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Found: 26 099
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Field assessment of genome-edited, low asparagine wheat: Europe's first CRISPR wheat field trial.
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A "Bare Hope of A Result": The Second CRISPR Patent Appeal.
On May 12, 2025, the US Court of Appeals for the Federal Circuit issued its second decision in the long-running CRISPR patent dispute between the Regents of the University of California and related institutions (CVC) and the Broad Institute. This Perspective recounts the principal dispute to date, reviews the Federal Circuit's recent opinion, and provides a critique of its analysis. In particular, this Perspective highlights how the decision is self-contradictory and in tension with patent law's conception doctrine-when an inventor has formed a "definite and permanent" idea of an invention in the mind or whether the invention was little more than a "bare hope" of a result. This Perspective briefly concludes with the implications of this recent decision and where the underlying dispute is likely headed.
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Single-Step, High-Efficiency CRISPR-Cas9 Genome Editing in Primary Human Disease-Derived Fibroblasts.
Genome editing is a tool that has many applications, including the validation of potential drug targets. However, performing genome editing in low-passage primary human cells with the greatest physiological relevance is notoriously difficult. High editing efficiency is desired because it enables gene knockouts (KO) to be generated in bulk cellular populations and circumvents the problem of having to generate clonal cell isolates. Here, we describe a single-step workflow enabling >90% KO generation in primary human lung fibroblasts via CRISPR ribonucleoprotein delivery in the absence of antibiotic selection or clonal expansion. As proof of concept, we edited two SMAD family members and demonstrated that in response to transforming growth factor beta, SMAD3, but not SMAD2, is critical for deposition of type I collagen in the fibrotic response. The optimization of this workflow can be readily transferred to other primary cell types.
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Genome editing: A breakthrough in life science and medicine.
Genome editing technologies represent a major breakthrough that has dramatically altered strategies in a wide range of biological studies. Genome editing simplifies and accelerates the creation of animal disease models and enables construction of models in most animal species, even those that are not amenable to conventional gene targeting technology.
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Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments.
CRISPR (clustered regularly interspaced short palindromic repeats) genome-editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep-sequencing data resulting from genome-editing experiments. However, these tools are typically developed in isolation and oftentimes are not readily translatable into laboratory-based experiments. Here, we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome-editing experiment. This protocol provides instructions for sgRNA design with CRISPOR (computational tool for the design, evaluation, and cloning of sgRNA sequences), experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso (computational tool for analysis of genome-editing outcomes from deep-sequencing data). This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4-5 weeks by non-experts, as well as computational data analysis that can be performed in 1-2 d by both computational and noncomputational biologists alike using web-based and/or command-line versions.
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Developing Heritable Mutations in Arabidopsis thaliana Using a Modified CRISPR/Cas9 Toolkit Comprising PAM-Altered Cas9 Variants and gRNAs.
Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), comprising an RNA-guided DNA endonuclease and a programmable guide RNA (gRNA), is currently recognized to be a powerful genome-editing tool and is widely used in biological science. Despite the usefulness of the system, a protospacer-adjacent motif (PAM) immediately downstream of the target sequence needs to be taken into account in the design of the gRNA, a requirement which limits the flexibility of the CRISPR-based genome-editing system. To overcome this limitation, a Cas9 isolated from Streptococcus pyogenes, namely SpCas9, engineered to develop several variants of Cas9 nuclease, has been generated. SpCas9 recognizes the NGG sequence as the PAM, whereas its variants are capable of interacting with different PAMs. Despite the potential advantage of the Cas9 variants, their functionalities have not previously been tested in the widely used model plant, Arabidopsis thaliana. Here, we developed a plant-specific vector series harboring SpCas9-VQR (NGAN or NGNG) or SpCas9-EQR (NGAG) and evaluated their functionalities. These modified Cas9 nucleases efficiently introduced mutations into the CLV3 and AS1 target genes using gRNAs that were compatible with atypical PAMs. Furthermore, the generated mutations were passed on to their offspring. This study illustrated the usefulness of the SpCas9 variants because the ability to generate heritable mutations will be of great benefit in molecular genetic analyses. A greater number of potential SpCas9-variant-recognition sites in these genes are predicted, compared with those of conventional SpCas9. These results demonstrated the usefulness of the SpCas9 variants for genome editing in the field of plant science research.
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CRISPR/anti-CRISPR genome editing in Clostridium beijerinckii.
The development of CRISPR technologies has revolutionized genome editing. However, in bacteria, CRISPR-based methods can be difficult to implement due to the cytotoxicity of CRISPR-associated proteins, which often impair or entirely prevent transformation. In this work, we combine inducible expression of classical CRISPR-Cas9 components with the anti-CRISPR protein AcrIIA4 from Listeria monocytogenes to tightly regulate Cas9 activity. Using this approach, we demonstrate efficient and iterative genome editing in the genetically recalcitrant Clostridium beijerinckii DSM 6423. While deletion of upp alone was not sufficient to render the strain sensitive to 5-fluorouracil, the additional deletion of a second gene involved in the uracil salvage pathway conferred resistance to the drug and validated our gene editing strategy. Collectively, our results show that CRISPR/anti-CRISPR systems can overcome a key limitation of CRISPR-based genome editing and may offer a broadly applicable strategy for engineering otherwise intractable bacterial species.
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Regulation of CRISPR trans-cleavage activity by an overhanging activator.
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system exhibits extraordinary capability in the field of molecular diagnosis and biosensing, attributed to its trans-cleavage ability. The precise modulation of performance has emerged as a significant challenge in advancing CRISPR technology to the next stage of development. Herein, we reported a CRISPR/Cas12a regulation strategy based on an overhanging activator. The presence of overhanging domains in activators creates steric hindrances that have a substantial impact on the trans-cleavage activity and activation timing of Cas12a. The trans-cleavage activity of Cas12a can be finely tuned by adjusting the position, length, and complementarity of the overhanging domains. Moreover, specific structures exhibit characteristics of automatic delayed activation. The presence of overhanging domains enables precise and timely activation of Cas12a, facilitating multifunctional applications. This system effectively accomplishes dynamic regulation, programmable release of cargo, logical operations, and multi-enzyme detection. The flexibility and versatility of this simple and powerful CRISPR regulatory strategy will pave the way for expanded applications of CRISPR/Cas in biotechnology, bioengineering, and biomedicine.
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Risks associated with CRISPR homing gene drive.
CRISPR homing gene drive is a disruptive biotechnology developed over the past decade with potential applications in public health, agriculture, and conservation biology. This technology relies on an autonomous selfish genetic element able to spread in natural populations through the release of gene drive individuals. However, it has not yet been deployed in the wild. In this review, we examine the key risks associated with CRISPR homing gene drives. First, we explore technical limitations, where gene drives might not be as efficient as intended, and cases where mitigation strategies may not be able to block a gene drive. Then, we present four main categories of adverse effects: (a) ecological risks, corresponding to unintended consequences on ecosystems and non-target populations; (b) sociological risks, i.e. concerns over public perception, governance, and societal acceptance; (c) risks associated with research activities; and (d) risks associated with malevolent usage. Regulatory aspects are not addressed here. This article provides a foundation for evaluating gene drive risks to ensure responsible and informed decision-making.
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A CRISPR/Cas9-based genome-editing system for yam (Dioscorea spp.).
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