bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023–12–17
fiveteen papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Epinephrine inhibits PI3Kα via the Hippo kinases.
  2. Paradoxical dominant negative activity of an immunodeficiency-associated activating PIK3R1 variant.
  3. Massively parallel sample preparation for multiplexed single-cell proteomics using nPOP.
  4. FilGAP regulates tumor growth in Glioma through the regulation of mTORC1 and mTORC2.
  5. Cells use multiple mechanisms for cell-cycle arrest upon withdrawal of individual amino acids.
  6. Parallelized engineering of mutational models using piggyBac transposon delivery of CRISPR libraries.
  7. Secretome analysis: Reading cellular sign language to understand intercellular communication.
  8. Base-editing mutagenesis maps alleles to tune human T cell functions.
  9. Modeling cardiac fibroblast heterogeneity from human pluripotent stem cell-derived epicardial cells.
  10. Single-cell spatial metabolomics with cell-type specific protein profiling for tissue systems biology.
  11. Atg7 senses ATP levels and regulates AKT1-PDCD4 phosphorylation-ubiquitination axis to promote survival during metabolic stress.
  12. Automated human induced pluripotent stem cell culture and sample preparation for 3D live-cell microscopy.
  13. ROS-induced ribosome impairment underlies ZAKα-mediated metabolic decline in obesity and aging.
  14. Single cell multi-omics reveal intra-cell-line heterogeneity across human cancer cell lines.
  15. Efficient selection of knocked-in pluripotent stem cells using a dual cassette cellular elimination system.
  1. Cell Rep. 2023 Dec 04. pii: S2211-1247(23)01547-4. [Epub ahead of print]42(12): 113535
    Epinephrine inhibits PI3Kα via the Hippo kinases.
    Ting-Yu Lin, Shakti Ramsamooj, Tiffany Perrier, Katarina Liberatore, Louise Lantier, Neil Vasan, Kannan Karukurichi, Seo-Kyoung Hwang, Edward A Kesicki, Edward R Kastenhuber, Thorsten Wiederhold, Tomer M Yaron, Emily M Huntsman, Mengmeng Zhu, Yilun Ma, Marcia N Paddock, Guoan Zhang, Benjamin D Hopkins, Owen McGuinness, Robert E Schwartz, Baran A Ersoy, Lewis C Cantley, Jared L Johnson, Marcus D Goncalves.
      The phosphoinositide 3-kinase p110α is an essential mediator of insulin signaling and glucose homeostasis. We interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate p110α at T1061, which inhibits its activity. This inhibitory state corresponds to a conformational change of a membrane-binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases MST1/2 using forskolin or epinephrine is associated with phosphorylation of T1061 and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules or if the T1061 is mutated to alanine. Our study defines an inhibitory pathway of PI3K signaling and a link between epinephrine and insulin signaling.
    Keywords:  CP: Metabolism; CP: Molecular biology; Hippo kinases; PI3K signaling; epinephrine signaling; glucose metabolism; glycogen metabolism; insulin sensitivity; liver
    DOI:  https://doi.org/10.1016/j.celrep.2023.113535
  2. bioRxiv. 2023 Nov 02. pii: 2023.11.02.565250. [Epub ahead of print]
    Paradoxical dominant negative activity of an immunodeficiency-associated activating PIK3R1 variant.
    Patsy R Tomlinson, Rachel Knox, Olga Perisic, Helen C Su, Gemma V Brierley, Roger L Williams, Robert K Semple.
      PIK3R1 encodes three regulatory subunits of class IA phosphoinositide 3-kinase (PI3K), each associating with any of three catalytic subunits, namely p110α, p110β or p110δ. Constitutional PIK3R1 mutations cause diseases with a genotype-phenotype relationship not yet fully explained: heterozygous loss-of-function mutations cause SHORT syndrome, featuring insulin resistance and short stature attributed to reduced p110α function, while heterozygous activating mutations cause immunodeficiency, attributed to p110δ activation and known as APDS2. Surprisingly, APDS2 patients do not show features of p110α hyperactivation, but do commonly have short stature or SHORT syndrome, suggesting p110α hypofunction. We sought to investigate this. In dermal fibroblasts from an APDS2 patient, we found no increased PI3K signalling, with p110δ expression markedly reduced. In preadipocytes, the APDS2 variant was potently dominant negative, associating with Irs1 and Irs2 but failing to heterodimerise with p110α. This attenuation of p110α signalling by a p110δ-activating PIK3R1 variant potentially explains co-incidence of gain-of-function and loss-of-function PIK3R1 phenotypes.
    DOI:  https://doi.org/10.1101/2023.11.02.565250
  3. bioRxiv. 2023 Nov 28. pii: 2023.11.27.568927. [Epub ahead of print]
    Massively parallel sample preparation for multiplexed single-cell proteomics using nPOP.
    Andrew Leduc, Luke Koury, Joshua Cantlon, Nikolai Slavov.
      Single-cell proteomics by mass spectrometry (MS) allows quantifying proteins with high specificity and sensitivity. To increase its throughput, we developed nPOP, a method for parallel preparation of thousands of single cells in nanoliter volume droplets deposited on glass slides. Here, we describe its protocol with emphasis on its flexibility to prepare samples for different multiplexed MS methods. An implementation with plexDIA demonstrates accurate quantification of about 3,000 - 3,700 proteins per human cell. The protocol is implemented on the CellenONE instrument and uses readily available consumables, which should facilitate broad adoption. nPOP can be applied to all samples that can be processed to a single-cell suspension. It takes 1 or 2 days to prepare over 3,000 single cells. We provide metrics and software for quality control that can support the robust scaling of nPOP to higher plex reagents for achieving reliable high-throughput single-cell protein analysis.
    DOI:  https://doi.org/10.1101/2023.11.27.568927
  4. Sci Rep. 2023 Dec 08. 13(1): 20956
    FilGAP regulates tumor growth in Glioma through the regulation of mTORC1 and mTORC2.
    Koji Tsutsumi, Ayumi Nohara, Taiki Tanaka, Moe Murano, Yurina Miyagaki, Yasutaka Ohta.
      The mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that forms the two different protein complexes, known as mTORC1 and mTORC2. mTOR signaling is activated in a variety of tumors, including glioma that is one of the malignant brain tumors. FilGAP (ARHGAP24) is a negative regulator of Rac, a member of Rho family small GTPases. In this study, we found that FilGAP interacts with mTORC1/2 and is involved in tumor formation in glioma. FilGAP interacted with mTORC1 via Raptor and with mTORC2 via Rictor and Sin1. Depletion of FilGAP in KINGS-1 glioma cells decreased phosphorylation of S6K and AKT. Furthermore, overexpression of FilGAP increased phosphorylation of S6K and AKT, suggesting that FilGAP activates mTORC1/2. U-87MG, glioblastoma cells, showed higher mTOR activity than KINGS-1, and phosphorylation of S6K and AKT was not affected by suppression of FilGAP expression. However, in the presence of PI3K inhibitors, phosphorylation of S6K and AKT was also decreased in U-87MG by depletion of FilGAP, suggesting that FilGAP may also regulate mTORC2 in U-87MG. Finally, we showed that depletion of FilGAP in KINGS-1 and U-87MG cells significantly reduced spheroid growth. These results suggest that FilGAP may contribute to tumor growth in glioma by regulating mTORC1/2 activities.
    DOI:  https://doi.org/10.1038/s41598-023-47892-1
  5. Cell Rep. 2023 Dec 07. pii: S2211-1247(23)01551-6. [Epub ahead of print]42(12): 113539
    Cells use multiple mechanisms for cell-cycle arrest upon withdrawal of individual amino acids.
    Yao Rong, Alicia M Darnell, Kiera M Sapp, Matthew G Vander Heiden, Sabrina L Spencer.
      Amino acids are required for cell growth and proliferation, but it remains unclear when and how amino acid availability impinges on the proliferation-quiescence decision. Here, we used time-lapse microscopy and single-cell tracking of cyclin-dependent kinase 2 (CDK2) activity to assess the response of individual cells to withdrawal of single amino acids and found strikingly different cell-cycle effects depending on the amino acid. For example, upon leucine withdrawal, MCF10A cells complete two cell cycles and then enter a CDK2-low quiescence, whereas lysine withdrawal causes immediate cell-cycle stalling. Methionine withdrawal triggers a restriction point phenotype similar to serum starvation or Mek inhibition: upon methionine withdrawal, cells complete their current cell cycle and enter a CDK2-low quiescence after mitosis. Modulation of restriction point regulators p21/p27 or cyclin D1 enables short-term rescue of proliferation under methionine and leucine withdrawal, and to a lesser extent lysine withdrawal, revealing a checkpoint connecting nutrient signaling to cell-cycle entry.
    Keywords:  CDK2; CP: Cell biology; amino acid withdrawal; cyclin D1; leucine; lysine; methionine; p21; p27; proliferation-quiescence decision; restriction point
    DOI:  https://doi.org/10.1016/j.celrep.2023.113539
  6. Cell Rep Methods. 2023 Dec 05. pii: S2667-2375(23)00351-X. [Epub ahead of print] 100672
    Parallelized engineering of mutational models using piggyBac transposon delivery of CRISPR libraries.
    Xander Nuttle, Nicholas D Burt, Benjamin Currall, Mariana Moysés-Oliveira, Kiana Mohajeri, Riya Bhavsar, Diane Lucente, Rachita Yadav, Derek J C Tai, James F Gusella, Michael E Talkowski.
      New technologies and large-cohort studies have enabled novel variant discovery and association at unprecedented scale, yet functional characterization of these variants remains paramount to deciphering disease mechanisms. Approaches that facilitate parallelized genome editing of cells of interest or induced pluripotent stem cells (iPSCs) have become critical tools toward this goal. Here, we developed an approach that incorporates libraries of CRISPR-Cas9 guide RNAs (gRNAs) together with inducible Cas9 into a piggyBac (PB) transposon system to engineer dozens to hundreds of genomic variants in parallel against isogenic cellular backgrounds. This method empowers loss-of-function (LoF) studies through the introduction of insertions or deletions (indels) and copy-number variants (CNVs), though generating specific nucleotide changes is possible with prime editing. The ability to rapidly establish high-quality mutational models at scale will facilitate the development of isogenic cellular collections and catalyze comparative functional genomic studies investigating the roles of hundreds of genes and mutations in development and disease.
    Keywords:  CP: Genetics; CP: Stem cell; CRISPR; allelic series; functional genomics; gRNA library; genome editing; genome engineering; induced pluripotent stem cells; mutational modeling; piggyBac transposon
    DOI:  https://doi.org/10.1016/j.crmeth.2023.100672
  7. Mol Cell Proteomics. 2023 Dec 09. pii: S1535-9476(23)00203-7. [Epub ahead of print] 100692
    Secretome analysis: Reading cellular sign language to understand intercellular communication.
    Wei Wu, Jeroen Krijgsveld.
      A significant portion of mammalian proteomes is secreted to the extracellular space to fulfill crucial roles in cell-to-cell communication. To best recapitulate the intricate and multi-faceted crosstalk between cells in a live organism, there is an ever-increasing need for methods to study protein secretion in model systems that include multiple cell types. In addition, post-translational modifications further expand the complexity and versatility of cellular communication. This review aims to summarise recent strategies and model systems that employ cellular co-culture, chemical biology tools, protein enrichment and proteomic methods to characterize the composition and function of cellular secretomes. This is all geared towards gaining better understanding of organismal biology in vivo mediated by secretory signaling.
    DOI:  https://doi.org/10.1016/j.mcpro.2023.100692
  8. Nature. 2023 Dec 13.
    Base-editing mutagenesis maps alleles to tune human T cell functions.
    Ralf Schmidt, Carl C Ward, Rama Dajani, Zev Armour-Garb, Mineto Ota, Vincent Allain, Rosmely Hernandez, Madeline Layeghi, Galen Xing, Laine Goudy, Dmytro Dorovskyi, Charlotte Wang, Yan Yi Chen, Chun Jimmie Ye, Brian R Shy, Luke A Gilbert, Justin Eyquem, Jonathan K Pritchard, Stacie E Dodgson, Alexander Marson.
      CRISPR-enabled screening is a powerful tool for the discovery of genes that control T cell function and has nominated candidate targets for immunotherapies1-6. However, new approaches are required to probe specific nucleotide sequences within key genes. Systematic mutagenesis in primary human T cells could reveal alleles that tune specific phenotypes. DNA base editors are powerful tools for introducing targeted mutations with high efficiency7,8. Here we develop a large-scale base-editing mutagenesis platform with the goal of pinpointing nucleotides that encode amino acid residues that tune primary human T cell activation responses. We generated a library of around 117,000 single guide RNA molecules targeting base editors to protein-coding sites across 385 genes implicated in T cell function and systematically identified protein domains and specific amino acid residues that regulate T cell activation and cytokine production. We found a broad spectrum of alleles with variants encoding critical residues in proteins including PIK3CD, VAV1, LCP2, PLCG1 and DGKZ, including both gain-of-function and loss-of-function mutations. We validated the functional effects of many alleles and further demonstrated that base-editing hits could positively and negatively tune T cell cytotoxic function. Finally, higher-resolution screening using a base editor with relaxed protospacer-adjacent motif requirements9 (NG versus NGG) revealed specific structural domains and protein-protein interaction sites that can be targeted to tune T cell functions. Base-editing screens in primary immune cells thus provide biochemical insights with the potential to accelerate immunotherapy design.
    DOI:  https://doi.org/10.1038/s41586-023-06835-6
  9. Nat Commun. 2023 Dec 11. 14(1): 8183
    Modeling cardiac fibroblast heterogeneity from human pluripotent stem cell-derived epicardial cells.
    Ian Fernandes, Shunsuke Funakoshi, Homaira Hamidzada, Slava Epelman, Gordon Keller.
      Cardiac fibroblasts play an essential role in the development of the heart and are implicated in disease progression in the context of fibrosis and regeneration. Here, we establish a simple organoid culture platform using human pluripotent stem cell-derived epicardial cells and ventricular cardiomyocytes to study the development, maturation, and heterogeneity of cardiac fibroblasts under normal conditions and following treatment with pathological stimuli. We demonstrate that this system models the early interactions between epicardial cells and cardiomyocytes to generate a population of fibroblasts that recapitulates many aspects of fibroblast behavior in vivo, including changes associated with maturation and in response to pathological stimuli associated with cardiac injury. Using single cell transcriptomics, we show that the hPSC-derived organoid fibroblast population displays a high degree of heterogeneity that approximates the heterogeneity of populations in both the normal and diseased human heart. Additionally, we identify a unique subpopulation of fibroblasts possessing reparative features previously characterized in the hearts of model organisms. Taken together, our system recapitulates many aspects of human cardiac fibroblast specification, development, and maturation, providing a platform to investigate the role of these cells in human cardiovascular development and disease.
    DOI:  https://doi.org/10.1038/s41467-023-43312-0
  10. Nat Commun. 2023 Dec 13. 14(1): 8260
    Single-cell spatial metabolomics with cell-type specific protein profiling for tissue systems biology.
    Thomas Hu, Mayar Allam, Shuangyi Cai, Walter Henderson, Brian Yueh, Aybuke Garipcan, Anton V Ievlev, Maryam Afkarian, Semir Beyaz, Ahmet F Coskun.
      Metabolic reprogramming in cancer and immune cells occurs to support their increasing energy needs in biological tissues. Here we propose Single Cell Spatially resolved Metabolic (scSpaMet) framework for joint protein-metabolite profiling of single immune and cancer cells in male human tissues by incorporating untargeted spatial metabolomics and targeted multiplexed protein imaging in a single pipeline. We utilized the scSpaMet to profile cell types and spatial metabolomic maps of 19507, 31156, and 8215 single cells in human lung cancer, tonsil, and endometrium tissues, respectively. The scSpaMet analysis revealed cell type-dependent metabolite profiles and local metabolite competition of neighboring single cells in human tissues. Deep learning-based joint embedding revealed unique metabolite states within cell types. Trajectory inference showed metabolic patterns along cell differentiation paths. Here we show scSpaMet's ability to quantify and visualize the cell-type specific and spatially resolved metabolic-protein mapping as an emerging tool for systems-level understanding of tissue biology.
    DOI:  https://doi.org/10.1038/s41467-023-43917-5
  11. Commun Biol. 2023 Dec 11. 6(1): 1252
    Atg7 senses ATP levels and regulates AKT1-PDCD4 phosphorylation-ubiquitination axis to promote survival during metabolic stress.
    Chengsi Deng, Chunlu Li, Xiang Dong, Yang Yu, Wendong Guo, Yi Guan, Xun Sun, Liu Cao.
      We report that autophagy-related gene 7 (ATG7) modulates p53 activity to regulate cell cycle and survival during metabolic stress, and that indicates Atg7 is functionally involved in cellular homeostasis in autophagy independent fashion. As a protein translation inhibitor, Programmed cell death 4 (PDCD4) expression is regulated by AKT1 phosphorylation. Here, we find that Atg7 interacts with PDCD4 and AKT1 to regulate AKT1-PDCD4 phosphorylation-ubiquitination axis during metabolic stress. We demonstrate that Atg7 senses decrease of ATP levels to suppress AKT-mediated PDCD4 phosphorylation at Ser67, which inhibits PDCD4 ubiquitinating during metabolic stress. Finally, PDCD4 accumulates and functions as a protein translation inhibitor to conserve energy, thus reducing apoptosis and allowing cells to survive stress periods. These results suggest that the ATP-Atg7-PDCD4 axis acts as a metabolic adaptation pathway which dictates cells to overcome metabolic stress.
    DOI:  https://doi.org/10.1038/s42003-023-05656-7
  12. Nat Protoc. 2023 Dec 12.
    Automated human induced pluripotent stem cell culture and sample preparation for 3D live-cell microscopy.
    Benjamin W Gregor, Mackenzie E Coston, Ellen M Adams, Joy Arakaki, Antoine Borensztejn, Thao P Do, Margaret A Fuqua, Amanda Haupt, Melissa C Hendershott, Winnie Leung, Irina A Mueller, Aditya Nath, Angelique M Nelson, Susanne M Rafelski, Emmanuel E Sanchez, Madison J Swain-Bowden, W Joyce Tang, Derek J Thirstrup, Winfried Wiegraebe, Brian P Whitney, Calysta Yan, Ruwanthi N Gunawardane, Nathalie Gaudreault.
      To produce abundant cell culture samples to generate large, standardized image datasets of human induced pluripotent stem (hiPS) cells, we developed an automated workflow on a Hamilton STAR liquid handler system. This was developed specifically for culturing hiPS cell lines expressing fluorescently tagged proteins, which we have used to study the principles by which cells establish and maintain robust dynamic localization of cellular structures. This protocol includes all details for the maintenance, passage and seeding of cells, as well as Matrigel coating of 6-well plastic plates and 96-well optical-grade, glass plates. We also developed an automated image-based hiPS cell colony segmentation and feature extraction pipeline to streamline the process of predicting cell count and selecting wells with consistent morphology for high-resolution three-dimensional (3D) microscopy. The imaging samples produced with this protocol have been used to study the integrated intracellular organization and cell-to-cell variability of hiPS cells to train and develop deep learning-based label-free predictions from transmitted-light microscopy images and to develop deep learning-based generative models of single-cell organization. This protocol requires some experience with robotic equipment. However, we provide details and source code to facilitate implementation by biologists less experienced with robotics. The protocol is completed in less than 10 h with minimal human interaction. Overall, automation of our cell culture procedures increased our imaging samples' standardization, reproducibility, scalability and consistency. It also reduced the need for stringent culturist training and eliminated culturist-to-culturist variability, both of which were previous pain points of our original manual pipeline workflow.
    DOI:  https://doi.org/10.1038/s41596-023-00912-w
  13. Science. 2023 Dec 08. 382(6675): eadf3208
    ROS-induced ribosome impairment underlies ZAKα-mediated metabolic decline in obesity and aging.
    Goda Snieckute, Laura Ryder, Anna Constance Vind, Zhenzhen Wu, Frederic Schrøder Arendrup, Mark Stoneley, Sébastien Chamois, Ana Martinez-Val, Marion Leleu, René Dreos, Alexander Russell, David Michael Gay, Aitana Victoria Genzor, Beatrice So-Yun Choi, Astrid Linde Basse, Frederike Sass, Morten Dall, Lucile Chantal Marie Dollet, Melanie Blasius, Anne E Willis, Anders H Lund, Jonas T Treebak, Jesper Velgaard Olsen, Steen Seier Poulsen, Mary Elizabeth Pownall, Benjamin Anderschou Holbech Jensen, Christoffer Clemmensen, Zach Gerhart-Hines, David Gatfield, Simon Bekker-Jensen.
      The ribotoxic stress response (RSR) is a signaling pathway in which the p38- and c-Jun N-terminal kinase (JNK)-activating mitogen-activated protein kinase kinase kinase (MAP3K) ZAKα senses stalling and/or collision of ribosomes. Here, we show that reactive oxygen species (ROS)-generating agents trigger ribosomal impairment and ZAKα activation. Conversely, zebrafish larvae deficient for ZAKα are protected from ROS-induced pathology. Livers of mice fed a ROS-generating diet exhibit ZAKα-activating changes in ribosomal elongation dynamics. Highlighting a role for the RSR in metabolic regulation, ZAK-knockout mice are protected from developing high-fat high-sugar (HFHS) diet-induced blood glucose intolerance and liver steatosis. Finally, ZAK ablation slows animals from developing the hallmarks of metabolic aging. Our work highlights ROS-induced ribosomal impairment as a physiological activation signal for ZAKα that underlies metabolic adaptation in obesity and aging.
    DOI:  https://doi.org/10.1126/science.adf3208
  14. Nat Commun. 2023 Dec 09. 14(1): 8170
    Single cell multi-omics reveal intra-cell-line heterogeneity across human cancer cell lines.
    Qionghua Zhu, Xin Zhao, Yuanhang Zhang, Yanping Li, Shang Liu, Jingxuan Han, Zhiyuan Sun, Chunqing Wang, Daqi Deng, Shanshan Wang, Yisen Tang, Yaling Huang, Siyuan Jiang, Chi Tian, Xi Chen, Yue Yuan, Zeyu Li, Tao Yang, Tingting Lai, Yiqun Liu, Wenzhen Yang, Xuanxuan Zou, Mingyuan Zhang, Huanhuan Cui, Chuanyu Liu, Xin Jin, Yuhui Hu, Ao Chen, Xun Xu, Guipeng Li, Yong Hou, Longqi Liu, Shiping Liu, Liang Fang, Wei Chen, Liang Wu.
      Human cancer cell lines have long served as tools for cancer research and drug discovery, but the presence and the source of intra-cell-line heterogeneity remain elusive. Here, we perform single-cell RNA-sequencing and ATAC-sequencing on 42 and 39 human cell lines, respectively, to illustrate both transcriptomic and epigenetic heterogeneity within individual cell lines. Our data reveal that transcriptomic heterogeneity is frequently observed in cancer cell lines of different tissue origins, often driven by multiple common transcriptional programs. Copy number variation, as well as epigenetic variation and extrachromosomal DNA distribution all contribute to the detected intra-cell-line heterogeneity. Using hypoxia treatment as an example, we demonstrate that transcriptomic heterogeneity could be reshaped by environmental stress. Overall, our study performs single-cell multi-omics of commonly used human cancer cell lines and offers mechanistic insights into the intra-cell-line heterogeneity and its dynamics, which would serve as an important resource for future cancer cell line-based studies.
    DOI:  https://doi.org/10.1038/s41467-023-43991-9
  15. Cell Rep Methods. 2023 Dec 01. pii: S2667-2375(23)00339-9. [Epub ahead of print] 100662
    Efficient selection of knocked-in pluripotent stem cells using a dual cassette cellular elimination system.
    Koji Nakade, Satomi Tsukamoto, Kenichi Nakashima, Yuri An, Iori Sato, Jingyue Li, Yuzuno Shimoda, Yasuko Hemmi, Yoshihiro Miwa, Yohei Hayashi.
      Although recent advances in genome editing technology with homology-directed repair have enabled the insertion of various reporter genes into the genome of mammalian cells, the efficiency is still low due to the random insertion of donor vectors into the host genome. To efficiently select knocked-in cells without random insertion, we developed the "double-tk donor vector system," in which the expression units of the thymidine kinase of herpes simplex virus (HSV-tk) are placed on both outer sides of homology arms. This system is superior in enriching knocked-in human induced pluripotent stem cells (hiPSCs) than conventional donor vector systems with a single or no HSV-tk cassette. Using this system, we efficiently generated fluorescent reporter knockin hiPSCs targeting POU5F1 (OCT3/4), EEF1A1, H2BC21 (H2B clustered histone 21), ISL1, and MYH7 genes. These results indicate that the double-tk donor vector system enables efficient selection of knocked-in hiPSCs carrying reporter proteins.
    Keywords:  CP: Stem cell; HDR; HSV-tk; donor vectors; fluorescent proteins; genome editing; hiPSCs; homology-directed repair; human induced pluripotent stem cells; knock-in; thymidine kinase of Herpes-simplex virus
    DOI:  https://doi.org/10.1016/j.crmeth.2023.100662
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