CRISPR Journal最新文献_第4页

Implementation of an Undergraduate Laboratory-Based Mammalian Genome Editing Course. 基于实验室的本科哺乳动物基因组编辑课程的实施。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2025-07-10 DOI: 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":"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.","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}

引用次数: 0

Advances in Gene Therapy for Sickle Cell Disease: From Preclinical Innovations to Clinical Implementation and Access Challenges. 镰状细胞病基因治疗的进展：从临床前创新到临床实施和获取挑战。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2025-06-01 Epub Date: 2025-05-12 DOI: 10.1089/crispr.2024.0101

Henna Butt, Mamatha Mandava, David Jacobsohn

引用次数: 0

CRISPR2025 New Zealand: Innovation and Collaboration. CRISPR2025新西兰：创新与合作。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2025-06-01 Epub Date: 2025-05-29 DOI: 10.1089/crispr.2025.0026

Katharina G Wandera, Jeremy Dubrulle, Russell Greene, Meric Ozturk, Gavin Knott, Dipali G Sashital, Peter C Fineran

引用次数: 0

Metagenome-Derived CRISPR-Cas12a Mining and Characterization. 宏基因组衍生的CRISPR-Cas12a挖掘和表征。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2025-06-01 Epub Date: 2025-05-21 DOI: 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":"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 in silico 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 in vivo in Escherichia coli 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.","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}

引用次数: 0

Facing Systemic Uncertainty, Can the CRISPR Community Stay the Course? 面对系统的不确定性，CRISPR社区能坚持到底吗？

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2025-06-01 DOI: 10.1089/crispr.2025.0050

Rodolphe Barrangou

引用次数: 0

Green Listed v2.0: A Web Application for Streamlined Design of Custom CRISPR Screens. 绿色列表v2.0：自定义CRISPR屏幕的流线型设计的Web应用程序。

IF 4 4区生物学

CRISPR Journal Pub Date : 2025-06-01 Epub Date: 2025-05-07 DOI: 10.1089/crispr.2025.0023

Esbjörn Henkel, Zhaojun Li, Daniel Uvehag, Bernhard Schmierer, Martin Henkel, Fredrik Wermeling

引用次数: 0

Optimizing Genome Editing in Mollusks (Crassostrea gigas) in Vitro Validation of sgRNA and Identifying Key Factors Influencing Efficiency. 优化软体动物（长牡蛎）基因组编辑体外验证sgRNA及确定影响效率的关键因素

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2025-06-01 Epub Date: 2025-02-28 DOI: 10.1089/crispr.2024.0086

Qian Li, Hong Yu, Shaojun Du, Qi Li

{"title":"Optimizing Genome Editing in Mollusks (Crassostrea gigas) in Vitro 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":"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 in vitro validation protocol for sgRNAs to streamline the gene editing process, capitalizing on the limited breeding season of the Pacific oyster (Crassostrea gigas). We evaluated the efficiency of 11 sgRNAs targeting four genes both in vitro and in vivo in C. gigas. 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 C. gigas, providing a robust framework for future gene editing endeavors in mollusks and other marine invertebrates.","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}

引用次数: 0

Discovery of Diverse CRISPR Leader Motifs, Putative Functions, and Applications for Enhanced CRISPR Detection and Subtype Annotation. 发现多种CRISPR先导基序，推测功能，以及增强CRISPR检测和亚型注释的应用。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2025-04-01 Epub Date: 2025-01-08 DOI: 10.1089/crispr.2024.0093

Murat Buyukyoruk, Pushya Krishna, Andrew Santiago-Frangos, Blake Wiedenheft

{"title":"Discovery of Diverse CRISPR Leader Motifs, Putative Functions, and Applications for Enhanced CRISPR Detection and Subtype Annotation.","authors":"Murat Buyukyoruk, Pushya Krishna, Andrew Santiago-Frangos, Blake Wiedenheft","doi":"10.1089/crispr.2024.0093","DOIUrl":"10.1089/crispr.2024.0093","url":null,"abstract":"Bacteria and archaea acquire resistance to genetic parasites by preferentially integrating short fragments of foreign DNA at one end of a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR). \"Leader\" DNA upstream of CRISPR loci regulates transcription and foreign DNA integration into the CRISPR. Here, we analyze 37,477 CRISPRs from 39,277 bacterial and 556 archaeal genomes to identify conserved sequence motifs in CRISPR leaders. A global analysis of all leader sequences fails to identify universally conserved motifs. However, an analysis of leader sequences that have been grouped by 16S rRNA-based taxonomy and CRISPR subtype reveals 87 specific motifs in type I, II, III, and V CRISPR leaders. Fourteen of these leader motifs have biochemically demonstrated roles in CRISPR biology including integration, transcription, and CRISPR RNA processing. Another 28 motifs are related to DNA binding sites for proteins with functions that are consistent with regulating CRISPR activity. In addition, we show that these leader motifs can be used to improve existing CRISPR detection methods and enhance the accuracy of CRISPR classification.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"137-148"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12171695/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142962553","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Strategies for Interdisciplinary Human Gene Editing Research: Insights from a Swiss Project. 跨学科人类基因编辑研究的策略：来自瑞士项目的见解。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2025-04-01 Epub Date: 2025-04-02 DOI: 10.1089/crispr.2024.0041

Agnes Kandlbinder, Marie-Hélène Peter-Spiess, Brigitte Leeners, Amina Mollaysa, Tommaso Cavazza, Anina Meier, Michael Braunschweig, Eleonora Ioannidi, Gerald Schwank, Michael Krauthammer

{"title":"Strategies for Interdisciplinary Human Gene Editing Research: Insights from a Swiss Project.","authors":"Agnes Kandlbinder, Marie-Hélène Peter-Spiess, Brigitte Leeners, Amina Mollaysa, Tommaso Cavazza, Anina Meier, Michael Braunschweig, Eleonora Ioannidi, Gerald Schwank, Michael Krauthammer","doi":"10.1089/crispr.2024.0041","DOIUrl":"10.1089/crispr.2024.0041","url":null,"abstract":"CRISPR gene editing is a cutting-edge technology that has advanced tremendously in recent years. The first clinical CRISPR applications have been approved, and more gene editing therapies are to be expected in human medicine. Consequently, continuous basic research is needed to assess possibilities and prime future clinical applications. Because this technology not only offers new possibilities for treating diseases but also raises important ethical and societal questions, collaboration between human, life, biomedical, and medical sciences is needed. In this article, we discuss the practical challenges of such interdisciplinary projects and present strategies for addressing them based on our experience of conducting an interdisciplinary project on CRISPR. This work aims to help and encourage interdisciplinary collaborations and discussions on modern scientific endeavors that, such as gene editing, tend to blur the lines between traditional disciplines. The strategies suggested include realistic expectations, shared goals, space setting, and expert and lay dialogue.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"79-88"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7617730/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143774789","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Initial Characterization of 12 New Subtypes and Variants of Type V CRISPR Systems. V型CRISPR系统的12个新亚型和变体的初步表征

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2025-04-01 Epub Date: 2025-03-31 DOI: 10.1089/crispr.2024.0100

Jesse Tordoff, Lauren E Alfonse, Kira S Makarova, Alexa Ornstein, Anthony J Garrity, Winston X Yan, David A Scott, Eugene V Koonin, David R Cheng

{"title":"Initial Characterization of 12 New Subtypes and Variants of Type V CRISPR Systems.","authors":"Jesse Tordoff, Lauren E Alfonse, Kira S Makarova, Alexa Ornstein, Anthony J Garrity, Winston X Yan, David A Scott, Eugene V Koonin, David R Cheng","doi":"10.1089/crispr.2024.0100","DOIUrl":"10.1089/crispr.2024.0100","url":null,"abstract":"Type V CRISPR systems are highly diverse in sequence, mechanism, and function. Although recent efforts have greatly expanded our understanding of their evolution, the diversity of type V systems remains to be completely explored, and many clades have not been experimentally characterized. In this work, we mined metagenomic databases to identify three new subtypes and nine new variants of Cas12, the effector of Type V systems, and provide experimental and computational characterization of their Protospacer-Adjacent Motif (PAM), interference activity, loci architecture, and tracrRNA dependence. Half of the new Cas12s are found in phages or prophages. New subtypes Cas12o and Cas12p lack the canonical RuvC catalytic residues, suggesting they interfere with the target without cleavage, possibly by blocking transcription or replication. One variant, Cas12f10, displays substantial activity on PAM-less targets. Our work expands the diversity of the functionally characterized Cas12 effectors and provides some promising candidates for genome engineering tools.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"149-154"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12171694/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143755334","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0