowfly,Lucilia cuprina. Using Tet-Off–regulated expression of Cas9 and dCas9, we targeted the sex-determining genetransformerwith the goal of converting females into males. Conditional Cas9 expression enabled knockout of a visual marker gene, confirming inducible genome editing. However, strains expressingtransformer-targeting sgRNA arrays did not undergo sex transformation. Embryonic microinjection oftransformer-targeting sgRNAs into Cas9-expressing embryos produced intersex individuals, indicating that sgRNA expression from the integrated arrays was insufficient to disrupt the sex determination pathway. In contrast, high-level dCas9 expression was associated with developmental delays, reduced body weight, and lethality. These findings establish the first conditional CRISPR expression system inL. cuprinaand demonstrate that Cas9 is compatible with inducible gene editing, whereas dCas9 is poorly tolerated at high expression levels. 

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3. Genome-wide CRISPR guide RNA design and specificity analysis with GuideScan2

   https://doi.org/10.1186/s13059-025-03488-8  

   Schmidt, Henri; Zhang, Minsi; Chakarov, Dimitar; Bansal, Vineet; Mourelatos, Haralambos; Sánchez-Rivera, Francisco J; Lowe, Scott W; Ventura, Andrea; Leslie, Christina S; Pritykin, Yuri (February 2025, Genome Biology)

   Abstract We present GuideScan2 for memory-efficient, parallelizable construction of high-specificity CRISPR guide RNA (gRNA) databases and user-friendly design and analysis of individual gRNAs and gRNA libraries for targeting coding and non-coding regions in custom genomes. GuideScan2 analysis identifies widespread confounding effects of low-specificity gRNAs in published CRISPR screens and enables construction of a gRNA library that reduces off-target effects in a gene essentiality screen. GuideScan2 also enables the design and experimental validation of allele-specific gRNAs in a hybrid mouse genome. GuideScan2 will facilitate CRISPR experiments across a wide range of applications. 

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4. Computational Tools and Resources for CRISPR/Cas Genome Editing

   https://doi.org/10.1016/j.gpb.2022.02.006  

   Li, Chao; Chu, Wen; Gill, Rafaqat Ali; Sang, Shifei; Shi, Yuqin; Hu, Xuezhi; Yang, Yuting; Zaman, Qamar U; Zhang, Baohong (February 2023, Genomics, Proteomics & Bioinformatics)

   Abstract The past decade has witnessed a rapid evolution in identifying more versatile clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) nucleases and their functional variants, as well as in developing precise CRISPR/Cas-derived genome editors. The programmable and robust features of the genome editors provide an effective RNA-guided platform for fundamental life science research and subsequent applications in diverse scenarios, including biomedical innovation and targeted crop improvement. One of the most essential principles is to guide alterations in genomic sequences or genes in the intended manner without undesired off-target impacts, which strongly depends on the efficiency and specificity of single guide RNA (sgRNA)-directed recognition of targeted DNA sequences. Recent advances in empirical scoring algorithms and machine learning models have facilitated sgRNA design and off-target prediction. In this review, we first briefly introduce the different features of CRISPR/Cas tools that should be taken into consideration to achieve specific purposes. Secondly, we focus on the computer-assisted tools and resources that are widely used in designing sgRNAs and analyzing CRISPR/Cas-induced on- and off-target mutations. Thirdly, we provide insights into the limitations of available computational tools that would help researchers of this field for further optimization. Lastly, we suggest a simple but effective workflow for choosing and applying web-based resources and tools for CRISPR/Cas genome editing. 

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5. Lentiviral CRISPRa/i in the adult prairie vole brain: modulating neuronal gene expression without DNA cleavage

   https://doi.org/10.3389/fgeed.2025.1602983  

   Loth, Meredith K; Mesch, Kendall T; Herrera-Garcia, Celine; Brusman, Liza E; Donaldson, Zoe R (May 2025, Frontiers in Genome Editing)

   Prairie voles (Microtus ochrogaster) are a powerful model for studying the neurobiology of social bonding, yet tools for region- and cell type-specific gene regulation remain underdeveloped in this species. Here, we present a lentivirus-mediated CRISPR activation and interference (CRISPRa/i) platform for somatic gene modulation in the prairie vole brain. This system enables non-mutagenic, titratable regulation of gene expression in the adult brain without germline modification. Our dual-vector system includes one construct expressing dCas9-VPR (VP64-p65-Rta) referred to as CRISPRa or dCas9-KRAB-MeCP2 (Kruppel-associated box-methyl CpG binding protein 2), referred to as CRISPRi under a neuron-specific promoter, and a second construct delivering a U6-driven sgRNA (single guide RNA) alongside an elongation factor 1 alpha (EF1α)-driven mCherry reporter. We detail the design, production, and stereotaxic delivery of these tools and demonstrate their application by targeting four genes implicated in social behavior (Oxtr, Avpr1a, Drd1,andDrd2) across two mesolimbic brain regions: the nucleus accumbens and ventral pallidum. Gene expression analyses confirmed robust, bidirectional transcriptional modulation for selected targets, establishing a proof of concept for CRISPRa/i in this non-traditional model. The dual-vector design is readily adaptable to other gene targets, cell types, and brain regions, and can be multiplexed to provide a flexible and scalable framework for investigating gene function in behaviorally relevant circuits. These advances represent the first successful implementation of somatic CRISPRa/i in prairie voles and expand the genetic toolkit available for this species. 

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