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CRISPR/Cas9-mediated gene replacement in the basidiomycetous yeast Pseudozyma antarctica.
The basidiomycetous yeast, Pseudozyma antarctica, has the ability to express industrially beneficial biodegradable plastic-degrading enzyme (PaE) and glycolipids. In this study, we developed a highly efficient gene-targeting method in P. antarctica using a CRISPR/Cas9 gene-editing approach. Transformation of protoplast cells was achieved by incubation with a ribonucleoprotein (RNP) complex prepared by mixing the Cas9 protein with a single-guide RNA together with donor DNA (dDNA) containing a selectable marker in vitro. The PaE gene was selected as the targeted locus for gene disruption and gene-disrupted colonies were readily detected by their ability to degrade polybutylene succinate-co-adipate on solid media. The accuracy of the gene conversion event was confirmed by colony PCR. An increase in the RNP mix increased both transformation and gene disruption efficiencies. Examining the effect of the homology arm length of the dDNA revealed that dDNA with homology arms longer than 0.1 kb induced efficient homologous recombination in our system. Furthermore, this system was successful in another targeted locus, PaADE2. Following the creation of RNP-induced double-strand break of the chromosomal DNA, dDNA could be inserted into the target locus even in the absence of homology arms.
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Fields of dreams.
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Engineering Abiotic Stress Tolerance in Crop Plants through CRISPR Genome Editing.
Environmental abiotic stresses challenge food security by depressing crop yields often exceeding 50% of their annual production. Different methods, including conventional as well as genomic-assisted breeding, mutagenesis, and genetic engineering have been utilized to enhance stress resilience in several crop species. Plant breeding has been partly successful in developing crop varieties against abiotic stresses owning to the complex genetics of the traits as well as the narrow genetic base in the germplasm. Irrespective of the fact that genetic engineering can transfer gene(s) from any organism(s), transgenic crops have become controversial mainly due to the potential risk of transgene-outcrossing. Consequently, the cultivation of transgenic crops is banned in certain countries, particularly in European countries. In this scenario, the discovery of the CRISPR tool provides a platform for producing transgene-free genetically edited plants-similar to the mutagenized crops that are not extensively regulated such as genetically modified organisms (GMOs). Thus, the genome-edited plants without a transgene would likely go into the field without any restriction. Here, we focused on the deployment of CRISPR for the successful development of abiotic stress-tolerant crop plants for sustaining crop productivity under changing environments.
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Development of Genome Editing Approaches against Herpes Simplex Virus Infections.
Herpes simplex virus 1 (HSV-1) is a herpesvirus that may cause cold sores or keratitis in healthy or immunocompetent individuals, but can lead to severe and potentially life-threatening complications in immune-immature individuals, such as neonates or immune-compromised patients. Like all other herpesviruses, HSV-1 can engage in lytic infection as well as establish latent infection. Current anti-HSV-1 therapies effectively block viral replication and infection. However, they have little effect on viral latency and cannot completely eliminate viral infection. These issues, along with the emergence of drug-resistant viral strains, pose a need to develop new compounds and novel strategies for the treatment of HSV-1 infection. Genome editing methods represent a promising approach against viral infection by modifying or destroying the genetic material of human viruses. These editing methods include homing endonucleases (HE) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein (Cas) RNA-guided nuclease system. Recent studies have showed that both HE and CRISPR/Cas systems are effective in inhibiting HSV-1 infection in cultured cells in vitro and in mice in vivo. This review, which focuses on recently published progress, suggests that genome editing approaches could be used for eliminating HSV-1 latent and lytic infection and for treating HSV-1 associated diseases.
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CRISPR-based engineering of gene knockout cells by homology-directed insertion in polyploid Drosophila S2R+ cells.
Precise and efficient genome modifications provide powerful tools for biological studies. Previous CRISPR gene knockout methods in cell lines have relied on frameshifts caused by stochastic insertion/deletion in all alleles. However, this method is inefficient for genes with high copy number due to polyploidy or gene amplification because frameshifts in all alleles can be difficult to generate and detect. Here we describe a homology-directed insertion method to knockout genes in the polyploid Drosophila S2R+ cell line. This protocol allows generation of homozygous mutant cell lines using an insertion cassette which autocatalytically generates insertion mutations in all alleles. Knockout cells generated using this method can be directly identified by PCR without a need for DNA sequencing. This protocol takes 2-3 months and can be applied to other polyploid cell lines or high-copy-number genes.
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A modality-aware CRISPR actionability framework for functional prioritization of genome-wide significant type 2 diabetes loci.
Genome-wide association studies (GWAS) have identified numerous loci associated with Type 2 Diabetes (T2D), yet translating statistical signals into experimentally testable hypotheses remains a central challenge in post-GWAS biology. The predominance of non-coding regulatory variants complicates target gene assignment and raises uncertainty regarding optimal clustered regularly interspaced short palindromic repeats (CRISPR) perturbation strategy. Here, we present a structured CRISPR Actionability Framework that integrates genomic context, pancreatic islet enhancer overlap, tissue-specific expression validation, and locus clarity into a quantitative CRISPR Actionability Score (CAS). We applied this framework to ten genome-wide significant T2D loci and assigned modality-aware CRISPR strategies (knockout versus CRISPR interference). CAS values ranged from 4 to 10, enabling tiered prioritization into high, moderate, and lower experimental priority classes. High-priority loci included SLC30A8, TCF7L2, and KCNJ11, which demonstrated strong regulatory or coding evidence combined with islet expression support. By explicitly linking genomic architecture to perturbation modality, this framework provides a transparent and reproducible bridge between statistical genetics and functional genome editing. This approach establishes a scalable template for rational CRISPR target selection in complex disease research.
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Precision harvest: path to genetically modified organism-free crops with CRISPR by 2035.
Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR) technology enable precise genetic modifications and produce genetically modified organism -free crops that match consumer preferences. By 2035, we will be able to consume CRISPR-edited crops, addressing food security issues and boosting economies for individual countries. This review highlights the progress of genetically modified crops and the regulatory challenges involved in bringing CRISPR-edited crops to market based on product- and process-based approaches across different regions. We also examine public preferences regarding these technologies and the current status of CRISPR-edited crops in terms of market availability. Furthermore, we stress the importance of establishing clear safety standards, effective patent management, and guidance on regulatory pathways for crop approval, as well as exploring future directions for integrating these technologies with artificial intelligence.
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CRISPR-PCD and CRISPR-PCRep: Two novel technologies for simultaneous multiple segmental chromosomal deletion/replacement in Saccharomyces cerevisiae.
Genome manipulation, especially the deletion or replacement of chromosomal regions, is a salient tool for the analysis of genome function. Because of low homologous recombination activity, however, current methods are limited to manipulating only one chromosomal region in a single transformation, making the simultaneous deletion or replacement of multiple chromosomal regions difficult, laborious, and time-consuming. Here, we have developed two highly efficient and versatile genome engineering technologies, named clustered regularly interspaced short palindromic repeats (CRISPR)-PCR-mediated chromosomal deletion (PCD) (CRISPR-PCD) and PCR-mediated chromosomal replacement (CRISPR-PCRep), that integrate the CRISPR-associated protein 9 (Cas9) genome editing system (CRISPR/Cas9) into, respectively, the PCD method for chromosomal deletion and our newly developed PCRep method for chromosomal replacement. Integration of CRISPR induces double strand breaks to activate homologous recombination, and thus enhances the efficiency of deletion by PCD and replacement by PCRep, enabling multiple chromosomal regions to be manipulated simultaneously for the first time. Our data show that CRISPR-PCD can delete two internal or terminal chromosomal regions, while CRISPR-PCRep can replace triple chromosomal regions simultaneously in a single transformation. Colony PCR analysis of structural alterations showed that triple replacement of four different sets of chromosomal regions was successful in 83%-100% of transformants analyzed. These novel genome engineering technologies, which greatly reduce time and labor for genome manipulation, will provide powerful tools to facilitate the simultaneous multiple deletion and replacement of chromosomal regions, enabling the rapid analysis of genome function and breeding of useful industrial yeast strains.
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Expanding the Scope of CRISPR/Cpf1-Mediated Genome Editing in Rice.
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WHO sticks to 2020 governance plan for human-genome editing.
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