Статті в журналах: "Système CRISPR-Cas9"

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Ballouhey, Océane, Marc Bartoli, and Nicolas Levy. "CRISP(R)ation musculaire." médecine/sciences 36, no. 4 (April 2020): 358–66. http://dx.doi.org/10.1051/medsci/2020081.

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Les dystrophies musculaires sont un ensemble de pathologies musculaires rares, caractérisées par une faiblesse et une dégénérescence progressive du muscle. Ce sont des maladies d’origine génétique causées par la mutation d’un ou de plusieurs gènes impliqués dans les fonctions musculaires. Malgré des progrès significatifs réalisés dans le champ des biothérapies au cours des dernières années, il n’existe pas, à ce jour, de traitement curatif disponible pour ces pathologies. Les études menées depuis la découverte de l’outil d’édition génomique CRISPR-Cas9 ont néanmoins permis des avancées significatives et prometteuses dans le traitement des dystrophies musculaires. Le système CRISPR-Cas9 permet une édition stable et permanente du génome et doit permettre d’éviter les traitements longs et répétitifs. Dans cette revue, nous aborderons les dernières avancées thérapeutiques utilisant le système CRISPR-Cas9 dans le cadre des dystrophies musculaires d’origine génétique.

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Croteau, Félix R., Geneviève M. Rousseau, and Sylvain Moineau. "Le système CRISPR-Cas." médecine/sciences 34, no. 10 (October 2018): 813–19. http://dx.doi.org/10.1051/medsci/2018215.

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CRISPR-Cas est un système immunitaire adaptatif utilisé par de nombreux microbes pour se défendre contre l’invasion d’acides nucléiques tels que les génomes viraux et autres éléments génétiques mobiles. Le système microbien utilise son locus CRISPR pour stocker de l’information génétique afin de produire des ARN guides. Ces derniers, de concert avec des endonucléases (Cas), empêchent des invasions futures. Des parties de ce système microbien ont été exploitées pour développer un puissant outil d’édition des génomes dans une panoplie d’organismes. La capacité de CRISPR-Cas9 à couper efficacement et à des endroits très précis de l’ADN pourrait peut-être permettre un jour de guérir certaines maladies génétiques humaines. La malléabilité de cet outil d’édition rend possible une variété d’applications allant de la modulation de l’expression de gènes à des modifications épigénétiques. Les locus CRISPR représentent également une mine d’informations pouvant servir de méthode de typage de souches microbiennes ou encore une façon d’étudier les interactions entre les bactéries et leurs habitats.

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Cohen, O., P. Maru, Q. Liang, and J. Saeij. "Toxoplasma : Identification d'une protéine impliquée dans l'échappement immunitaire grâce au système CRISPR/Cas9." Médecine et Maladies Infectieuses Formation 3, no. 2 (June 2024): S107. http://dx.doi.org/10.1016/j.mmifmc.2024.04.316.

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Brusson, Megane, та Annarita Miccio. "Une approche CRISPR/Cas pour traiter les β-hémoglobinopathies". médecine/sciences 41, № 1 (січень 2025): 33–39. https://doi.org/10.1051/medsci/2024191.

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Les β-hémoglobinopathies sont des anémies génétiques graves dues à des mutations affectant l’hémoglobine adulte. Pour y remédier, le système CRISPR/Cas9 a été utilisé pour modifier génétiquement les cellules souches/progénitrices hématopoïétiques des patients ex vivo, et réactiver l’expression de l’hémoglobine fœtale dans la lignée érythroïde. Plus de 70 patients atteints de β-thalassémie ou de drépanocytose ont reçu la thérapie Casgevy®. La plupart de ces patients ont présenté une amélioration notable de leur phénotype clinique, avec une grande efficacité d’édition et des taux d’hémoglobine normaux ou presque. Bien que la sécurité et l’efficacité à long terme doivent encore être évaluées, des stratégies sont en développement pour améliorer les résultats, réduire la génotoxicité potentielle et diminuer les coûts.

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Dekeyzer, Blanche, Marie Hoareau, and Gabriel Laghlali. "Utiliser le système CRISPR/Cas9 SAM (synergic activation mediator) pour identifier des facteurs de restriction antiviraux par criblage génomique." médecine/sciences 34, no. 5 (May 2018): 401–3. http://dx.doi.org/10.1051/medsci/20183405010.

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Chaudhry, Ahsen Tahir, and Daud Akhtar. "Gene Therapy and Modification as a Therapeutic Strategy for Cancer." University of Ottawa Journal of Medicine 6, no. 1 (May 11, 2016): 44–48. http://dx.doi.org/10.18192/uojm.v6i1.1564.

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Gene therapy is an exciting new field of personalized medicine, allowing for medical procedures that can target diseases such as cancer in novel ways. Technologies that involve gene transfer treatments allow for the insertion of foreign DNA into tumour cells, resulting in restored protein expression or altered function. Gene therapy can also be used as a form of immunotherapy, either by modifying cancer cells to make them better targeted by the immune system, or by modifying the body’s immune cells to make them more aggressive towards tumours. Additionally, oncolytic virotherapy uses classes of genetically modified viruses that can specifically target and interfere with tumour cells. The ongoing development of the CRISPR/Cas9 gene editing tool may also have promise in future therapeutic applications, with the tool being capable of removing cancer-causing, latent viral infections, such as HPV, from afflicted cells. Nonetheless, there are still many questions of safety, efficacy, and commercial viability which remain to be resolved with many gene therapy procedures. There is also emerging controversy over the ethical, legal, and moral implications that modifying the genetic content of human beings will have on society. These concerns must be confronted and addressed if the benefits promised by gene therapy are to be properly realized. La thérapie génétique est un nouveau domaine d’étude médicale personnalisée qui permet de cibler des maladies spécifiques comme le cancer de façon innovatrice. Cette thérapie utilise le transfert de gènes avec une insertion d’ADN étrangère dans les cellules cancéreuses dans le but de restaurer l’expression des protéines et de retrouver la fonction cellulaire. La thérapie génétique peut aussi être utilisée comme une forme d’immunothérapie, soit en modifiant les cellules cancéreuses pour qu’elles soient mieux ciblées par le système immunitaire ou en modifiant les cellules immunitaires du corps pour les rendre plus agressives envers les tumeurs. De plus, une virothérapie oncolytique utilise des virus génétiquement modifiés qui peuvent cibler spécifiquement et interférer avec des cellules cancéreuses. Le développement du système d’édition génétique CRISPR/Cas9 s’avère prometteur pour les applications thérapeutiques futures. Cet outil est capable d’enlever les infections virales latentes dans les cellules affectées qui peuvent causer le cancer, tel que l’HPV. Malgré ces découvertes, plusieurs questions importantes demeurent quant à la sécurité et à l’efficacité de leur application. Il s’agit d’un domaine controversé avec des implications éthiques, légales, et morales, car le tout implique une modification du contenu génétique humain. Ces inquiétudes doivent être adressées afin de pouvoir continuer à explorer les bienfaits de cette thérapie génétique. En poursuivant la recherche dans ce domaine, il serait possible de valider cette thérapie et optimiser ses bienfaits.

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Reboud-Ravaux, Michèle. "Dégradation induite des protéines par des molécules PROTAC et stratégies apparentées : développements à visée thérapeutique." Biologie Aujourd’hui 215, no. 1-2 (2021): 25–43. http://dx.doi.org/10.1051/jbio/2021007.

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Alors que, pour la plupart, les médicaments actuels sont de petites molécules inhibant l’action d’une protéine en bloquant un site d’interaction, la dégradation ciblée des protéines, découverte il y a une vingtaine d’années via les petites molécules PROTAC, connaît aujourd’hui un très grand développement, aussi bien au niveau universitaire qu’industriel. Cette dégradation ciblée permet de contrôler la concentration intracellulaire d’une protéine spécifique comme peuvent le faire les techniques basées sur les acides nucléiques (oligonucléotides antisens, ARNsi, CRISPR-Cas9). Les molécules PROTAC sont des chimères hétéro-bifonctionnelles capables de lier simultanément une protéine spécifique devant être dégradée et une E3 ubiquitine ligase. Les PROTAC sont donc capables de provoquer l’ubiquitinylation de la protéine ciblée et sa dégradation par le protéasome 26S. De nature peptidique, puis non peptidique, les PROTAC sont maintenant administrables par voie orale. Ce détournement du système ubiquitine protéasome permet aux molécules PROTAC d’élargir considérablement le champ des applications thérapeutiques puisque l’élimination de protéines dépourvues de poches ou de crevasses bien définies, dites difficiles à cibler, devient possible. Cette technologie versatile a conduit à la dégradation d’une grande variété de protéines comme des facteurs de transcription, des sérine/thréonine/tyrosine kinases, des protéines de structure, des protéines cytosoliques, des lecteurs épigénétiques. Certaines ligases telles que VHL, MDM2, cereblon et IAP sont couramment utilisées pour être recrutées par les PROTAC. Actuellement, le nombre de ligases pouvant être utilisées ainsi que la nature des protéines dégradées sont en constante augmentation. Deux PROTAC sont en étude clinique pour les cancers du sein (ARV471) et de la prostate (ARV110). La dégradation spécifique d’une protéine par le protéasome peut aussi être induite par d’autres types de molécules synthétiques : colles moléculaires, marqueurs hydrophobes, HaloPROTAC, homo-PROTAC. D’autres constituants cellulaires sont aussi éligibles à une dégradation induite : ARN-PROTAC pour les protéines se liant à l’ARN et RIBOTAC pour la dégradation de l’ARN lui-même comme celui du SARS-CoV-2. Des dégradations induites en dehors du protéasome sont aussi connues : LYTAC, pour des chimères détournant la dégradation de protéines extracellulaires vers les lysosomes, et MADTAC, pour des chimères détournant la dégradation par macroautophagie. Plusieurs techniques, en particulier des plates-formes de criblage, la modélisation mathématique et la conception computationnelle sont utilisées pour le développement de nouveaux PROTAC efficaces.

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Kang Yue, 康玥, 廖雪瑶 Liao Xueyao, 谭向宇 Tan Xiangyu, 郭萍 Guo Ping та 田训 Tian Xun. "CRISPR/Cas9系统活细胞成像技术进展（特邀）". Infrared and Laser Engineering 51, № 11 (2022): 20220597. http://dx.doi.org/10.3788/irla20220597.

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Kwon, Deok-Ho, Joong-Hee Park, Deok Yeol Jeong, Jae-Bum Park, Dong-Min Park, Kyoung-Gon Kang, Seo-Young Choi, Soo Rin Kim, and Suk-Jin Ha. "Application of Genome Editing Method on Kluyveromyces marxianus 17694-DH2 using CRISPR-Cas9 System for Enhanced Xylose Utilization." KSBB Journal 34, no. 4 (December 31, 2019): 243–47. http://dx.doi.org/10.7841/ksbbj.2019.34.4.243.

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Klein, Nathalie, Selina Rust, and Lennart Randau. "CRISPR-Cas-Systeme der Klasse 1: Genome Engineering und Silencing." BIOspektrum 28, no. 4 (June 2022): 370–73. http://dx.doi.org/10.1007/s12268-022-1775-9.

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AbstractClass 1 CRISPR-Cas systems are prevalent among prokaryotes and are characterized by effector complexes that consist of multiple Cas protein subunits. Type I systems recruit the DNA nuclease Cas3 for target DNA degradation. Type IV systems exhibit CRISPR interference in the absence of DNA cleavage. These mechanisms allow for versatile genome engineering and silencing approaches. Here, we indicate advantages and drawbacks in comparison to more commonly employed Cas9-based tools.

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Khan, Sehrish, Muhammad Mahmood, Sajjad Rahman, Farzana Rizvi, and Aftab Ahmad. "Evaluation of the CRISPR/Cas9 system for the development of resistance against Cotton leaf curl virus in model plants." Plant Protection Science 56, No. 3 (June 11, 2020): 154–62. http://dx.doi.org/10.17221/105/2019-pps.

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Over the last decade, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR/Cas9 system has been used by biologists in various fields. In plant biology, the technology is being utilised to manufacture transgenic plants resistant to different diseases. In Pakistan, the Cotton leaf curl virus (CLCuV) (a begomovirus) affects cotton plants causing significant loss to the economy of this agriculturally based country. In the present study, we use the CRISPR/Cas9 system in Nicotiana benthamiana Domin (a model plant) to develop resistance against CLCuV. An interesting facet of the study was the comparison of two constructs (pHSE401 and pKSE401) with regards to their efficacy in the virus inhibition. The pKSE401 vector contained a Cas9 nuclease and two guide RNAs (gRNAs), one targeting the Replication associated protein (Rep) gene and the other targeted the βC1 gene of the Betasatellite. The vector pHSE401 had only one sgRNA that targeted the (Rep) gene. Both genes that are intended to be targeted play important roles in the replication of CLCuV. Plants infiltrated with pKSE401 exhibited a delay in the development of the symptoms of the disease and showed lower virus titres. Our proposed multiplexing approach gave efficient results of the resistance in the model plants, and the results in this communication may be extended to the CRISPR/Cas9 based editing of cotton plants.

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Wu, Wenyi, Luosheng Tang, Patricia A. D'Amore, and Hetian Lei. "Application of CRISPR-Cas9 in eye disease." Experimental Eye Research 161 (August 2017): 116–23. http://dx.doi.org/10.1016/j.exer.2017.06.007.

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Burnight, Erin R., Joseph C. Giacalone, Jessica A. Cooke, Jessica R. Thompson, Laura R. Bohrer, Kathleen R. Chirco, Arlene V. Drack, et al. "CRISPR-Cas9 genome engineering: Treating inherited retinal degeneration." Progress in Retinal and Eye Research 65 (July 2018): 28–49. http://dx.doi.org/10.1016/j.preteyeres.2018.03.003.

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Schmierer, Bernhard, Sandeep K. Botla, Jilin Zhang, Mikko Turunen, Teemu Kivioja, and Jussi Taipale. "CRISPR/Cas9 screening using unique molecular identifiers." Molecular Systems Biology 13, no. 10 (October 2017): 945. http://dx.doi.org/10.15252/msb.20177834.

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Değirmenci, Laura, Dietmar Geiger, Fábio Luiz Rogé Ferreira, Alexander Keller, Beate Krischke, Martin Beye, Ingolf Steffan-Dewenter, and Ricarda Scheiner. "CRISPR/Cas 9-Mediated Mutations as a New Tool for Studying Taste in Honeybees." Chemical Senses 45, no. 8 (September 24, 2020): 655–66. http://dx.doi.org/10.1093/chemse/bjaa063.

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Abstract Honeybees rely on nectar as their main source of carbohydrates. Sucrose, glucose, and fructose are the main components of plant nectars. Intriguingly, honeybees express only 3 putative sugar receptors (AmGr1, AmGr2, and AmGr3), which is in stark contrast to many other insects and vertebrates. The sugar receptors are only partially characterized. AmGr1 detects different sugars including sucrose and glucose. AmGr2 is assumed to act as a co-receptor only, while AmGr3 is assumedly a fructose receptor. We show that honeybee gustatory receptor AmGr3 is highly specialized for fructose perception when expressed in Xenopus oocytes. When we introduced nonsense mutations to the respective AmGr3 gene using CRISPR/Cas9 in eggs of female workers, the resulting mutants displayed almost a complete loss of responsiveness to fructose. In contrast, responses to sucrose were normal. Nonsense mutations introduced by CRISPR/Cas9 in honeybees can thus induce a measurable behavioral change and serve to characterize the function of taste receptors in vivo. CRISPR/Cas9 is an excellent novel tool for characterizing honeybee taste receptors in vivo. Biophysical receptor characterization in Xenopus oocytes and nonsense mutation of AmGr3 in honeybees unequivocally demonstrate that this receptor is highly specific for fructose.

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Hay, Elizabeth A., Christopher Knowles, Andreas Kolb, and Alasdair MacKenzie. "Using the CRISPR/Cas9 system to understand neuropeptide biology and regulation." Neuropeptides 64 (August 2017): 19–25. http://dx.doi.org/10.1016/j.npep.2016.11.010.

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Pérez-Sosa, Camilo, Maximiliano S. Pérez, Alexander Paolo Vallejo-Janeta, Shekhar Bhansali, Santiago Miriuka, and Betiana Lerner. "Droplets for Gene Editing Using CRISPR-Cas9 and Clonal Selection Improvement Using Hydrogels." Micromachines 15, no. 3 (March 19, 2024): 413. http://dx.doi.org/10.3390/mi15030413.

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Gene editing tools have triggered a revolutionary transformation in the realms of cellular and molecular physiology, serving as a fundamental cornerstone for the evolution of disease models and assays in cell culture reactions, marked by various enhancements. Concurrently, microfluidics has emerged over recent decades as a versatile technology capable of elevating performance and reducing costs in daily experiments across diverse scientific disciplines, with a pronounced impact on cell biology. The amalgamation of these groundbreaking techniques holds the potential to amplify the generation of stable cell lines and the production of extracellular matrix hydrogels. These hydrogels, assuming a pivotal role in isolating cells at the single-cell level, facilitate a myriad of analyses. This study presents a novel method that seamlessly integrates CRISPR-Cas9 gene editing techniques with single-cell isolation methods in induced pluripotent stem cell (hiPSC) lines, utilizing the combined power of droplets and hydrogels. This innovative approach is designed to optimize clonal selection, thereby concurrently reducing costs and the time required for generating a stable genetically modified cell line. By bridging the advancements in gene editing and microfluidic technologies, our approach not only holds significant promise for the development of disease models and assays but also addresses the crucial need for efficient single-cell isolation. This integration contributes to streamlining processes, making it a transformative method with implications for enhancing the efficiency and cost-effectiveness of stable cell line generation. As we navigate the intersection of gene editing and microfluidics, our study marks a significant stride toward innovative methodologies in the dynamic landscape of cellular and molecular physiology research.

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Tripathi, Ratnakar, Nishant R. Sinha, Duraisamy Kempuraj, Praveen K. Balne, James R. Landreneau, Ankit Juneja, Aaron D. Webel, and Rajiv R. Mohan. "Evaluation of CRISPR/Cas9 mediated TGIF gene editing to inhibit corneal fibrosis in vitro." Experimental Eye Research 220 (July 2022): 109113. http://dx.doi.org/10.1016/j.exer.2022.109113.

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Halmi, Muhammad Farid Azlan, Mohd Amirul Faiz Zulkifli, and Kamal Hisham Kamarul Zaman. "CRISPR-Cas9 Genome Editing: A Brief Scientometric Insight on Scientific Production and Knowledge Structure." Journal of Scientometric Research 12, no. 2 (August 6, 2023): 395–402. http://dx.doi.org/10.5530/jscires.12.2.035.

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Sheets, Lavinia, Melanie Holmgren, and Katie S. Kindt. "How Zebrafish Can Drive the Future of Genetic-based Hearing and Balance Research." Journal of the Association for Research in Otolaryngology 22, no. 3 (April 28, 2021): 215–35. http://dx.doi.org/10.1007/s10162-021-00798-z.

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AbstractOver the last several decades, studies in humans and animal models have successfully identified numerous molecules required for hearing and balance. Many of these studies relied on unbiased forward genetic screens based on behavior or morphology to identify these molecules. Alongside forward genetic screens, reverse genetics has further driven the exploration of candidate molecules. This review provides an overview of the genetic studies that have established zebrafish as a genetic model for hearing and balance research. Further, we discuss how the unique advantages of zebrafish can be leveraged in future genetic studies. We explore strategies to design novel forward genetic screens based on morphological alterations using transgenic lines or behavioral changes following mechanical or acoustic damage. We also outline how recent advances in CRISPR-Cas9 can be applied to perform reverse genetic screens to validate large sequencing datasets. Overall, this review describes how future genetic studies in zebrafish can continue to advance our understanding of inherited and acquired hearing and balance disorders.

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Horie, Kengo, Kiyoshi Inoue, Shingo Suzuki, Saki Adachi, Saori Yada, Takashi Hirayama, Shizu Hidema, Larry J. Young, and Katsuhiko Nishimori. "Oxytocin receptor knockout prairie voles generated by CRISPR/Cas9 editing show reduced preference for social novelty and exaggerated repetitive behaviors." Hormones and Behavior 111 (May 2019): 60–69. http://dx.doi.org/10.1016/j.yhbeh.2018.10.011.

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Hay, Elizabeth Anne, Abdulla Razak Khalaf, Pietro Marini, Andrew Brown, Karyn Heath, Darrin Sheppard, and Alasdair MacKenzie. "An analysis of possible off target effects following CAS9/CRISPR targeted deletions of neuropeptide gene enhancers from the mouse genome." Neuropeptides 64 (August 2017): 101–7. http://dx.doi.org/10.1016/j.npep.2016.11.003.

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Zeeshan, Saman, Ruoyun Xiong, Bruce T. Liang, and Zeeshan Ahmed. "100 Years of evolving gene–disease complexities and scientific debutants." Briefings in Bioinformatics 21, no. 3 (April 11, 2019): 885–905. http://dx.doi.org/10.1093/bib/bbz038.

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AbstractIt’s been over 100 years since the word `gene’ is around and progressively evolving in several scientific directions. Time-to-time technological advancements have heavily revolutionized the field of genomics, especially when it’s about, e.g. triple code development, gene number proposition, genetic mapping, data banks, gene–disease maps, catalogs of human genes and genetic disorders, CRISPR/Cas9, big data and next generation sequencing, etc. In this manuscript, we present the progress of genomics from pea plant genetics to the human genome project and highlight the molecular, technical and computational developments. Studying genome and epigenome led to the fundamentals of development and progression of human diseases, which includes chromosomal, monogenic, multifactorial and mitochondrial diseases. World Health Organization has classified, standardized and maintained all human diseases, when many academic and commercial online systems are sharing information about genes and linking to associated diseases. To efficiently fathom the wealth of this biological data, there is a crucial need to generate appropriate gene annotation repositories and resources. Our focus has been how many gene–disease databases are available worldwide and which sources are authentic, timely updated and recommended for research and clinical purposes. In this manuscript, we have discussed and compared 43 such databases and bioinformatics applications, which enable users to connect, explore and, if possible, download gene–disease data.

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Rojano, Elena, Pedro Seoane, Juan A. G. Ranea, and James R. Perkins. "Regulatory variants: from detection to predicting impact." Briefings in Bioinformatics 20, no. 5 (June 8, 2018): 1639–54. http://dx.doi.org/10.1093/bib/bby039.

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Abstract Variants within non-coding genomic regions can greatly affect disease. In recent years, increasing focus has been given to these variants, and how they can alter regulatory elements, such as enhancers, transcription factor binding sites and DNA methylation regions. Such variants can be considered regulatory variants. Concurrently, much effort has been put into establishing international consortia to undertake large projects aimed at discovering regulatory elements in different tissues, cell lines and organisms, and probing the effects of genetic variants on regulation by measuring gene expression. Here, we describe methods and techniques for discovering disease-associated non-coding variants using sequencing technologies. We then explain the computational procedures that can be used for annotating these variants using the information from the aforementioned projects, and prediction of their putative effects, including potential pathogenicity, based on rule-based and machine learning approaches. We provide the details of techniques to validate these predictions, by mapping chromatin–chromatin and chromatin–protein interactions, and introduce Clustered Regularly Interspaced Short Palindromic Repeats-Associated Protein 9 (CRISPR-Cas9) technology, which has already been used in this field and is likely to have a big impact on its future evolution. We also give examples of regulatory variants associated with multiple complex diseases. This review is aimed at bioinformaticians interested in the characterization of regulatory variants, molecular biologists and geneticists interested in understanding more about the nature and potential role of such variants from a functional point of views, and clinicians who may wish to learn about variants in non-coding genomic regions associated with a given disease and find out what to do next to uncover how they impact on the underlying mechanisms.

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Wang, Yi, Hui Wang, Ying Gao, Ding Zhang, Yan Zheng, Xingxing Hu, Qiuying Gao, et al. "A Feasibility and Safety Study of Non-Viral Genome Targeting Anti-CD19 CAR-T in Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia." Blood 136, Supplement 1 (November 5, 2020): 19–20. http://dx.doi.org/10.1182/blood-2020-139190.

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Introduction: It has been made great clinical progresses in hematological malignancies by chimeric antigen receptor (CAR) T cell therapy which utilizes virus vector for manufacture. However, there're still issues unresolved, for instance, sophisticated virus production process, deadly Cytokine Release Syndrome (CRS) side-effect, and high recurrence rate, which probably limit the availability of CAR-T therapy. Non-viral Genome Targeting CAR-T (nvGT CAR-T) may provide a feasible solution to those unmet needs mentioned above. We used CRISPR-Cas9 and non-viral vector to insert anti-CD19 CAR DNA to a specific genome locus in human T cells, which in theory, produces more moderate CAR-T cells compared with conventional CAR-T cells. The efficacy of anti-CD19 nvGT CAR-T cells had been demonstrated in our previous pre-clinical studies, and in this Phase I clinical trial (ChiCTR2000031942), its safety and efficacy in relapsed/refractory B-Cell Acute Lymphoblastic Leukemia (r/r B-ALL) patients were explored. Objective: The primary objective of this Phase I trial is to assess safety, including evaluation of adverse events (AEs) and AEs of special interest, such as CRS and neurotoxicity. Secondary objective is to evaluate efficacy as measured by the ratio of complete remission (CR). Method: Peripheral blood mononuclear cells were collected from patients or allogeneic donors, then CD3+ T cells were selected and modified by nvGT vector to produce anti-CD19 CAR-T, then administrated to patients with r/r B-ALL. Up to July 2020, twelve patients with r/r B-ALL had been enrolled in this study and 8 patients completed their treatments and entered follow-up period. For 8 patients with follow-up data, the median age was 33 years (range, 13 to 61), and the median number of previous regimens was 5 (range, 2 to 11). The median baseline percentage of bone marrow (BM) blast is 72% (range, 24.5% to 99%). Among those subjects, 2 patients once have been conducted autologous or allogeneic hematopoietic stem cell transplantation (Auto-HSCT or Allo-HSCT), and 2 patients experienced serious infection before CAR-T infusion. No patient has been treated by any other CAR-T therapy before enrollment. Baseline characteristics refer to Table 1. Administering a lymphodepleting chemotherapy regimen of cyclophosphamide 450-750 mg/m2 intravenously and fludarabine 25-45 mg/m2 intravenously on the fifth, fourth, and third day before infusion of anti-CD19 nvGT CAR-T, all patients received an infusion at dose of 0.55-8.21×106/kg (Table 1). Result: Until day 30 post CAR-T cell infusion, 8/8 (100%) cases achieved CR and 7/8 (87.5%) had minimal residual disease (MRD)-negative CR (Table 1). Anti-bacterial and anti-fungal were performed in patients SC-3, SC-4 and SC-5 after CAR-T cell infusion, which seems no influence on efficacy. Patient SC-7 was diagnosed as T-cell Acute Lymphoblastic Leukemia before Allo-HSCT but with recent recurrence of B-ALL, which was MRD-negative CR on day 21 post nvGT CAR-T therapy. Up to July 2020, all cases remain CR status. CRS occurred in all patients (100%) receiving anti-CD19 nvGT CAR-T cell, including 1 patient (12.5%) with grade 3 (Lee grading system1) CRS, two (25%) with grade 2 CRS, and 5 (62.5%) with grade 1 CRS. There were no cases of grade 4 or higher CRS (Table 1). The median time to onset CRS was 9 days (range, 1 to 12 days) and the median duration of CRS was 6 days (range, 2 to 9 days). None developed neurotoxicity. No fatal or life-threatening reactions happened and no Tocilizumab and Corticosteroids administered following CAR-T treatment. Data including body temperature (Figure 1), CAR-positive T cell percentage (Figure 2), Interleukin-6 (IL-6) and Interleukin-8 (IL-8) (Figure 3 and 4), C-reactive Protein (CRP) (Figure 5), Lactate Dehydrogenase (LDH) (Figure 6), and Procalcitonin (PCT) (Figure 7), are in accordance with the trend of CRS. Conclusion: This Phase I clinical trial primarily validates the efficacy of this novel CAR-T therapy, however, it still needs time to prove its durability. Surprisingly, we find that nvGT CAR-T therapy is seemingly superior than viral CAR-T therapy in terms of safety. All subjects which are high-risk patients with high tumor burden had low grade CRS, even a few patients sent home for observation post infusion with limited time of in-patient care. Furthermore, patients could tolerate a higher dose without severe adverse events, which probably bring a better dose-related efficacy. Disclosures No relevant conflicts of interest to declare.

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"Special issue: Discovery and development of the CRISPR–Cas9 system / Numéro spécial : Découverte et mise au point du système CRISPR–Cas9." Biochemistry and Cell Biology 95, no. 2 (April 2017): 185. http://dx.doi.org/10.1139/bcb-2017-0050.

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Crispo, Martina, Alejo Menchaca, Géraldine Schlapp, and María Noel Meikle. "Génération d’animaux génétiquement modifiés en Amérique du Sud." Bulletin de l'Académie vétérinaire de France 174 (2021). http://dx.doi.org/10.3406/bavf.2021.70957.

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La création d'animaux génétiquement modifiés a permis de réaliser de grandes avancées scientifiques dans des domaines variés. En 2006 nous avons mis en place à l'Institut Pasteur de Montevideo une Unité Technologique capable de conduire un programme de recherche propre et de fournir aux chercheurs d’Uruguay et de la région des modèles de souris génétiquement modifiés. En 2014, nous avons mis en œuvre le système CRISPR/Cas9, ce qui nous a permis d'élargir le cadre de notre activité et de développer d'autres projets impliquant la souris et le mouton, nous positionnant ainsi comme plateforme technologique de référence en Amérique du Sud. La législation relative aux Organismes Génétiquement Modifiés (OGMs) dans plusieurs pays d’Amérique du Sud ne prend en compte que les modifications comportant l'addition d'ADN exogène et en exclut donc l’édition génomique. Cette situation procure un avantage régional favorisant le développement rapide de modèles génétiquement éditées y compris dans des espèces animales de rentes. Mots-Clés : CRISPR, édition génomique, rongeurs, mouton.

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ZHANG, Cunfang, Linyong HU, Sijia LIU, Wen WANG, and Kai ZHAO. "CRISPR/Cas9 Sistemi Kullanılarak Ectodysplasin A (eda) İfade Etmeyen Zebra Balığı Üretimi." Kafkas Universitesi Veteriner Fakultesi Dergisi, 2020. http://dx.doi.org/10.9775/kvfd.2019.23252.

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Sherkatghanad, Zeinab, Moloud Abdar, Jeremy Charlier, and Vladimir Makarenkov. "Using traditional machine learning and deep learning methods for on- and off-target prediction in CRISPR/Cas9: a review." Briefings in Bioinformatics, April 20, 2023. http://dx.doi.org/10.1093/bib/bbad131.

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Abstract CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9) is a popular and effective two-component technology used for targeted genetic manipulation. It is currently the most versatile and accurate method of gene and genome editing, which benefits from a large variety of practical applications. For example, in biomedicine, it has been used in research related to cancer, virus infections, pathogen detection, and genetic diseases. Current CRISPR/Cas9 research is based on data-driven models for on- and off-target prediction as a cleavage may occur at non-target sequence locations. Nowadays, conventional machine learning and deep learning methods are applied on a regular basis to accurately predict on-target knockout efficacy and off-target profile of given single-guide RNAs (sgRNAs). In this paper, we present an overview and a comparative analysis of traditional machine learning and deep learning models used in CRISPR/Cas9. We highlight the key research challenges and directions associated with target activity prediction. We discuss recent advances in the sgRNA–DNA sequence encoding used in state-of-the-art on- and off-target prediction models. Furthermore, we present the most popular deep learning neural network architectures used in CRISPR/Cas9 prediction models. Finally, we summarize the existing challenges and discuss possible future investigations in the field of on- and off-target prediction. Our paper provides valuable support for academic and industrial researchers interested in the application of machine learning methods in the field of CRISPR/Cas9 genome editing.

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Bhattacharjee, Rudrarup, Lopamudra Das Roy, and Amarendranath Choudhury. "Understanding on CRISPR/Cas9 mediated cutting-edge approaches for cancer therapeutics." Discover Oncology 13, no. 1 (June 8, 2022). http://dx.doi.org/10.1007/s12672-022-00509-x.

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AbstractThe research focus on CRISPR/Cas9 has gained substantial concentration since the discovery of ‘an unusual repeat sequence’ reported by Ishino et al. (J Bacteriol 169:5429–5433, 1987) and the journey comprises the recent Nobel Prize award (2020), conferred to Emmanuelle Charpentier and Jennifer Doudna. Cumulatively, the CRISPR has a short, compact, and most discussed success of its application in becoming one of the most versatile and paradigm shifting technologies of Biological Research. Today, the CRISPR/Cas9 genome editing system is almost ubiquitously utilized in many facets of biological research where its tremendous gene manipulation capability has been harnessed to create miracles. From 2012, the CRISPR/Cas 9 system has been showcased in almost 15,000 research articles in the PubMed database, till date. Backed by some strong molecular evidence, the CRISPR system has been utilized in a few clinical trials targeted towards various pathologies. While the area covered by CRISPR is cosmic, this review will focus mostly on the utilization of CRISPR/Cas9 technology in the field of cancer therapy.

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Zhang, Tengbo, Yaxu Li, Yanrong Yang, Linjun Weng, Zhiqiang Wu, Jiali Zhu, Jieling Qin, Qi Liu, and Ping Wang. "iCRISEE: an integrative analysis of CRISPR screen by reducing false positive hits." Briefings in Bioinformatics 23, no. 1 (December 15, 2021). http://dx.doi.org/10.1093/bib/bbab505.

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Abstract Clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas9) technology has become a popular tool for the study of genome function, and the use of this technology can achieve large-scale screening studies of specific phenotypes. Several analysis tools for CRISPR/Cas9 screening data have been developed, while high false positive rate remains a great challenge. To this end, we developed iCRISEE, an integrative analysis of CRISPR ScrEEn by reducing false positive hits. iCRISEE can dramatically reduce false positive hits and it is robust to different single guide RNA (sgRNA) library by introducing precise data filter and normalization, model selection and valid sgRNA number correction in data preprocessing, sgRNA ranking and gene ranking. Furthermore, a powerful web server has been presented to automatically complete the whole CRISPR/Cas9 screening analysis, where we integrated the main hypothesis in multiple algorithms as a full workflow, including quality control, sgRNA extracting, sgRNA alignment, sgRNA ranking, gene ranking and pathway enrichment. In addition, output of iCRISEE, including result mapping, sample clustering, sgRNA ranking and gene ranking, can be easily visualized and downloaded for publication. Taking together, iCRISEE presents to be the state-of-the-art and user-friendly tool for CRISPR screening data analysis. iCRISEE is available at https://www.icrisee.com.

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Yang, Zitian, Zexin Zhang, Jing Li, Wen Chen, and Changning Liu. "CRISPRlnc: a machine learning method for lncRNA-specific single-guide RNA design of CRISPR/Cas9 system." Briefings in Bioinformatics 25, no. 2 (January 22, 2024). http://dx.doi.org/10.1093/bib/bbae066.

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Abstract CRISPR/Cas9 is a promising RNA-guided genome editing technology, which consists of a Cas9 nuclease and a single-guide RNA (sgRNA). So far, a number of sgRNA prediction softwares have been developed. However, they were usually designed for protein-coding genes without considering that long non-coding RNA (lncRNA) genes may have different characteristics. In this study, we first evaluated the performances of a series of known sgRNA-designing tools in the context of both coding and non-coding datasets. Meanwhile, we analyzed the underpinnings of their varied performances on the sgRNA’s specificity for lncRNA including nucleic acid sequence, genome location and editing mechanism preference. Furthermore, we introduce a support vector machine-based machine learning algorithm named CRISPRlnc, which aims to model both CRISPR knock-out (CRISPRko) and CRISPR inhibition (CRISPRi) mechanisms to predict the on-target activity of targets. CRISPRlnc combined the paired-sgRNA design and off-target analysis to achieve one-stop design of CRISPR/Cas9 sgRNAs for non-coding genes. Performance comparison on multiple datasets showed that CRISPRlnc was far superior to existing methods for both CRISPRko and CRISPRi mechanisms during the lncRNA-specific sgRNA design. To maximize the availability of CRISPRlnc, we developed a web server (http://predict.crisprlnc.cc) and made it available for download on GitHub.

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Е.М., Колоскова, Езерский В.А., Белова Н.В., Кутьин И.В., Рябых В.П., Трубицина Т.П. та Максименко С.В. "ГЕННО-ИНЖЕНЕРНАЯ КОНСТРУКЦИЯ ДЛЯ ЗАМЕЩЕНИЯ ГЕНА МЫШИ ПОСЛЕДОВАТЕЛЬНОСТЬЮ кДНК ЛАКТОФЕРРИНА ЧЕЛОВЕКА МЕТОДОМ HDR С ПРИМЕНЕНИЕМ СИСТЕМЫ CRISPR / Cas9". Проблемы биологии продуктивных животных, № 2(2) (31 серпня 2018). https://doi.org/10.25687/1996-6733.prodanimbiol.2018.2.19-30.

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По результатам рестриктного анализа потенциальных компонентов генно-инженерной конструкции основным вектором-носителем была выбрана плазмида рJET1.2/blunt . ПЦР-амплификат кДНК чЛФ клонировали в рJET1.2/blunt с получением плазмиды рJET1.2hLf . фрагменты 3’WAP и 5’WAP, вырезанные из полученных ранее промежуточных плазмид pTZ5’WAP и pTZ3’WAP, были последовательно клонированы в рjet1.2hlf . Рестриктный и ПЦР-анализы подтвердили состав созданной плазмиды рWAPhLf. Линейная генная конструкция размером 4061 п.н. вырезается рестриктазами EagI (NotI) и ClaI , содержит 2136 п.н. кДНК чЛФ, левое и правое плечи гомологии гену кислого сывороточного белка мыши размером 945 и 980 п.н. соответственно. Полученную плазмиду (или линеаризованную генную конструкцию) предполагается использовать в качестве матрицы для замещения кодирующей последовательности гена mWAP на последовательность кДНК чЛФ по механизму прямой гомологичной рекомбинации с применением системы CRISPR/Cas9.

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С.В., Максименко, Трубицина Т.П., Белова Н.В., Кутьин И.В. та Рябых В.П. "ПОЛУЧЕНИЕ БЛАСТОЦИСТ МЫШИ В КУЛЬТУРАЛЬНЫХ СРЕДАХ ПОСЛЕ МИКРОИНЪЕКЦИИ В ПРОНУКЛЕУСЫ ЗИГОТ СМЕСИ ПЛАЗМИД ДЛЯ ЭКСПРЕССИИ КОМПОНЕНТОВ CRISPR/Cas9 СИСТЕМЫ". Проблемы биологии продуктивных животных, № 3 (20 грудня 2018). https://doi.org/10.25687/1996-6733.prodanimbiol.2018.3.106-110.

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Цель работы - исследовать способность зигот к развитию до стадии бластоцисты после микроинъекции в их пронуклеусы смеси плазмид для экспрессии компонентов CRISPR/Cas9 системы и культивирования на коммерческих средах. Донорами клеток были самки мышей линии C57BL6/CBA в возрасте 6-9 недель. В качестве манипуляционной среды использовалась коммерческая среда Global with Hepes® с добавлением 10% заменителя сывороточного белка, а в качестве культуральной среды - Global® с добавлением 10% заменителя сывороточного белка. В результате получены данные, которые свидетельствуют о том, что исследованные среды могут быть успешно использованы для получения бластоцист. При культивировании после микроинъекции в течение суток вступили в развитие 84,5% эмбрионов, стадии компактной морулы достигли 52%, из них стадии бластоцисты - 92%. Заключили, что выход бластоцист от числа микроиъецированных зигот на уровне 44% достаточен для выполнения дальнейших работ по получению геномно-редактированных животных, продуцирующих рекомбинантные белки с молоком. The aim of the study is to investigate the ability of the zygotes to develop to the blastocyst stage after microinjection into their pronuclei of a mixture of plasmids for expressing the components of CRISPR/Cas9 system and cultivation on commercial media. Cell donors were female C57BL6/CBA mice aged 6-9 weeks. As a manipulation medium, the Global with Hepes® commercial medium was used with the addition of 10% whey protein substitute, and as a culture medium - Global® with the addition of 10% whey protein substitute. As a result, data have been obtained that indicate that the media studied can be successfully used to produce blastocysts. During cultivation after microinjection, 84.5% of embryos entered the development within a day, reaching 52% before the stage of compact morula, of which up to the stage of blastocyst - 92%. It was concluded that the yield of blastocysts from the number of micro-injected zygotes at the level of 44% is sufficient to carry out further work on obtaining genomic-edited animals that produce recombinant proteins with milk.

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Hu, Yunping, Baisong Lu, Zhiyong Deng, Fei Xing, and Wesley Hsu. "Virus-like particle-based delivery of Cas9/guide RNA ribonucleoprotein efficiently edits the brachyury gene and inhibits chordoma growth in vivo." Discover Oncology 14, no. 1 (May 18, 2023). http://dx.doi.org/10.1007/s12672-023-00680-9.

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Abstract Purpose Chordoma is a rare and aggressive bone cancer driven by the developmental transcription factor brachyury. Efforts to target brachyury are hampered by the absence of ligand-accessible small-molecule binding pockets. Genome editing with CRISPR systems provides an unprecedented opportunity to modulate undruggable transcription factor targets. However, delivery of CRISPR remains a bottleneck for in vivo therapy development. The aim was to investigate the in vivo therapeutic efficiency of Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) delivery through a novel virus-like particle (VLP) by fusing an aptamer-binding protein to the lentiviral nucleocapsid protein. Methods The p24 based ELISA and transmission electron microscopy were used to determine the characterization of engineered VLP-packaged Cas9/gRNA RNP. The deletion efficiency of brachyury gene in chordoma cells and tissues was measured by genome cleavage detection assay. RT-PCR, Western blot, immunofluorescence staining, and IHC were employed to test the function of brachyury deletion. Cell growth and tumor volume were measured to evaluate the therapeutic efficiency of brachyury deletion by VLP-packaged Cas9/gRNA RNP. Results Our “all-in-one” VLP-based Cas9/gRNA RNP system allows for transient expression of Cas9 in chordoma cells, but maintains efficient editing capacity leading to approximately 85% knockdown of brachyury with subsequent inhibition of chordoma cell proliferation and tumor progression. In addition, this VLP-packaged brachyury-targeting Cas9 RNP avoids systemic toxicities in vivo. Conclusion Our preclinical studies demonstrate the potential of VLP-based Cas9/gRNA RNP gene therapy for the treatment of brachyury-dependent chordoma.

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Zhang, Guishan, Ye Luo, Xianhua Dai, and Zhiming Dai. "Benchmarking deep learning methods for predicting CRISPR/Cas9 sgRNA on- and off-target activities." Briefings in Bioinformatics 24, no. 6 (September 22, 2023). http://dx.doi.org/10.1093/bib/bbad333.

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Abstract In silico design of single guide RNA (sgRNA) plays a critical role in clustered regularly interspaced, short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. Continuous efforts are aimed at improving sgRNA design with efficient on-target activity and reduced off-target mutations. In the last 5 years, an increasing number of deep learning-based methods have achieved breakthrough performance in predicting sgRNA on- and off-target activities. Nevertheless, it is worthwhile to systematically evaluate these methods for their predictive abilities. In this review, we conducted a systematic survey on the progress in prediction of on- and off-target editing. We investigated the performances of 10 mainstream deep learning-based on-target predictors using nine public datasets with different sample sizes. We found that in most scenarios, these methods showed superior predictive power on large- and medium-scale datasets than on small-scale datasets. In addition, we performed unbiased experiments to provide in-depth comparison of eight representative approaches for off-target prediction on 12 publicly available datasets with various imbalanced ratios of positive/negative samples. Most methods showed excellent performance on balanced datasets but have much room for improvement on moderate- and severe-imbalanced datasets. This study provides comprehensive perspectives on CRISPR/Cas9 sgRNA on- and off-target activity prediction and improvement for method development.

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Hu, Xumeng, Beibei Zhang, Xiaoli Li, Miao Li, Yange Wang, Handong Dan, Jiamu Zhou, et al. "The application and progression of CRISPR/Cas9 technology in ophthalmological diseases." Eye, August 1, 2022. http://dx.doi.org/10.1038/s41433-022-02169-1.

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Guan, Zengrui, and Zhenran Jiang. "Transformer-based anti-noise models for CRISPR-Cas9 off-target activities prediction." Briefings in Bioinformatics, April 17, 2023. http://dx.doi.org/10.1093/bib/bbad127.

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Abstract The off-target effect occurring in the CRISPR-Cas9 system has been a challenging problem for the practical application of this gene editing technology. In recent years, various prediction models have been proposed to predict potential off-target activities. However, most of the existing prediction methods do not fully exploit guide RNA (gRNA) and DNA sequence pair information effectively. In addition, available prediction methods usually ignore the noise effect in original off-target datasets. To address these issues, we design a novel coding scheme, which considers the key features of mismatch type, mismatch location and the gRNA-DNA sequence pair information. Furthermore, a transformer-based anti-noise model called CrisprDNT is developed to solve the noise problem that exists in the off-target data. Experimental results of eight existing datasets demonstrate that the method with the inclusion of the anti-noise loss functions is superior to available state-of-the-art prediction methods. CrisprDNT is available at https://github.com/gzrgzx/CrisprDNT.

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Huang, Xiaoqiang, Jun Zhou, Dongshan Yang, Jifeng Zhang, Xiaofeng Xia, Yuqing Eugene Chen, and Jie Xu. "Decoding CRISPR–Cas PAM recognition with UniDesign." Briefings in Bioinformatics, April 19, 2023. http://dx.doi.org/10.1093/bib/bbad133.

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Abstract The critical first step in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–associated (CRISPR–Cas) protein-mediated gene editing is recognizing a preferred protospacer adjacent motif (PAM) on target DNAs by the protein’s PAM-interacting amino acids (PIAAs). Thus, accurate computational modeling of PAM recognition is useful in assisting CRISPR–Cas engineering to relax or tighten PAM requirements for subsequent applications. Here, we describe a universal computational protein design framework (UniDesign) for designing protein–nucleic acid interactions. As a proof of concept, we applied UniDesign to decode the PAM–PIAA interactions for eight Cas9 and two Cas12a proteins. We show that, given native PIAAs, the UniDesign-predicted PAMs are largely identical to the natural PAMs of all Cas proteins. In turn, given natural PAMs, the computationally redesigned PIAA residues largely recapitulated the native PIAAs (74% and 86% in terms of identity and similarity, respectively). These results demonstrate that UniDesign faithfully captures the mutual preference between natural PAMs and native PIAAs, suggesting it is a useful tool for engineering CRISPR–Cas and other nucleic acid-interacting proteins. UniDesign is open-sourced at https://github.com/tommyhuangthu/UniDesign.

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Yu, Xiaowei, Nannan Sun, Xue Yang, Zhenni Zhao, Jiamin Zhang, Miao Zhang, Dandan Zhang, Jian Ge, and Zhigang Fan. "Myelin regulatory factor deficiency is associated with the retinal photoreceptor defects in mice." Visual Neuroscience 38 (2021). http://dx.doi.org/10.1017/s0952523821000043.

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Abstract Previously, we reported the myelin regulatory factor (MYRF) as a candidate gene for nanophthalmos. We have also produced Myrf knockdown (Myrf+/−) mouse strain to investigate the cellular and molecular phenotypes of reduced MYRF expression in the retina. Myrf+/− mouse strain was generated using the CRISPR/Cas9 system. Optomotor response system, electroretinogram (ERG), spectral-domain optical coherence tomography (SD-OCT), histology, and immunohistochemistry were performed to evaluate retinal spatial vision, electrophysiological function, retinal thickness, and pathological changes in cone or rod photoreceptors, respectively. RNA sequencing (RNA-seq) was performed to investigate the underlying molecular mechanism linking Myrf deficiency with photoreceptor defects. The genotype and phenotype of CRISPR/Cas9-induced Myrf+/− mice and their offspring were comprehensively investigated. Photoreceptor defects were detected in the retinas of Myrf+/− mice. Visual acuity and ERG responses were decreased in Myrf+/− mice compared with the control mice (Myrf+/+). The loss of cone and rod neurons was proportional to the decreased outer nuclear layer (ONL) thickness. Moreover, RNA-seq revealed that phototransduction and estrogen signaling pathways played important roles in the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Myrf+/− mouse strain provides a good model to investigate the function of the MYRF gene. Photoreceptor defects with impaired functions of spatial vision and retinal electrophysiology indicate an important role played by MYRF in retinal development. Alterations in phototransduction and estrogen signaling pathways play important roles in linking Myrf deficiency with retinal photoreceptor defects.

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Niu, Xiaohui, Kaixuan Deng, Lifen Liu, Kun Yang, and Xuehai Hu. "A statistical framework for predicting critical regions of p53-dependent enhancers." Briefings in Bioinformatics, May 11, 2020. http://dx.doi.org/10.1093/bib/bbaa053.

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Abstract P53 is the ‘guardian of the genome’ and is responsible for regulating cell cycle and apoptosis. The genomic p53 binding regions, where activating transcriptional factors and cofactors like p300 simultaneously bind, are called ‘p53-dependent enhancers’, which play an important role in tumorigenesis. Current experimental assays generally provide a broad peak of each enhancer element, leaving our knowledge about critical enhancer regions (CERs) limited. Under the inspiration of enhancer dissection by CRISPR-Cas9 screen library on genome-wide p53 binding sites, here we introduce a statistical framework called ‘Computational CRISPR Strategy’ (CCS), to predict whether a given DNA fragment will be a p53-dependent CER by employing 7-mer as feature extractions along with random forest as the regressor. When training on a p53 CRISPR enhancer dataset, CCS not only accurately fitted the top-ranked enriched single guide RNAs (sgRNAs) but also successfully reproduced two known CERs that were validated by experiments. When applying it to an independent testing dataset on a tilling of a 2K-b genomic region of CRISPR-deCDKN1A-Lib, the trained model shows great generalizability by identifying a CER containing five top-ranked sgRNAs. A feature importance analysis further indicates that top-ranked 7-mers are mapped onto informative TF motifs including POU5F1 and SOX5, which are differentially enriched in p53-dependent CERs and are potential factors to make a general p53 binding site to form a p53-dependent CER, providing the interpretability of the trained model. Our results demonstrate that CCS is an alternative way of the CRISPR experiment to screen the genome for mapping p53-dependent CERs.

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Neil, Kevin, Nancy Allard, Patricia Roy, Frédéric Grenier, Alfredo Menendez, Vincent Burrus, and Sébastien Rodrigue. "High‐efficiency delivery of CRISPR‐Cas9 by engineered probiotics enables precise microbiome editing." Molecular Systems Biology 17, no. 10 (October 2021). http://dx.doi.org/10.15252/msb.202110335.

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43

Choudhury, Alaksh, Jacob A. Fenster, Reilly G. Fankhauser, Joel L. Kaar, Olivier Tenaillon, and Ryan T. Gill. "CRISPR /Cas9 recombineering‐mediated deep mutational scanning of essential genes in Escherichia coli." Molecular Systems Biology 16, no. 3 (March 2020). http://dx.doi.org/10.15252/msb.20199265.

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44

Ma, Junze, Jinglei Li, and Zheng Lu. "Application of CRISPR/Cas9 Gene Editing Technology in Studies of Intestinal Anaerobic Bacteria." Life Science and Technology, December 15, 2022. http://dx.doi.org/10.57237/j.life.2022.01.002.

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45

Ollivier, Matthias, Joselyn S. Soto, Kay E. Linker, Stefanie L. Moye, Yasaman Jami-Alahmadi, Anthony E. Jones, Ajit S. Divakaruni, Riki Kawaguchi, James A. Wohlschlegel, and Baljit S. Khakh. "Crym-positive striatal astrocytes gate perseverative behaviour." Nature, February 28, 2024. http://dx.doi.org/10.1038/s41586-024-07138-0.

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AbstractAstrocytes are heterogeneous glial cells of the central nervous system1–3. However, the physiological relevance of astrocyte diversity for neural circuits and behaviour remains unclear. Here we show that a specific population of astrocytes in the central striatum expresses μ-crystallin (encoded by Crym in mice and CRYM in humans) that is associated with several human diseases, including neuropsychiatric disorders4–7. In adult mice, reducing the levels of μ-crystallin in striatal astrocytes through CRISPR–Cas9-mediated knockout of Crym resulted in perseverative behaviours, increased fast synaptic excitation in medium spiny neurons and dysfunctional excitatory–inhibitory synaptic balance. Increased perseveration stemmed from the loss of astrocyte-gated control of neurotransmitter release from presynaptic terminals of orbitofrontal cortex–striatum projections. We found that perseveration could be remedied using presynaptic inhibitory chemogenetics8, and that this treatment also corrected the synaptic deficits. Together, our findings reveal converging molecular, synaptic, circuit and behavioural mechanisms by which a molecularly defined and allocated population of striatal astrocytes gates perseveration phenotypes that accompany neuropsychiatric disorders9–12. Our data show that Crym-positive striatal astrocytes have key biological functions within the central nervous system, and uncover astrocyte–neuron interaction mechanisms that could be targeted in treatments for perseveration.

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46

Groot, Reinoud, Joel Lüthi, Helen Lindsay, René Holtackers, and Lucas Pelkmans. "Large‐scale image‐based profiling of single‐cell phenotypes in arrayed CRISPR‐Cas9 gene perturbation screens." Molecular Systems Biology 14, no. 1 (January 2018). http://dx.doi.org/10.15252/msb.20178064.

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47

Aoi, Yuki, Abdelilah Benamar, Luc Saulnier, Marie-Christine Ralet, and Helen M. North. "Biochemical data documenting variations in mucilage polysaccharides in a range of glycosyltransferase mutants." Scientific Data 10, no. 1 (October 14, 2023). http://dx.doi.org/10.1038/s41597-023-02604-2.

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AbstractDuring Arabidopsis seed coat development, copious amounts of mucilage polysaccharides are produced in the epidermal cells. When hydrated on imbibition, these polysaccharides expand and are released to encapsulate the seed as a two-layered hydrogel. Polysaccharides are synthesized from UDP-sugars by glycosyltransferases (GTs) and several GTs, with differing activities, have been identified that contribute to mucilage polysaccharide synthesis. How these GTs orchestrate production of the complex polysaccharides found in mucilage remains to be determined. In this study, we generated a range of multiple GT mutants using either CRISPR/Cas9 targeted mutation or genetic crosses of existing T-DNA insertion mutants. Four traits for mucilage amounts or macromolecular properties were examined for four replicate seed lots from 31 different GT mutant combinations. This data provides a valuable resource for future genetic, biochemical, structural, and functional studies of the roles and properties of polysaccharides present in Arabidopsis mucilage and the relative contributions of different GTs to mucilage production.

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48

Hassan, Arshia Zernab, Henry N. Ward, Mahfuzur Rahman, Maximilian Billmann, Yoonkyu Lee, and Chad L. Myers. "Dimensionality reduction methods for extracting functional networks from large‐scale CRISPR screens." Molecular Systems Biology, September 26, 2023. http://dx.doi.org/10.15252/msb.202311657.

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AbstractCRISPR‐Cas9 screens facilitate the discovery of gene functional relationships and phenotype‐specific dependencies. The Cancer Dependency Map (DepMap) is the largest compendium of whole‐genome CRISPR screens aimed at identifying cancer‐specific genetic dependencies across human cell lines. A mitochondria‐associated bias has been previously reported to mask signals for genes involved in other functions, and thus, methods for normalizing this dominant signal to improve co‐essentiality networks are of interest. In this study, we explore three unsupervised dimensionality reduction methods—autoencoders, robust, and classical principal component analyses (PCA)—for normalizing the DepMap to improve functional networks extracted from these data. We propose a novel “onion” normalization technique to combine several normalized data layers into a single network. Benchmarking analyses reveal that robust PCA combined with onion normalization outperforms existing methods for normalizing the DepMap. Our work demonstrates the value of removing low‐dimensional signals from the DepMap before constructing functional gene networks and provides generalizable dimensionality reduction‐based normalization tools.

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49

Zhou, Jianyuan, Yanshang Li, Haotian Cao, Min Yang, Lingyu Chu, Taisong Li, Zhengmin Yu, et al. "CATA: a comprehensive chromatin accessibility database for cancer." Database 2022, no. 2022 (January 1, 2022). http://dx.doi.org/10.1093/database/baab085.

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Abstract Accessible chromatin refers to the active regions of a chromosome that are bound by many transcription factors (TFs). Changes in chromatin accessibility play a critical role in tumorigenesis. With the emergence of novel methods like Assay for Transposase-accessible Chromatin Sequencing, a sequencing method that maps chromatin-accessible regions (CARs) and enables the computational analysis of TF binding at chromatin-accessible sites, the regulatory landscape in cancer can be dissected. Herein, we developed a comprehensive cancer chromatin accessibility database named CATA, which aims to provide available resources of cancer CARs and to annotate their potential roles in the regulation of genes in a cancer type-specific manner. In this version, CATA stores 2 991 163 CARs from 23 cancer types, binding information of 1398 TFs within the CARs, and provides multiple annotations about these regions, including common single nucleotide polymorphisms (SNPs), risk SNPs, copy number variation, somatic mutations, motif changes, expression quantitative trait loci, methylation and CRISPR/Cas9 target loci. Moreover, CATA supports cancer survival analysis of the CAR-associated genes and provides detailed clinical information of the tumor samples. Database URL: CATA is available at http://www.xiejjlab.bio/cata/.

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50

Abdul Rahman, Siti Fairus, Azali Azlan, Kwok-Wai Lo, Ghows Azzam, and Nethia Mohana-Kumaran. "Dual inhibition of anti-apoptotic proteins BCL-XL and MCL-1 enhances cytotoxicity of Nasopharyngeal carcinoma cells." Discover Oncology 13, no. 1 (February 3, 2022). http://dx.doi.org/10.1007/s12672-022-00470-9.

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AbstractOne of the many strategies that cancer cells evade death is through up-regulation of the BCL-2 anti-apoptotic proteins. Hence, these proteins have become attractive therapeutic targets. Given that different cell populations rely on different anti-apoptotic proteins for survival, it is crucial to determine which proteins are important for Nasopharyngeal carcinoma (NPC) cell survival. Here we determined the survival requirements for the NPC cells using a combination of the CRISPR/Cas9 technique and selective BH3-mimetics. A human apoptosis RT2 Profiler PCR Array was first employed to profile the anti-apoptotic gene expressions in NPC cell lines HK-1 and C666-1. The HK-1 cells expressed all the anti-apoptotic genes (MCL-1, BFL-1, BCL-2, BCL-XL, and BCL-w). Similarly, the C666-1 cells expressed all the anti-apoptotic genes except BFL-1 (undetectable level). Notably, both cell lines highly expressed MCL-1. Deletion of MCL-1 sensitized the NPC cells to BCL-XL selective inhibitor A-1331852, suggesting that MCL-1 and BCL-XL may be important for NPC cell survival. Co-inhibition of MCL-1 and BCL-2 with MCL-1 selective inhibitor S63845 and BCL-2 selective inhibitor ABT-199 inhibited NPC cell proliferation but the effect on cell viability was more profound with co-inhibition of MCL-1 and BCL-XL with S63845 and A-1331852, implying that MCL-1 and BCL-XL are crucial for NPC cell survival. Furthermore, co-inhibition of MCL-1 and BCL-XL inhibited the growth and invasion of NPC spheroids. Deletion of BFL-1 sensitized NPC cells to A-1331852 suggesting that BFL-1 may play a role in NPC cell survival. Taken together co-inhibition of BCL-XL and MCL-1/BFL-1 could be potential treatment strategies for NPC.

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