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Journal articles on the topic 'CRISPR/Cas9-tagging'

Author: Grafiati

Published: 1 April 2023

Last updated: 31 July 2025

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1

Modaffari, Domenico, Aimée Finlayson, Yuyang Miao, Edward W. J. Wallace, and Kenneth E. Sawin. "Improved gene editing and fluorescent-protein tagging in Aspergillus nidulans using a Golden Gate-based CRISPR-Cas9 plasmid system." Wellcome Open Research 9 (October 17, 2024): 602. http://dx.doi.org/10.12688/wellcomeopenres.23086.1.

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CRISPR-Cas9 systems can be used for precise genome editing in filamentous fungi, including Aspergillus nidulans. However, current CRISPR-Cas9 systems for A. nidulans rely on relatively complex or multi-step cloning methods to build a plasmid expressing both Cas9 and an sgRNA targeting a genomic locus. In this study we improve on existing plasmid-based CRISPR-Cas9 systems for Aspergilli by creating an extremely simple-to-use CRISPR-Cas9 system for A. nidulans genome editing. In our system, a plasmid containing both Cas9 and an sgRNA is assembled in a one-step Golden Gate reaction. We demonstrat

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2

Thöne, Fabian M. B., Nina S. Kurrle, Harald von Melchner, and Frank Schnütgen. "CRISPR/Cas9-mediated generic protein tagging in mammalian cells." Methods 164-165 (July 2019): 59–66. http://dx.doi.org/10.1016/j.ymeth.2019.02.018.

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3

Wang, Qiang, and Jeffrey J. Coleman. "CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum." Fungal Genetics and Biology 126 (May 2019): 17–24. http://dx.doi.org/10.1016/j.fgb.2019.02.002.

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4

Lin, Da-Wei, Benjamin P. Chung, Jia-Wei Huang, Xiaorong Wang, Lan Huang, and Peter Kaiser. "Microhomology-based CRISPR tagging tools for protein tracking, purification, and depletion." Journal of Biological Chemistry 294, no. 28 (2019): 10877–85. http://dx.doi.org/10.1074/jbc.ra119.008422.

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Work in yeast models has benefitted tremendously from the insertion of epitope or fluorescence tags at the native gene locus to study protein function and behavior under physiological conditions. In contrast, work in mammalian cells largely relies on overexpression of tagged proteins because high-quality antibodies are only available for a fraction of the mammalian proteome. CRISPR/Cas9-mediated genome editing has recently emerged as a powerful genome-modifying tool that can also be exploited to insert various tags and fluorophores at gene loci to study the physiological behavior of proteins i

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5

Lankford, Kaylee P., and John D. Hulleman. "Protocol for HiBiT tagging endogenous proteins using CRISPR-Cas9 gene editing." STAR Protocols 5, no. 2 (2024): 103000. http://dx.doi.org/10.1016/j.xpro.2024.103000.

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6

Beneke, Tom, Ulrich Dobramysl, Carolina Moura Costa Catta-Preta, Jeremy Charles Mottram, Eva Gluenz, and Richard Wheeler. "Genome sequence of Leishmania mexicana MNYC/BZ/62/M379 expressing Cas9 and T7 RNA polymerase." Wellcome Open Research 7 (December 5, 2022): 294. http://dx.doi.org/10.12688/wellcomeopenres.18575.1.

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We present the genome sequence of Leishmania mexicana MNYC/BZ/62/M379 modified to express Cas9 and T7 RNA-polymerase, revealing high similarity to the reference genome (MHOM/GT2001/U1103). Through RNAseq-based annotation of coding sequences and untranslated regions, we provide primer sequences for construct and sgRNA template generation for CRISPR-assisted gene deletion and endogenous tagging.

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Beneke, Tom, Ulrich Dobramysl, Carolina Moura Costa Catta-Preta, Jeremy Charles Mottram, Eva Gluenz, and Richard J. Wheeler. "Genome sequence of Leishmania mexicana MNYC/BZ/62/M379 expressing Cas9 and T7 RNA polymerase." Wellcome Open Research 7 (February 23, 2023): 294. http://dx.doi.org/10.12688/wellcomeopenres.18575.2.

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We present the genome sequence of Leishmania mexicana MNYC/BZ/62/M379 modified to express Cas9 and T7 RNA-polymerase, revealing high similarity to the reference genome (MHOM/GT2001/U1103). Through RNAseq-based annotation of coding sequences and untranslated regions, we provide primer sequences for construct and sgRNA template generation for CRISPR-assisted gene deletion and endogenous tagging.

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8

Li, Weicheng, Yaoyao Zhang, Katy Moffat, Venugopal Nair, and Yongxiu Yao. "V5 and GFP Tagging of Viral Gene pp38 of Marek’s Disease Vaccine Strain CVI988 Using CRISPR/Cas9 Editing." Viruses 14, no. 2 (2022): 436. http://dx.doi.org/10.3390/v14020436.

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Marek’s disease virus (MDV) is a member of alphaherpesviruses associated with Marek’s disease, a highly contagious neoplastic disease in chickens. The availability of the complete sequence of the viral genome allowed for the identification of major genes associated with pathogenicity using different techniques, such as bacterial artificial chromosome (BAC) mutagenesis and the recent powerful clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based editing system. Thus far, most studies on MDV genome editing using the CRISPR/Cas9 system have fo

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Dort, Erika N., and Richard C. Hamelin. "Heterogeneity in establishment of polyethylene glycol-mediated plasmid transformations for five forest pathogenic Phytophthora species." PLOS ONE 19, no. 9 (2024): e0306158. http://dx.doi.org/10.1371/journal.pone.0306158.

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Plasmid-mediated DNA transformation is a foundational molecular technique and the basis for most CRISPR-Cas9 gene editing systems. While plasmid transformations are well established for many agricultural Phytophthora pathogens, development of this technique in forest Phytophthoras is lacking. Given our long-term research objective to develop CRISPR-Cas9 gene editing in a forest pathogenic Phytophthora species, we sought to establish the functionality of polyethylene glycol (PEG)-mediated plasmid transformation in five species: P. cactorum, P. cinnamomi, P. cryptogea, P. ramorum, and P. syringa

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10

Torres-Garcia, Sito, Lorenza Di Pompeo, Luke Eivers, et al. "SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast." Wellcome Open Research 5 (November 24, 2020): 274. http://dx.doi.org/10.12688/wellcomeopenres.16405.1.

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The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast Schizosaccharomyces pombe rely on tedious and time-consuming cloning procedures to introduce a specific sgRNA target sequence into a Cas9-expressing plasmid. In addition, Cas9 endonuclease has been reported to be toxic to fission yeast when constitutively overexpressed from the strong adh1 promoter. To overcome these problems we have developed an improved system, SpEDIT, that uses a synthesised Cas9 sequence codon-optimised for S. pombe expressed from the medium strength adh15

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11

Beneke, Tom, Ross Madden, Laura Makin, Jessica Valli, Jack Sunter, and Eva Gluenz. "A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids." Royal Society Open Science 4, no. 5 (2017): 170095. http://dx.doi.org/10.1098/rsos.170095.

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Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA t

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12

Calverley, Ben C., Karl E. Kadler, and Adam Pickard. "Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging." Cells 9, no. 9 (2020): 2070. http://dx.doi.org/10.3390/cells9092070.

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The ability to quantitate a protein of interest temporally and spatially at subcellular resolution in living cells would generate new opportunities for research and drug discovery, but remains a major technical challenge. Here, we describe dynamic, high-sensitivity protein quantitation technique using NanoLuciferase (NLuc) tagging, which is effective across microscopy and multiwell platforms. Using collagen as a test protein, the CRISPR-Cas9-mediated introduction of nluc (encoding NLuc) into the Col1a2 locus enabled the simplification and miniaturisation of procollagen-I (PC-I) quantitation. C

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Kovářová, Julie, Markéta Novotná, Joana Faria, et al. "CRISPR/Cas9-based precision tagging of essential genes in bloodstream form African trypanosomes." Molecular and Biochemical Parasitology 249 (May 2022): 111476. http://dx.doi.org/10.1016/j.molbiopara.2022.111476.

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14

Blaeser, Anthony R., Pei Lu, and Qi Long Lu. "347. Tagging FKRP and LARGE by CRISPR/Cas9 for Monitoring Expression and Localization." Molecular Therapy 23 (May 2015): S138. http://dx.doi.org/10.1016/s1525-0016(16)33956-9.

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Nitika and Andrew W. Truman. "Endogenous epitope tagging of heat shock protein 70 isoform Hsc70 using CRISPR/Cas9." Cell Stress and Chaperones 23, no. 3 (2017): 347–55. http://dx.doi.org/10.1007/s12192-017-0845-2.

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16

Roberts, Brock, Amanda Haupt, Andrew Tucker, et al. "Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization." Molecular Biology of the Cell 28, no. 21 (2017): 2854–74. http://dx.doi.org/10.1091/mbc.e17-03-0209.

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We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid c

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17

Wege, Sarah-Maria, Katharina Gejer, Fabienne Becker, Michael Bölker, Johannes Freitag, and Björn Sandrock. "Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis." Journal of Fungi 7, no. 2 (2021): 149. http://dx.doi.org/10.3390/jof7020149.

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The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require

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18

Nemeth, Tibor, Andrea Zarnocki, Anett Ladanyi, et al. "PCR-based CRISPR/Cas9 system for fluorescent tagging: A tool for studying Candida parapsilosis virulence." PLOS ONE 20, no. 2 (2025): e0312948. https://doi.org/10.1371/journal.pone.0312948.

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Candida parapsilosis is persistent in a hospital environment hence it is often associated with nosocomial infections especially amongst low-birth weight neonates. Genetic modification is therefore important to characterise the physiological and virulence related properties of this fungus. A PCR-based CRISPR/Cas9 system has been adopted to facilitate the generation of fluorescent tagged prototroph isolates. We examined a total of eight fluorescent protein coding genes, out of which three were found to be applicable for simultaneous utilisation. We investigated three clinical isolates of C. para

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19

Morrow, Christopher S., Tiaira J. Porter, and Darcie L. Moore. "Fluorescent tagging of endogenous proteins with CRISPR/Cas9 in primary mouse neural stem cells." STAR Protocols 2, no. 3 (2021): 100744. http://dx.doi.org/10.1016/j.xpro.2021.100744.

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20

Lyu, Qing, Vidhi Dhagia, Yu Han, et al. "CRISPR-Cas9–Mediated Epitope Tagging Provides Accurate and Versatile Assessment of Myocardin—Brief Report." Arteriosclerosis, Thrombosis, and Vascular Biology 38, no. 9 (2018): 2184–90. http://dx.doi.org/10.1161/atvbaha.118.311171.

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21

Cheng, Tian-Lin, and Zilong Qiu. "Long non-coding RNA tagging and expression manipulation via CRISPR/Cas9-mediated targeted insertion." Protein & Cell 9, no. 9 (2017): 820–25. http://dx.doi.org/10.1007/s13238-017-0464-9.

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22

Kong, Nannan, and Ying Wai Chan. "Protocol for biallelic tagging of an endogenous gene using CRISPR-Cas9 in human cells." STAR Protocols 4, no. 2 (2023): 102286. http://dx.doi.org/10.1016/j.xpro.2023.102286.

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23

Matsuda, Takahiko, and Izumi Oinuma. "Imaging endogenous synaptic proteins in primary neurons at single-cell resolution using CRISPR/Cas9." Molecular Biology of the Cell 30, no. 22 (2019): 2838–55. http://dx.doi.org/10.1091/mbc.e19-04-0223.

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Fluorescence imaging at single-cell resolution is a crucial approach to analyzing the spatiotemporal regulation of proteins within individual cells of complex neural networks. Here we present a nonviral strategy that enables the tagging of endogenous loci by CRISPR/Cas9-mediated genome editing combined with a nucleofection technique. The method allowed expression of fluorescently tagged proteins at endogenous levels, and we successfully achieved tagging of a presynaptic protein, synaptophysin (Syp), and a postsynaptic protein, PSD-95, in cultured postmitotic neurons. Superresolution fluorescen

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Li, James, Sandra Arest, Bartlomiej Olszowy, John Gordon, Carlos A. Barrero, and Oscar Perez-Leal. "CRISPR/Cas9-Based Screening of FDA-Approved Drugs for NRF2 Activation: A Novel Approach to Discover Therapeutics for Non-Alcoholic Fatty Liver Disease." Antioxidants 12, no. 7 (2023): 1363. http://dx.doi.org/10.3390/antiox12071363.

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With the rising prevalence of obesity, non-alcoholic fatty liver disease (NAFLD) now affects 20–25% of the global population. NAFLD, a progressive condition associated with oxidative stress, can result in cirrhosis and liver cancer in 10% and 3% of patients suffering NAFLD, respectively. Therapeutic options are currently limited, emphasizing the need for novel treatments. In this study, we examined the potential of activating the transcription factor NRF2, a crucial player in combating oxidative stress, as an innovative approach to treating NAFLD. Utilizing a CRISPR/Cas9-engineered human HEK29

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Yamashita, Kensuke, and Tetsuya Muramoto. "Efficient endogenous protein labelling in Dictyostelium using CRISPR/Cas9 knock-in and split fluorescent proteins." PLOS One 20, no. 6 (2025): e0326577. https://doi.org/10.1371/journal.pone.0326577.

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Fluorescent protein tagging is a powerful technique for visualising protein dynamics; however, full-length fluorescent protein knock-in can be inefficient at certain genomic loci, making it challenging to achieve stable and uniform expression. To address this issue, we used CRISPR/Cas9-mediated knock-in strategies with split fluorescent proteins in Dictyostelium discoideum. This approach enabled efficient integration of the short mNeonGreen2 (mNG2) fragment, mNG211, particularly at functionally critical loci such as major histone h2bv3, where full-length tagging was unsuccessful. Our analysis

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Papasavva, Panayiota L., Petros Patsali, Constantinos C. Loucari, et al. "CRISPR Editing Enables Consequential Tag-Activated MicroRNA-Mediated Endogene Deactivation." International Journal of Molecular Sciences 23, no. 3 (2022): 1082. http://dx.doi.org/10.3390/ijms23031082.

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Molecular therapies and functional studies greatly benefit from spatial and temporal precision of genetic intervention. We therefore conceived and explored tag-activated microRNA (miRNA)-mediated endogene deactivation (TAMED) as a research tool and potential lineage-specific therapy. For proof of principle, we aimed to deactivate γ-globin repressor BCL11A in erythroid cells by tagging the 3′ untranslated region (UTR) of BCL11A with miRNA recognition sites (MRSs) for the abundant erythromiR miR-451a. To this end, we employed nucleofection of CRISPR/Cas9 ribonucleoprotein (RNP) particles alongsi

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Aoto, Kazushi, Shuji Takabayashi, Hiroki Mutoh та Hirotomo Saitsu. "Generation of Flag/DYKDDDDK Epitope Tag Knock-In Mice Using i-GONAD Enables Detection of Endogenous CaMKIIα and β Proteins". International Journal of Molecular Sciences 23, № 19 (2022): 11915. http://dx.doi.org/10.3390/ijms231911915.

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Specific antibodies are necessary for cellular and tissue expression, biochemical, and functional analyses of protein complexes. However, generating a specific antibody is often time-consuming and effort-intensive. The epitope tagging of an endogenous protein at an appropriate position can overcome this problem. Here, we investigated epitope tag position using AlphaFold2 protein structure prediction and developed Flag/DYKDDDDK tag knock-in CaMKIIα and CaMKIIβ mice by combining CRISPR-Cas9 genome editing with electroporation (i-GONAD). With i-GONAD, it is possible to insert a small fragment of

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Wu, Lipeng, Dezhong Yin, Hua Su, et al. "Abstract 5607: A highly efficient donor system for CRISPR knock-in editing." Cancer Research 84, no. 6_Supplement (2024): 5607. http://dx.doi.org/10.1158/1538-7445.am2024-5607.

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Abstract CRISPR is a potent tool widely used for genome editing. While CRISPR knock-out, achieved through in-del mutations via cellular repair mechanisms, has proven remarkably effective, the knock-in system for exogenous fragment insertion encounters challenges due to limited specificity and efficiency. Two primary methods for exogenous fragment insertion, namely NHEJ (Non-Homologous End Joining) and HR (Homologous Recombination), exist. HR allows for the construction of DNA insertions with precise junctions but is comparatively less efficient than NHEJ. To address these limitations, our syst

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Li, Qingyun, Scott Barish, Sumie Okuwa, and Pelin C. Volkan. "Examination of Endogenous Rotund Expression and Function in DevelopingDrosophilaOlfactory System Using CRISPR-Cas9–Mediated Protein Tagging." G3: Genes|Genomes|Genetics 5, no. 12 (2015): 2809–16. http://dx.doi.org/10.1534/g3.115.021857.

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Willems, Jelmer, Arthur P. H. de Jong, Nicky Scheefhals, et al. "ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons." PLOS Biology 18, no. 4 (2020): e3000665. http://dx.doi.org/10.1371/journal.pbio.3000665.

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Zuo, Yamei, Xue Mei, and Andrew Singson. "CRISPR/Cas9 Mediated Fluorescent Tagging of Caenorhabditis elegans SPE-38 Reveals a Complete Localization Pattern in Live Spermatozoa." Biomolecules 13, no. 4 (2023): 623. http://dx.doi.org/10.3390/biom13040623.

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The Caenorhabditis elegans spe-38 gene encodes a four-pass transmembrane molecule that is required in sperm for fertilization. In previous work, the localization of the SPE-38 protein was examined using polyclonal antibodies on spermatids and mature amoeboid spermatozoa. SPE-38 is localized to unfused membranous organelles (MOs) in nonmotile spermatids. Different fixation conditions revealed that SPE-38 either localized to fused MOs and the cell body plasma membrane or the pseudopod plasma membrane of mature sperm. To address this localization paradox in mature sperm, CRISPR/Cas9 genome editin

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Hou, Yuqing, Xi Cheng, and George B. Witman. "Direct in situ protein tagging in Chlamydomonas reinhardtii utilizing TIM, a method for CRISPR/Cas9-based targeted insertional mutagenesis." PLOS ONE 17, no. 12 (2022): e0278972. http://dx.doi.org/10.1371/journal.pone.0278972.

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Chlamydomonas reinhardtii is an important model organism for the study of many cellular processes, and protein tagging is an increasingly indispensable tool for these studies. To circumvent the disadvantages of conventional approaches in creating a tagged cell line, which involve transforming either a wild-type or null-mutant cell line with an exogenous DNA construct that inserts randomly into the genome, we developed a strategy to tag the endogenous gene in situ. The strategy utilizes TIM, a CRISPR/Cas9-based method for targeted insertional mutagenesis in C. reinhardtii. We have tested the st

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Leong, Shwee Khuan, Jye-Chian Hsiao, and Jiun-Jie Shie. "A Multiscale Molecular Dynamic Analysis Reveals the Effect of Sialylation on EGFR Clustering in a CRISPR/Cas9-Derived Model." International Journal of Molecular Sciences 23, no. 15 (2022): 8754. http://dx.doi.org/10.3390/ijms23158754.

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Bacterial and viral pathogens can modulate the glycosylation of key host proteins to facilitate pathogenesis by using various glycosidases, particularly sialidases. Epidermal growth factor receptor (EGFR) signaling is activated by ligand-induced receptor dimerization and oligomerization. Ligand binding induces conformational changes in EGFR, leading to clusters and aggregation. However, information on the relevance of EGFR clustering in the pattern of glycosylation during bacterial and viral invasion remains unclear. In this study, (1) we established CRISPR/Cas9-mediated GFP knock-in (EGFP-KI)

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Mészár, Zoltán, Éva Kókai, Rita Varga, et al. "CRISPR/Cas9-Based Mutagenesis of Histone H3.1 in Spinal Dynorphinergic Neurons Attenuates Thermal Sensitivity in Mice." International Journal of Molecular Sciences 23, no. 6 (2022): 3178. http://dx.doi.org/10.3390/ijms23063178.

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Burn injury is a trauma resulting in tissue degradation and severe pain, which is processed first by neuronal circuits in the spinal dorsal horn. We have recently shown that in mice, excitatory dynorphinergic (Pdyn) neurons play a pivotal role in the response to burn-injury-associated tissue damage via histone H3.1 phosphorylation-dependent signaling. As Pdyn neurons were mostly associated with mechanical allodynia, their involvement in thermonociception had to be further elucidated. Using a custom-made AAV9_mutH3.1 virus combined with the CRISPR/cas9 system, here we provide evidence that bloc

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Lander, Noelia, Miguel A. Chiurillo, Melissa Storey, Anibal E. Vercesi, and Roberto Docampo. "CRISPR/Cas9-mediated endogenous C-terminal Tagging ofTrypanosoma cruziGenes Reveals the Acidocalcisome Localization of the Inositol 1,4,5-Trisphosphate Receptor." Journal of Biological Chemistry 291, no. 49 (2016): 25505–15. http://dx.doi.org/10.1074/jbc.m116.749655.

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Kuri, Paola, Nicole L. Schieber, Thomas Thumberger, Joachim Wittbrodt, Yannick Schwab, and Maria Leptin. "Dynamics of in vivo ASC speck formation." Journal of Cell Biology 216, no. 9 (2017): 2891–909. http://dx.doi.org/10.1083/jcb.201703103.

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Activated danger or pathogen sensors trigger assembly of the inflammasome adaptor ASC into specks, large signaling platforms considered hallmarks of inflammasome activation. Because a lack of in vivo tools has prevented the study of endogenous ASC dynamics, we generated a live ASC reporter through CRISPR/Cas9 tagging of the endogenous gene in zebrafish. We see strong ASC expression in the skin and other epithelia that act as barriers to insult. A toxic stimulus triggered speck formation and rapid pyroptosis in keratinocytes in vivo. Macrophages engulfed and digested that speck-containing, pyro

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Thakare, Swapnil S., Navita Bansal, S. Vanchinathan, et al. "GFP tagging based method to analyze the genome editing efficiency of CRISPR/Cas9-gRNAs through transient expression in N. benthamiana." Journal of Plant Biochemistry and Biotechnology 29, no. 2 (2019): 183–92. http://dx.doi.org/10.1007/s13562-019-00540-0.

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Naeimi Kararoudi, Meisam, Shibi Likhite, Ezgi Elmas, et al. "CD33 Targeting Primary CAR-NK Cells Generated By CRISPR Mediated Gene Insertion Show Enhanced Anti-AML Activity." Blood 136, Supplement 1 (2020): 3. http://dx.doi.org/10.1182/blood-2020-142494.

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Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. Recently, we described an efficient method for gene targeting in NK cells using Cas9/ribonucleoprotein complexes (PMID: 29985369 and 32603414). Here we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus using a wide variety of

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Alok, Anshu, Hanny Chauhan, Santosh Kumar Upadhyay, Ashutosh Pandey, Jitendra Kumar, and Kashmir Singh. "Compendium of Plant-Specific CRISPR Vectors and Their Technical Advantages." Life 11, no. 10 (2021): 1021. http://dx.doi.org/10.3390/life11101021.

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CRISPR/Cas mediated genome editing is a revolutionary approach for manipulating the plant genome. However, the success of this technology is highly dependent on selection of a specific vector and the other components. A plant-specific CRISPR/Cas vector usually consists of a Cas gene, target-specific gRNA, leader sequence, selectable marker gene, precise promoters, and other accessories. It has always been challenging to select the specific vector for each study due to a lack of comprehensive information on CRISPR vectors in one place. Herein, we have discussed every technical aspect of various

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He, Wei, Hao Cui, Na Li, et al. "Involvement of INS15 in the development and pathogenicity of the zoonotic pathogen Cryptosporidium parvum." PLOS Neglected Tropical Diseases 18, no. 10 (2024): e0012569. http://dx.doi.org/10.1371/journal.pntd.0012569.

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Background Cryptosporidium parvum is a common protozoan pathogen responsible for moderate to severe diarrhea in humans and animals. The C. parvum genome contains 22 genes encoding insulinase-like M16 proteases (INS) with diverse structures and sequences, suggesting that members of the protein family may have distinct biological functions in the life cycle of parasites. Here, we investigated the role of INS15 and INS16, two proteases encoded by neighboring genes with high sequence identity, in the growth and development of C. parvum in vivo and in vitro. Methodology/Principal findings INS15 and

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Kou, Yong-Jie, Jin Gao, Rui Li, et al. "Functional Characterization of Six Eukaryotic Translation Initiation Factors of Toxoplasma gondii Using the CRISPR-Cas9 System." International Journal of Molecular Sciences 25, no. 14 (2024): 7834. http://dx.doi.org/10.3390/ijms25147834.

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Eukaryotic translation initiation factors (eIFs) are crucial for initiating protein translation and ensuring the correct assembly of mRNA-ribosomal subunit complexes. In this study, we investigated the effects of deleting six eIFs in the apicomplexan parasite Toxoplasma gondii using the CRISPR-Cas9 system. We determined the subcellular localization of these eIFs using C-terminal endogenous tagging and immunofluorescence analysis. Four eIFs (RH::315150-6HA, RH::286090-6HA, RH::249370-6HA, and RH::211410-6HA) were localized in the cytoplasm, while RH::224235-6HA was localized in the apicoplast.

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Donlin-Asp, Paul G., Claudio Polisseni, Robin Klimek, Alexander Heckel, and Erin M. Schuman. "Differential regulation of local mRNA dynamics and translation following long-term potentiation and depression." Proceedings of the National Academy of Sciences 118, no. 13 (2021): e2017578118. http://dx.doi.org/10.1073/pnas.2017578118.

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Decades of work have demonstrated that messenger RNAs (mRNAs) are localized and translated within neuronal dendrites and axons to provide proteins for remodeling and maintaining growth cones or synapses. It remains unknown, however, whether specific forms of plasticity differentially regulate the dynamics and translation of individual mRNA species. To address this, we targeted three individual synaptically localized mRNAs, CamkIIa, β-actin, Psd95, and used molecular beacons to track endogenous mRNA movements. We used reporters and CRISPR/Cas9 gene editing to track mRNA translation in cultured

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Kesavan, Gokul, Anja Machate, and Michael Brand. "CRISPR/Cas9-Based Split Fluorescent Protein Tagging." Zebrafish, September 7, 2021. http://dx.doi.org/10.1089/zeb.2021.0031.

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Lackner, Daniel H., Alexia Carré, Paloma M. Guzzardo, et al. "A generic strategy for CRISPR-Cas9-mediated gene tagging." Nature Communications 6, no. 1 (2015). http://dx.doi.org/10.1038/ncomms10237.

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Shinkado, Sota, Hiroki Saito, Masaya Yamazaki, et al. "Genome editing using a versatile vector-based CRISPR/Cas9 system in Fusarium species." Scientific Reports 12, no. 1 (2022). http://dx.doi.org/10.1038/s41598-022-20697-4.

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AbstractFusarium species include important filamentous fungal pathogens that can infect plants, animals, and humans. Meanwhile, some nonpathogenic Fusarium species are promising biocontrol agents against plant pathogens. Here, we developed a genome editing technology using a vector-based CRISPR/Cas9 system for Fusarium oxysporum f. sp. lycopersici (Fol). This optimized CRISPR/Cas9 system, harboring an endogenous U6 small nuclear RNA promoter for the expression of single-guide RNA and an endogenous H2B nuclear localization signal for the localization of Cas9, enabled efficient targeted gene kno

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Lander, Noelia, Miguel Chiurillo, Aníbal Vercesi, and Roberto Docampo. "Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi." BIO-PROTOCOL 7, no. 10 (2017). http://dx.doi.org/10.21769/bioprotoc.2299.

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Kuang, Dexuan, Jichen Qiao, Zhou Li, et al. "Tagging to endogenous genes of Plasmodium falciparum using CRISPR/Cas9." Parasites & Vectors 10, no. 1 (2017). http://dx.doi.org/10.1186/s13071-017-2539-0.

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Wu, Szu-Hsien Sam, Heetak Lee, Réka Szép-Bakonyi, et al. "SCON—a Short Conditional intrON for conditional knockout with one-step zygote injection." Experimental & Molecular Medicine, December 9, 2022. http://dx.doi.org/10.1038/s12276-022-00891-0.

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AbstractThe generation of conditional alleles using CRISPR technology is still challenging. Here, we introduce a Short Conditional intrON (SCON, 189 bp) that enables the rapid generation of conditional alleles via one-step zygote injection. In this study, a total of 13 SCON mouse lines were successfully generated by 2 different laboratories. SCON has conditional intronic functions in various vertebrate species, and its target insertion is as simple as CRISPR/Cas9-mediated gene tagging.

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Li, Xiang, Lu Mu, Hongzhe Peng, Sun Nyunt Wai, Longjun Pu, and Bo Dong. "Development of cell labeling and gene editing tools in urochordate Ciona." Marine Life Science & Technology, June 3, 2025. https://doi.org/10.1007/s42995-025-00300-1.

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Abstract Urochordate Ciona spp. are ideal marine model organisms for studying embryogenesis and developmental and evolutionary biology. However, the effective implementation of genetic labeling and CRISPR/Cas9-based editing tools at cellular resolution remains challenging. This study successfully developed and validated a collection of Gateway-based vectors for cell labeling in Ciona spp. The destination vector sets contained two Gateway cassettes flanked by Minos sites, allowing the N- or C-terminal tagging of a protein of interest with various fluorescent markers. In addition, we optimized t

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Dewari, Pooran Singh, Benjamin Southgate, Katrina Mccarten, et al. "An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein." eLife 7 (April 11, 2018). http://dx.doi.org/10.7554/elife.35069.

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CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5–30%, were

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