CRISPR JournalPub Date : 2025-07-17DOI: 10.1177/25731599251361362
Jacob S Sherkow
{"title":"A \"Bare Hope of A Result\": The Second CRISPR Patent Appeal.","authors":"Jacob S Sherkow","doi":"10.1177/25731599251361362","DOIUrl":"https://doi.org/10.1177/25731599251361362","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144661069","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
CRISPR JournalPub Date : 2025-07-14DOI: 10.1177/25731599251358852
Valentin A Manuvera, Pavel A Bobrovsky, Daria D Kharlampieva, Ekaterina N Grafskaia, Ksenia A Brovina, Maria Y Serebrennikova, Vassili N Lazarev
{"title":"Bacterial Expression System with Deep Repression and Activation via CRISPR-Cas9.","authors":"Valentin A Manuvera, Pavel A Bobrovsky, Daria D Kharlampieva, Ekaterina N Grafskaia, Ksenia A Brovina, Maria Y Serebrennikova, Vassili N Lazarev","doi":"10.1177/25731599251358852","DOIUrl":"https://doi.org/10.1177/25731599251358852","url":null,"abstract":"<p><p>Incomplete repression of recombinant genes encoding toxic polypeptides can suppress cell growth even in the absence of a transcription inducer. To address this issue, we developed a CRISPR-Cas9-based genome editing approach that directly modifies the plasmid encoding the toxic peptide during <i>Escherichia coli</i> cultivation. The constructed plasmids contained a transcription terminator between the promoter and coding region, preventing full gene expression through abortive transcription. Upon CRISPR-Cas9 activation, this region is excised, thus restoring the functional gene. To implement this approach, we modified widely used pET-series expression plasmids by adding extra terminators in the 5'-untranslated region of the recombinant gene. Four antimicrobial peptides with strong bactericidal properties served as toxic gene products, while green fluorescent protein was used to assess the efficiency of expression repression. As a result, we developed an expression system with strong repression, which is activated by CRISPR-Cas9-mediated excision of a DNA fragment from the plasmids.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144638679","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
CRISPR JournalPub Date : 2025-07-10DOI: 10.1089/crispr.2025.0017
Isabelle Guerra, Karin Jensen, Pablo Perez-Pinera
{"title":"Implementation of an Undergraduate Laboratory-Based Mammalian Genome Editing Course.","authors":"Isabelle Guerra, Karin Jensen, Pablo Perez-Pinera","doi":"10.1089/crispr.2025.0017","DOIUrl":"https://doi.org/10.1089/crispr.2025.0017","url":null,"abstract":"<p><p>Genome engineering methods can be utilized to perform complex genetic manipulations in living cells with remarkable efficiency and precision. Given the transformative potential of these enabling technologies, their applications are steadily expanding into most biology and biomedical fields where they play a central role in many experimental frameworks. For these reasons, in order to effectively prepare future generations of biologists and bioengineers for successful careers, there is a high need to incorporate courses teaching genome editing fundamentals into existing curricula. To accomplish this objective, lecture-based courses are rapidly integrating genome editing concepts; however, there are few laboratory courses that teach the practical skills needed to successfully perform genome editing experiments. Here, we describe the development and implementation of a semester-long laboratory course that teaches students not only the techniques needed to perform gene knockout, gene activation, gene repression, and base editing in mammalian cells but also prepares them to design and troubleshoot experiments, write scientific manuscripts, as well as prepare and deliver scientific presentations. Course evaluations demonstrate that this class effectively equips students with the knowledge and hands-on experience needed to succeed in careers related to genome engineering, cell and tissue engineering, and, more broadly, biology.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144602242","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
CRISPR JournalPub Date : 2025-06-01Epub Date: 2025-05-12DOI: 10.1089/crispr.2024.0101
Henna Butt, Mamatha Mandava, David Jacobsohn
{"title":"Advances in Gene Therapy for Sickle Cell Disease: From Preclinical Innovations to Clinical Implementation and Access Challenges.","authors":"Henna Butt, Mamatha Mandava, David Jacobsohn","doi":"10.1089/crispr.2024.0101","DOIUrl":"10.1089/crispr.2024.0101","url":null,"abstract":"<p><p>Sickle cell disease (SCD) is a hereditary blood disorder caused by a specific mutation in the β-globin gene, leading to the production of hemoglobin S, which deforms red blood cells, causing occlusion in small blood vessels. This results in pain, anemia, organ damage, infections, and increased stroke risk. Treatment options, including disease-modifying therapies and curative hematopoietic stem cell transplants, have limited accessibility. Recently, autologous gene therapy has emerged as a promising curative option, particularly for SCD. Gene editing techniques such as CRISPR, base editing, and prime editing offer potential to correct this mutation. In this review, we discuss recent preclinical studies and clinical trials of gene and cell therapies, focusing on the progress of FDA-approved treatments like Lyfgenia and Casgevy. We also examine the many challenges, including accessibility, safety, and long-term efficacy, which continue to shape the future of SCD gene therapy.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"174-188"},"PeriodicalIF":3.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144008525","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
CRISPR JournalPub Date : 2025-06-01Epub Date: 2025-05-29DOI: 10.1089/crispr.2025.0026
Katharina G Wandera, Jeremy Dubrulle, Russell Greene, Meric Ozturk, Gavin Knott, Dipali G Sashital, Peter C Fineran
{"title":"CRISPR2025 New Zealand: Innovation and Collaboration.","authors":"Katharina G Wandera, Jeremy Dubrulle, Russell Greene, Meric Ozturk, Gavin Knott, Dipali G Sashital, Peter C Fineran","doi":"10.1089/crispr.2025.0026","DOIUrl":"10.1089/crispr.2025.0026","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"166-173"},"PeriodicalIF":3.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144163702","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
CRISPR JournalPub Date : 2025-06-01Epub Date: 2025-05-21DOI: 10.1089/crispr.2024.0099
Kalani Gast, Sydney Baker, Adair L Borges, Stephanie Ward, Jillian F Banfield, Rodolphe Barrangou
{"title":"Metagenome-Derived CRISPR-Cas12a Mining and Characterization.","authors":"Kalani Gast, Sydney Baker, Adair L Borges, Stephanie Ward, Jillian F Banfield, Rodolphe Barrangou","doi":"10.1089/crispr.2024.0099","DOIUrl":"10.1089/crispr.2024.0099","url":null,"abstract":"<p><p>The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies has revolutionized genome editing, with continued interest in expanding the CRISPR-associated proteins (Cas) toolbox with diverse, efficient, and specific effectors. CRISPR-Cas12a is a potent, programmable RNA-guided dual nickase, broadly used for genome editing. Here, we mined dairy cow microbial metagenomes for CRISPR-Cas systems, unraveling novel Cas12a enzymes. Using <i>in silico</i> pipelines, we characterized and predicted key drivers of CRISPR-Cas12a activity, encompassing guides and protospacer adjacent motifs for five systems. We next assessed their functional potential in cell-free transcription-translation assays with GFP-based fluorescence readouts. Lastly, we determined their genome editing potential <i>in vivo</i> in <i>Escherichia coli</i> by generating 1 kb knockouts. Unexpectedly, we observed natural sequence variation in the bridge-helix domain of the best-performing candidate and used mutagenesis to alter the activity of Cas12a orthologs, resulting in increased gene editing capabilities of a relatively inefficient candidate. This study illustrates the potential of underexplored metagenomic sequence diversity for the development and refinement of genome editing effectors.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"189-204"},"PeriodicalIF":3.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144121294","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
CRISPR JournalPub Date : 2025-06-01DOI: 10.1089/crispr.2025.0050
Rodolphe Barrangou
{"title":"Facing Systemic Uncertainty, Can the CRISPR Community Stay the Course?","authors":"Rodolphe Barrangou","doi":"10.1089/crispr.2025.0050","DOIUrl":"https://doi.org/10.1089/crispr.2025.0050","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"8 3","pages":"165"},"PeriodicalIF":3.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144276658","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
CRISPR JournalPub Date : 2025-06-01Epub Date: 2025-05-07DOI: 10.1089/crispr.2025.0023
Esbjörn Henkel, Zhaojun Li, Daniel Uvehag, Bernhard Schmierer, Martin Henkel, Fredrik Wermeling
{"title":"Green Listed v2.0: A Web Application for Streamlined Design of Custom CRISPR Screens.","authors":"Esbjörn Henkel, Zhaojun Li, Daniel Uvehag, Bernhard Schmierer, Martin Henkel, Fredrik Wermeling","doi":"10.1089/crispr.2025.0023","DOIUrl":"10.1089/crispr.2025.0023","url":null,"abstract":"<p><p>Custom CRISPR screens are powerful tools for rapid, hypothesis-driven discovery, but their design is often complex and time-consuming. Green Listed v2.0 simplifies this process with an intuitive workflow for designing custom CRISPR spacer libraries and supports downstream analysis for all users, irrespective of their computational experience. The web application features a user-friendly graphical interface freely accessible at https://greenlisted.cmm.se. Version 2.0 includes significant upgrades to the original 2016 version that were implemented based on user feedback. This includes a new gene synonym tool, expanded library options, optimized output lists, performance improvements, and linked scripts for the rational design of custom CRISPR screen gene sets.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"216-223"},"PeriodicalIF":3.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144040571","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
CRISPR JournalPub Date : 2025-06-01Epub Date: 2025-02-28DOI: 10.1089/crispr.2024.0086
Qian Li, Hong Yu, Shaojun Du, Qi Li
{"title":"Optimizing Genome Editing in Mollusks (<i>Crassostrea gigas</i>) <i>in Vitro</i> Validation of sgRNA and Identifying Key Factors Influencing Efficiency.","authors":"Qian Li, Hong Yu, Shaojun Du, Qi Li","doi":"10.1089/crispr.2024.0086","DOIUrl":"10.1089/crispr.2024.0086","url":null,"abstract":"<p><p>CRISPR-Cas9 genome editing holds tremendous potential for accelerating genetic improvements in aquaculture. The success of the CRISPR-Cas9 system relies on the specificity and efficiency of engineered single-guide RNAs (sgRNAs). In this study, we optimized an <i>in vitro</i> validation protocol for sgRNAs to streamline the gene editing process, capitalizing on the limited breeding season of the Pacific oyster (<i>Crassostrea gigas</i>). We evaluated the efficiency of 11 sgRNAs targeting four genes both <i>in vitro</i> and <i>in vivo</i> in <i>C. gigas</i>. In addition, we found that Cas9 protein differs from Cas9 mRNA in gene editing efficiency at various stages of early development. Cas9 protein proved particular efficacy in achieving early and efficient gene knockout, functioning effectively during the first cell division and facilitating biallelic gene knockouts. Statistical analysis showed that in the protein group, the biallelic editing frequency ranged from 12.5% to 57.8%, and the overall editing frequency reached as high as 75-90.6%. The mRNA group exhibited a biallelic editing frequency of 3.1-14.0% and the overall editing frequency spanning 65.6-78.1%. Contrary to expectations, low-temperature incubation (20°C) of oyster embryos prolonged the time window for the first cell division but did not improve gene editing efficiency, likely due to the high temperature sensitivity of Cas9 enzyme activity. Together, this study provides a comprehensive analysis of factors affecting the efficiency of CRISPR-Cas9 gene editing in <i>C. gigas</i>, providing a robust framework for future gene editing endeavors in mollusks and other marine invertebrates.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"205-215"},"PeriodicalIF":3.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143528008","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
CRISPR JournalPub Date : 2025-05-21DOI: 10.1089/crispr.2025.0054
Kaushik Sunder Rajan
{"title":"Biotechnologies in the World: On Global Asymmetries and the Need for Cosmopolitanism.","authors":"Kaushik Sunder Rajan","doi":"10.1089/crispr.2025.0054","DOIUrl":"https://doi.org/10.1089/crispr.2025.0054","url":null,"abstract":"<p><p>Conversations regarding genome editing are not simply about the transformative science involved. They touch upon fundamental moral questions concerning the human condition, indeed what it means to be human itself. The recent approval of a gene therapy for sickle cell disease encapsulates the relationship between scientific innovation and health care access and the relations of power and political economy that structure the world of biotech and biomedicine. Globally transformative biotechnologies must ethically situate themselves if they are not merely to reproduce longstanding historical and structural asymmetries. The time has come to embrace a cosmopolitan ethic that is attuned to the varied constitutionalisms through which debates about public good, healthy societies, and social compacts materialize around the world.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144112739","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}