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https://read.qxmd.com/read/38255224/increasing-gene-editing-efficiency-via-crispr-cas9-or-cas12a-mediated-knock-in-in-primary-human-t-cells
#21
REVIEW
Natalia Kruglova, Mikhail Shepelev
T lymphocytes represent a promising target for genome editing. They are primarily modified to recognize and kill tumor cells or to withstand HIV infection. In most studies, T cell genome editing is performed using the CRISPR/Cas technology. Although this technology is easily programmable and widely accessible, its efficiency of T cell genome editing was initially low. Several crucial improvements were made in the components of the CRISPR/Cas technology and their delivery methods, as well as in the culturing conditions of T cells, before a reasonable editing level suitable for clinical applications was achieved...
January 6, 2024: Biomedicines
https://read.qxmd.com/read/38224812/one-step-knock-in-of-two-antimicrobial-peptide-transgenes-at-multiple-loci-of-catfish-by-crispr-cas9-mediated-multiplex-genome-engineering
#22
JOURNAL ARTICLE
Jinhai Wang, Indira Medina Torres, Mei Shang, Jacob Al-Armanazi, Hamza Dilawar, Darshika U Hettiarachchi, Abel Paladines-Parrales, Barrett Chambers, Kate Pottle, Misha Soman, Baofeng Su, Rex A Dunham
CRISPR/Cas9-mediated multiplex genome editing (MGE) conventionally uses multiple single-guide RNAs (sgRNAs) for gene-targeted mutagenesis via the non-homologous end joining (NHEJ) pathway. MGE has been proven to be highly efficient for functional gene disruption/knockout (KO) at multiple loci in mammalian cells or organisms. However, in the absence of a DNA donor, this approach is limited to small indels without transgene integration. Here, we establish the linear double-stranded DNA (dsDNA) and double-cut plasmid (dcPlasmid) combination-assisted MGE in channel catfish (Ictalurus punctatus), allowing combinational deletion mutagenesis and transgene knock-in (KI) at multiple sites through NHEJ/homology-directed repair (HDR) pathway in parallel...
January 13, 2024: International Journal of Biological Macromolecules
https://read.qxmd.com/read/38169468/transient-inhibition-of-53bp1-increases-the-frequency-of-targeted-integration-in-human-hematopoietic-stem-and-progenitor-cells
#23
JOURNAL ARTICLE
Ron Baik, M Kyle Cromer, Steve E Glenn, Christopher A Vakulskas, Kay O Chmielewski, Amanda M Dudek, William N Feist, Julia Klermund, Suzette Shipp, Toni Cathomen, Daniel P Dever, Matthew H Porteus
Genome editing by homology directed repair (HDR) is leveraged to precisely modify the genome of therapeutically relevant hematopoietic stem and progenitor cells (HSPCs). Here, we present a new approach to increasing the frequency of HDR in human HSPCs by the delivery of an inhibitor of 53BP1 (named "i53") as a recombinant peptide. We show that the use of i53 peptide effectively increases the frequency of HDR-mediated genome editing at a variety of therapeutically relevant loci in HSPCs as well as other primary human cell types...
January 2, 2024: Nature Communications
https://read.qxmd.com/read/38168153/impact-of-crispr-hdr-editing-versus-lentiviral-transduction-on-long-term-engraftment-and-clonal-dynamics-of-hspcs-in-rhesus-macaques
#24
Byung-Chul Lee, Ashley Gin, Chuanfeng Wu, Komudi Singh, Max Grice, Ryland Mortlock, Diana Abraham, Xing Fan, Yifan Zhou, Aisha AlJanahi, Uimook Choi, Suk See de Ravin, Taehoon Shin, Sogun Hong, Cynthia E Dunbar
For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) requires both sufficient HDR efficiency and protection of LT-HSC function and number. The impact of HDR on true LT-HSCs clonal dynamics in a relevant large animal model has not previously been studied. To track the HDR-edited cells, autologous rhesus macaque (RM) CD34 + cells were electroporated with the gRNA/Cas9 ribonucleoprotein (RNP) and HDR cassette barcode library structure and reinfused into RMs following myeloablation...
December 13, 2023: bioRxiv
https://read.qxmd.com/read/38147210/protein-tyrosine-phosphatase-studies-in-zebrafish
#25
JOURNAL ARTICLE
Daniëlle T J Woutersen, Jisca Majolée, Jeroen den Hertog
The zebrafish is an ideal model for functional analysis of genes at the molecular, protein, cell, organ, and organism levels. We have used zebrafish to analyze the function of members of the protein tyrosine phosphatase (PTP) superfamily for more than two decades. The molecular genetic toolbox has significantly improved over the years. Currently, generating mutant lines that lack the function of a PTP gene is relatively straightforward by CRISPR/Cas9 technology-mediated generation of insertions or deletions in the target gene...
2024: Methods in Molecular Biology
https://read.qxmd.com/read/38144710/progress-in-gene-editing-tools-implications-and-success-in-plants-a-review
#26
REVIEW
Suman Jyoti Bhuyan, Manoj Kumar, Pandurang Ramrao Devde, Avinash Chandra Rai, Amit Kumar Mishra, Prashant Kumar Singh, Kadambot H M Siddique
Genetic modifications are made through diverse mutagenesis techniques for crop improvement programs. Among these mutagenesis tools, the traditional methods involve chemical and radiation-induced mutagenesis, resulting in off-target and unintended mutations in the genome. However, recent advances have introduced site-directed nucleases (SDNs) for gene editing, significantly reducing off-target changes in the genome compared to induced mutagenesis and naturally occurring mutations in breeding populations. SDNs have revolutionized genetic engineering, enabling precise gene editing in recent decades...
2023: Frontiers in genome editing
https://read.qxmd.com/read/38108963/approaches-to-therapeutic-gene-editing-in-alpha-1-antitrypsin-deficiency
#27
JOURNAL ARTICLE
Alisha M Gruntman, Wen Xue, Terence R Flotte
Five distinct gene therapy approaches have been developed for treating AATD. These approaches include knockout of the mutant (PiZ) allele by introduction of double-strand breaks (DSBs) and subsequent creation of insertions and deletions (indels) by DSB repair, homology-directed repair (HDR) targeted to the mutation site, base editing, prime editing, and alternatively targeted knock-in techniques. Each approach will be discussed and a brief summary of a standard CRISPR-Cas9 targeting method will be presented...
2024: Methods in Molecular Biology
https://read.qxmd.com/read/38069573/leveraging-crispr-cas9-for-accurate-detection-of-aav-neutralizing-antibodies-the-aav-hdr-method
#28
JOURNAL ARTICLE
Guohua Li, Saining Tian, Xin-Yu Sun, Mei Zhao, Feng Zhang, Jianping Zhang, Tao Cheng, Xiao-Bing Zhang
The effectiveness of AAV-based gene therapy is frequently constrained by the presence of AAV-neutralizing antibodies (NAbs). Existing detection techniques have shown inconsistencies across laboratories and cellular dependencies, challenging their universal applicability. Here, we redefine the NAb titer concept to represent the capability to neutralize a specific number of AAV virions per milliliter of serum. We present the AAV-HDR assay, which harnesses the CRISPR-Cas9 system, offering a precise and sensitive means of detecting AAV NAbs...
December 9, 2023: Human Gene Therapy
https://read.qxmd.com/read/38036077/targeted-genome-engineering-based-on-crispr-cas9-system-to-enhance-fviii-expression-in-vitro
#29
JOURNAL ARTICLE
Lidong Zhao, Shuai Fang, Yanchun Ma, Juan Ren, Lixia Hao, Lei Wang, Jia Yang, Xiaomei Lu, Linhua Yang, Gang Wang
BACKGROUND: Hemophilia A is caused by a deficiency of coagulation factor VIII in the body due to a defect in the F8 gene. The emergence of CRISPR/Cas9 gene editing technology will make it possible to alter the expression of the F8 gene in hemophiliacs, while achieving a potential cure for the disease. METHODS: Initially, we identified high-activity variants of FVIII and constructed donor plasmids using enzymatic digestion and ligation techniques. Subsequently, the donor plasmids were co-transfected with sgRNA-Cas9 protein into mouse Neuro-2a cells, followed by flow cytometry-based cell sorting and puromycin selection...
November 28, 2023: Gene
https://read.qxmd.com/read/38023927/crispr-enabled-investigation-of-fitness-costs-associated-with-the-e198a-mutation-in-%C3%AE-tubulin-of-colletotrichum-siamense
#30
JOURNAL ARTICLE
Scott D Cosseboom, Chiti Agarwal, Mengjun Hu
INTRODUCTION: Understanding fitness costs associated with fungicide resistance is critical to improve resistance management strategies. E198A in b-tubulin confers resistance to the fungicide thiophanate-methyl and has been widely reported in several plant pathogens including Colletotrichum siamense . METHOD: To better understand potential fitness costs associated with the resistance, a ribonucleoprotein (RNP) complex mediated CRISPR/Cas9 system was used to create a point mutation (E198A) through homology directed repair (HDR) in each of the sensitive (E198) C...
2023: Frontiers in Plant Science
https://read.qxmd.com/read/38014175/lung-and-liver-editing-by-lipid-nanoparticle-delivery-of-a-stable-crispr-cas9-rnp
#31
Kai Chen, Hesong Han, Sheng Zhao, Bryant Xu, Boyan Yin, Marena Trinidad, Benjamin W Burgstone, Niren Murthy, Jennifer A Doudna
Lipid nanoparticle (LNP) delivery of CRISPR ribonucleoproteins (RNPs) has the potential to enable high-efficiency in vivo genome editing with low toxicity and an easily manufactured technology, if RNP efficacy can be maintained during LNP production. In this study, we engineered a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) using directed evolution to generate iGeoCas9 evolved variants capable of robust genome editing of cells and organs. iGeoCas9s were significantly better at editing cells than wild-type GeoCas9, with genome editing levels >100X greater than those induced by the native GeoCas9 enzyme...
November 15, 2023: bioRxiv
https://read.qxmd.com/read/38012557/genome-editing-approaches-with-crispr-cas9-the-association-of-nox4-expression-in-breast-cancer-patients-and-effectiveness-evaluation-of-different-strategies-of-crispr-cas9-to-knockout-nox4-in-cancer-cells
#32
JOURNAL ARTICLE
Marzieh Javadi, Hossein Sazegar, Abbas Doosti
BACKGROUND: The increasing prevalence of cancer detection necessitated practical strategies to deliver highly accurate, beneficial, and dependable processed information together with experimental results. We deleted the cancer biomarker NOX4 using three novel genetic knockout (KO) methods. Homology-directed repair (HDR), Dual allele HITI (Du-HITI) and CRISPR-excision were utilized in this study. METHODS: The predictive value of the NOX4 expression profile was assessed using a combined hazard ratio (HR) with a 95% confidence interval (CI)...
November 27, 2023: BMC Cancer
https://read.qxmd.com/read/38007480/modulation-of-cell-cycle-increases-crispr-mediated-homology-directed-dna-repair
#33
JOURNAL ARTICLE
Guoling Li, Xiaohui Yang, Xinxin Luo, Zhenfang Wu, Huaqiang Yang
BACKGROUND: Gene knock-in (KI) in animal cells via homology-directed repair (HDR) is an inefficient process, requiring a laborious work for screening from few modified cells. HDR tends to occur in the S and G2/M phases of cell cycle; therefore, strategies that enhance the proportion of cells in these specific phases could improve HDR efficiency. RESULTS: We used various types of cell cycle inhibitors to synchronize the cell cycle in S and G2/M phases in order to investigate their effect on regulating CRISPR/Cas9-mediated HDR...
November 25, 2023: Cell & Bioscience
https://read.qxmd.com/read/37983505/suppression-of-pinoid-mutant-phenotypes-by-mutations-in-pin-formed-1-and-pin1-gfp-fusion
#34
JOURNAL ARTICLE
Michael Mudgett, Zhouxin Shen, Xinhua Dai, Steven P Briggs, Yunde Zhao
Disruption of either the auxin transporter PIN-FORMED 1 (PIN1) or the protein kinase PINOID (PID) leads to the development of pin-like inflorescences. Previous studies have shown that phosphoregulation of PIN1 by AGC kinases including PID directs auxin flux to drive organ initiation. Here, we report unexpected findings on the genetic interactions between these two genes. We deleted the first 2/3 of the PIN1 coding sequence using CRISPR/Cas9, and the resulting pin1 mutant ( pin1-27 ) was a strong allele. Surprisingly, heterozygous pin1-27 suppressed two independent pid null mutants, whereas homozygous pin1-27 enhanced the phenotypes of the pid mutants during embryogenesis...
November 28, 2023: Proceedings of the National Academy of Sciences of the United States of America
https://read.qxmd.com/read/37977144/integration-of-xeno-free-single-cell-cloning-in-crispr-mediated-dna-editing-of%C3%A2-human-ipscs-improves-homogeneity-and-methodological-efficiency-of%C3%A2-cellular-disease-modeling
#35
JOURNAL ARTICLE
Atefeh Namipashaki, Kealan Pugsley, Xiaodong Liu, Kirra Abrehart, Sue Mei Lim, Guizhi Sun, Marco J Herold, Jose M Polo, Mark A Bellgrove, Ziarih Hawi
The capability to generate induced pluripotent stem cell (iPSC) lines, in tandem with CRISPR-Cas9 DNA editing, offers great promise to understand the underlying genetic mechanisms of human disease. The low efficiency of available methods for homogeneous expansion of singularized CRISPR-transfected iPSCs necessitates the coculture of transfected cells in mixed populations and/or on feeder layers. Consequently, edited cells must be purified using labor-intensive screening and selection, culminating in inefficient editing...
November 8, 2023: Stem Cell Reports
https://read.qxmd.com/read/37896260/amphipathic-cell-penetrating-peptide-aided-delivery-of-cas9-rnp-for-in-vitro-gene-editing-and-correction
#36
JOURNAL ARTICLE
Mert Öktem, Enrico Mastrobattista, Olivier G de Jong
The therapeutic potential of the CRISPR-Cas9 gene editing system in treating numerous genetic disorders is immense. To fully realize this potential, it is crucial to achieve safe and efficient delivery of CRISPR-Cas9 components into the nuclei of target cells. In this study, we investigated the applicability of the amphipathic cell-penetrating peptide LAH5, previously employed for DNA delivery, in the intracellular delivery of spCas9:sgRNA ribonucleoprotein (RNP) and the RNP/single-stranded homology-directed repair (HDR) template...
October 20, 2023: Pharmaceutics
https://read.qxmd.com/read/37877400/-a-novel-crispr-cas9-hlaci-donor-adapting-system-for-dsdna-templated-gene-editing
#37
JOURNAL ARTICLE
Baoxia Ma, Jieyu Cui, Hongrun Qian, Xiaojun Zhang, Sen Yang, Qijing Zhang, Yifan Han, Zhiying Zhang, Jiangang Wang, Kun Xu
During the gene editing process mediated by CRISPR/Cas9, precise genome editing and gene knock-in can be achieved by the homologous recombination of double-stranded DNA (dsDNA) donor template. However, the low-efficiency of homologous recombination in eukaryotic cells hampers the development and application of this gene editing strategy. Here, we developed a novel CRISPR/Cas9-hLacI donor adapting system (DAS) to enhance the dsDNA-templated gene editing, taking the advantage of the specific binding of the LacI repressor protein and the LacO operator sequence derived for the Escherichia coli lactose operon...
October 25, 2023: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
https://read.qxmd.com/read/37833064/application-of-crispr-cas9-technology-in-diabetes-research
#38
REVIEW
Malihe Lotfi, Alexandra E Butler, Vasily N Sukhorukov, Amirhossein Sahebkar
Diabetes is a chronic disorder with rapidly increasing prevalence that is a major global issue of our current era. There are two major types of diabetes. Polygenic forms of diabetes include type 1 diabetes (T1D) and type 2 diabetes (T2D) and its monogenic forms are maturity-onset diabetes of the young (MODY) and neonatal diabetes mellitus (NDM). There are no permanent therapeutic approaches for diabetes and current therapies rely on regular administration of various drugs or insulin injection. Recently, gene editing strategies have offered new promise for treating genetic disorders...
January 2024: Diabetic Medicine: a Journal of the British Diabetic Association
https://read.qxmd.com/read/37806084/electroporation-based-easi-crispr-yields-biallelic-insertions-of-egfp-hibit-cassette-in-immortalized-chicken-oviduct-epithelial-cells
#39
JOURNAL ARTICLE
Lingkang Liu, Jinyu Wei, Chen Chen, Qianxue Liang, Boyong Wang, Wende Wu, Gonghe Li, Xibang Zheng
Laying hens are an excellent experimental oviduct model for studying reproduction biology. Because chicken oviduct epithelial cells (cOECs) have a crucial role in synthesizing and secreting ovalbumin, laying hens have been regarded an ideal bioreactor for producing pharmaceuticals in egg white through transgene or gene editing of the ovalbumin (OVA) gene. However, related studies in cOECs are largely limited because of the lack of immortalized model cells. In addition, the editing efficiency of conventional CRISPR-HDR knock-in in chicken cells is suboptimal (ranging from 1 to 10%) and remains elevated...
September 14, 2023: Poultry Science
https://read.qxmd.com/read/37788867/mosquito-embryo-microinjection
#40
JOURNAL ARTICLE
Robert A Harrell
Genetically modified (GM) mosquitoes are an important tool in the fight against mosquito-borne disease, both indirectly through their use in research investigating host-pathogen interaction, mosquito olfaction, and anthropomorphic behavior and in future direct uses for suppression and possibly eradication through sterile insect technique (SIT) and/or gene-drive programs. Successful creation of GM mosquitoes depends on microinjection procedures that precisely deliver injection materials while causing as little damage to mosquito embryos as possible...
October 3, 2023: Cold Spring Harbor Protocols
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