bims-pideca 2024-11-03 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2024‒11‒03
thirteen papers selected by
Ralitsa Radostinova Madsen, MRC-PPU

ATP-competitive inhibitors of PI3K enzymes demonstrate an isoform selective dual action by controlling membrane binding.
A multiplex single-cell RNA-Seq pharmacotranscriptomics pipeline for drug discovery.
PI3K/AKT signaling controls ICM maturation and proper epiblast and primitive endoderm specification in mice.
Genetically-Encoded Fluorescence Barcodes for Single-Cell Analysis.
A multi-modal single-cell and spatial expression map of metastatic breast cancer biopsies across clinicopathological features.
STRAIGHT-IN Dual: a platform for dual, single-copy integrations of DNA payloads and gene circuits into human induced pluripotent stem cells.
Defining the transcriptome of PIK3CA-altered cells in a human capillary malformation using single cell long-read sequencing.
Method of moments framework for differential expression analysis of single-cell RNA sequencing data.
Senescence suppresses the integrated stress response and activates a stress-remodeled secretory phenotype.
Inavolisib-Based Therapy in PIK3CA-Mutated Advanced Breast Cancer.
Downregulation of PIK3IP1/TrIP on T cells is controlled by TCR signal strength, PKC and metalloprotease-mediated cleavage.
The oncogenic axis YAP/MYC/EZH2 impairs PTEN tumor suppression activity enhancing lung tumorigenicity.
CRISPR associated enzymes are mislocalized to the cytoplasm in iPSC-derived neurons resulting in KRAB-specific degradation.

Biochem J. 2024 Nov 01. pii: BCJ20240479. [Epub ahead of print]

ATP-competitive inhibitors of PI3K enzymes demonstrate an isoform selective dual action by controlling membrane binding.

Grace Q Gong, Glenn Robert Masson, Woo-Jeong Jeong Lee, James Mj Dickson, Jackie D Kendall, Manoj K Rathinaswamy, Christina M Buchanan, Martin Middleditch, Brady Owen, Julie A Spicer, Gordon W Rewcastle, William A Denny, John E Burke, Peter R Shepherd, Roger L Williams, Jack U Flanagan.

  PI3Kα, consisting of the p110α isoform of the catalytic subunit of PI 3-kinase (encoded by PIK3CA) and the p85α regulatory subunit (encoded by PI3KR1) is activated by growth factor receptors. The identification of common oncogenic mutations in PIK3CA has driven the development of many inhibitors that bind to the ATP-binding site in the p110α subunit. Upon activation, PI3Kα undergoes conformational changes that promote its membrane interaction and catalytic activity, yet the effects of ATP-site directed inhibitors on the PI3Kα membrane interaction are unknown. Using FRET and Biolayer Interferometry assays, we show that a class of ATP-site directed inhibitors represented by GSK2126458 block the growth factor activated PI3KαWT membrane interaction, an activity dependent on the ligand forming specific ATP-site interactions. The membrane interaction for hot spot oncogenic mutations that bypass normal p85α regulatory mechanisms was insensitive to GSK2126458, while GSK2126458 could regulate mutations found outside of these hot spot regions. Our data show that the effect of GSK126458 on the membrane interaction requires the enzyme to revert from its growth factor activated state to a basal state. We find that an ATP substrate analogue can increase the wild type PI3Kα membrane interaction, uncovering a substrate based regulatory event that can be mimicked by different inhibitor chemotypes. Our findings, together with the discovery of small molecule allosteric activators of PI3Kα illustrate that PI3Kα membrane interactions can be modulated by factors related to ligand binding both within the ATP site and at allosteric sites.

Keywords:  PIK3CA; lipid kinase; membrane proteins; phosphoinositide 3-kinase; protein conformation; small molecules

DOI:  https://doi.org/10.1042/BCJ20240479
Nat Chem Biol. 2024 Oct 31.

A multiplex single-cell RNA-Seq pharmacotranscriptomics pipeline for drug discovery.

Alice Dini, Harlan Barker, Emilia Piki, Subodh Sharma, Juuli Raivola, Astrid Murumägi, Daniela Ungureanu.

The gene-regulatory dynamics governing drug responses in cancer are yet to be fully understood. Here, we report a pipeline capable of producing high-throughput pharmacotranscriptomic profiling through live-cell barcoding using antibody-oligonucleotide conjugates. This pipeline combines drug screening with 96-plex single-cell RNA sequencing. We show the potential of this approach by exploring the heterogeneous transcriptional landscape of primary high-grade serous ovarian cancer (HGSOC) cells after treatment with 45 drugs, with 13 distinct classes of mechanisms of action. A subset of phosphatidylinositol 3-OH kinase (PI3K), protein kinase B (AKT) and mammalian target of rapamycin (mTOR) inhibitors induced the activation of receptor tyrosine kinases, such as the epithelial growth factor receptor (EGFR), and this was mediated by the upregulation of caveolin 1 (CAV1). This drug resistance feedback loop could be mitigated by the synergistic action of agents targeting PI3K-AKT-mTOR and EGFR for HGSOC with CAV1 and EGFR expression. Using this workflow could enable the personalized testing of patient-derived tumor samples at single-cell resolution.

DOI: https://doi.org/10.1038/s41589-024-01761-8
Dev Cell. 2024 Oct 22. pii: S1534-5807(24)00601-4. [Epub ahead of print]

PI3K/AKT signaling controls ICM maturation and proper epiblast and primitive endoderm specification in mice.

Anna Geiselmann, Adèle Micouin, Sandrine Vandormael-Pournin, Vincent Laville, Almira Chervova, Sébastien Mella, Pablo Navarro, Michel Cohen-Tannoudji.

  The inner cell mass (ICM) of early mouse embryos is specified into epiblast (Epi) and primitive endoderm (PrE) lineages during blastocyst formation. The antagonistic transcription factors (TFs) NANOG and GATA-binding protein 6 (GATA6) in combination with fibroblast growth factor (FGF)/extracellular-signal-regulated kinase (ERK) signaling are central actors in ICM fate choice. However, what initiates the specification of ICM progenitors into Epi or PrE and whether other factors are involved in this process has not been fully understood yet. Here, we show that phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) is constitutively active during preimplantation development. Using pharmacological inhibition, we demonstrate that PI3K/AKT enables the formation of a functional ICM capable of giving rise to both the Epi and the PrE: it maintains the expression of the TF NANOG, which specifies the Epi, and confers responsiveness to FGF4, which is essential for PrE specification. Our work thus identifies PI3K/AKT signaling as an upstream regulator controlling the molecular events required for both Epi and PrE specification.

Keywords:  FGF4; FOXO3; GSK3; NANOG; epiblast; inner cell mass; lineage specification; mTOR; mouse preimplantation embryo; primitive endoderm

DOI:  https://doi.org/10.1016/j.devcel.2024.10.001
bioRxiv. 2024 Oct 24. pii: 2024.10.23.619855. [Epub ahead of print]

Genetically-Encoded Fluorescence Barcodes for Single-Cell Analysis.

Xiaoming Lu, Daniel J Pritko, Megan E Abravanel, Jonah R Huggins, Feranmi Ogunleye, Tirthankar Biswas, Katia C Ashy, Semaj K Woods, Mariclaire Wt Livingston, Mark Blenner, Marc R Birtwistle.

Genetically-encoded, single-cell barcodes are broadly useful for experimental tasks such as lineage tracing or genetic screens. For such applications, a barcode library would ideally have high diversity (many unique barcodes), non-destructive identification (repeated measurements in the same cells or population), and fast, inexpensive readout (many cells and conditions). Current nucleic acid barcoding methods generate high diversity but require destructive and slow/expensive readout, and current fluorescence barcoding methods are non-destructive, fast, and inexpensive to readout but lack high diversity. We recently proposed theory for how fluorescent protein combinations may generate a high-diversity barcode library with non-destructive, fast and inexpensive identification. Here, we present an initial experimental proof-of-concept by generating a library of ~150 barcodes from two-way combinations of 18 fluorescent proteins. We use a pooled cloning strategy to generate a barcode library that is validated to contain every possible combination of the 18 fluorescent proteins. Experimental results using single mammalian cells and spectral flow cytometry demonstrate excellent classification performance of individual fluorescent proteins, with the exception of mTFP1, and of most evaluated barcodes, with many true positive rates >99%. The library is compatible with genetic screening for hundreds of genes (or gene pairs) and lineage tracing hundreds of clones. This work lays a foundation for greater diversity libraries (potentially ~10 5 and more) generated from hundreds of spectrally-resolvable tandem fluorescent protein probes.

DOI: https://doi.org/10.1101/2024.10.23.619855
Nat Med. 2024 Oct 30.

A multi-modal single-cell and spatial expression map of metastatic breast cancer biopsies across clinicopathological features.

Johanna Klughammer, Daniel L Abravanel, Åsa Segerstolpe, Timothy R Blosser, Yury Goltsev, Yi Cui, Daniel R Goodwin, Anubhav Sinha, Orr Ashenberg, Michal Slyper, Sébastien Vigneau, Judit Jané-Valbuena, Shahar Alon, Chiara Caraccio, Judy Chen, Ofir Cohen, Nicole Cullen, Laura K DelloStritto, Danielle Dionne, Janet Files, Allison Frangieh, Karla Helvie, Melissa E Hughes, Stephanie Inga, Abhay Kanodia, Ana Lako, Colin MacKichan, Simon Mages, Noa Moriel, Evan Murray, Sara Napolitano, Kyleen Nguyen, Mor Nitzan, Rebecca Ortiz, Miraj Patel, Kathleen L Pfaff, Caroline B M Porter, Asaf Rotem, Sarah Strauss, Robert Strasser, Aaron R Thorner, Madison Turner, Isaac Wakiro, Julia Waldman, Jingyi Wu, Jorge Gómez Tejeda Zañudo, Diane Zhang, Nancy U Lin, Sara M Tolaney, Eric P Winer, Edward S Boyden, Fei Chen, Garry P Nolan, Scott J Rodig, Xiaowei Zhuang, Orit Rozenblatt-Rosen, Bruce E Johnson, Aviv Regev, Nikhil Wagle.

Although metastatic disease is the leading cause of cancer-related deaths, its tumor microenvironment remains poorly characterized due to technical and biospecimen limitations. In this study, we assembled a multi-modal spatial and cellular map of 67 tumor biopsies from 60 patients with metastatic breast cancer across diverse clinicopathological features and nine anatomic sites with detailed clinical annotations. We combined single-cell or single-nucleus RNA sequencing for all biopsies with a panel of four spatial expression assays (Slide-seq, MERFISH, ExSeq and CODEX) and H&E staining of consecutive serial sections from up to 15 of these biopsies. We leveraged the coupled measurements to provide reference points for the utility and integration of different experimental techniques and used them to assess variability in cell type composition and expression as well as emerging spatial expression characteristics across clinicopathological and methodological diversity. Finally, we assessed spatial expression and co-localization features of macrophage populations, characterized three distinct spatial phenotypes of epithelial-to-mesenchymal transition and identified expression programs associated with local T cell infiltration versus exclusion, showcasing the potential of clinically relevant discovery in such maps.

DOI: https://doi.org/10.1038/s41591-024-03215-z
bioRxiv. 2024 Oct 17. pii: 2024.10.17.616637. [Epub ahead of print]

STRAIGHT-IN Dual: a platform for dual, single-copy integrations of DNA payloads and gene circuits into human induced pluripotent stem cells.

Albert Blanch-Asensio, Deon S Ploessl, Nathan B Wang, Christine L Mummery, Kate E Galloway, Richard P Davis.

Targeting DNA payloads into human (h)iPSCs involves multiple time-consuming, inefficient steps that must be repeated for each construct. Here, we present STRAIGHT-IN Dual, which enables simultaneous, allele-specific, single-copy integration of two DNA payloads with 100% efficiency within one week. Notably, STRAIGHT-IN Dual leverages the STRAIGHT-IN platform to allow near-scarless cargo integration, facilitating the recycling of components for subsequent cellular modifications. Using STRAIGHT-IN Dual, we investigated how promoter choice and gene syntax influences transgene silencing, and demonstrate the impact of these design features on forward programming of hiPSCs into neurons. Furthermore, we designed a grazoprevir-inducible synZiFTR system to complement the widely-used tetracycline-inducible system, providing independent, tunable, and temporally controlled expression of both transcription factors and functional reporters. The unprecedented efficiency and speed with which STRAIGHT-IN Dual generates homogenous genetically engineered hiPSC populations represents a major advancement for synthetic biology in stem cell applications and opens opportunities for precision cell engineering.

DOI: https://doi.org/10.1101/2024.10.17.616637
Sci Rep. 2024 10 25. 14(1): 25440

Defining the transcriptome of PIK3CA-altered cells in a human capillary malformation using single cell long-read sequencing.

Michelle A Wedemeyer, Tianli Ding, Elizabeth A R Garfinkle, Jesse J Westfall, Jaye B Navarro, Maria Elena Hernandez Gonzalez, Elizabeth A Varga, Patricia Witman, Elaine R Mardis, Catherine E Cottrell, Anthony R Miller, Katherine E Miller.

PIK3CA-related overgrowth spectrum (PROS) disorders are caused by somatic mosaic variants that result in constitutive activation of the phosphatidylinositol-3-kinase/AKT/mTOR pathway. Promising responses to molecularly targeted therapy have been reported, although identification of an appropriate agent can be hampered by the mosaic nature and corresponding low variant allele frequency of the causal variant. Moreover, our understanding of the molecular consequences of these variants-for example how they affect gene expression profiles-remains limited. Here we describe in vitro expansion of a human capillary malformation followed by molecular characterization using exome sequencing, single cell gene expression, and targeted long-read single cell RNA-sequencing in a patient with clinical features consistent with Megalencephaly-Capillary Malformation Syndrome (MCAP, a PROS condition). These approaches identified a targetable PIK3CA variant with expression restricted to PAX3+ fibroblast and undifferentiated keratinocyte populations. This study highlights the innovative combination of next-generation single cell sequencing methods to better understand unique transcriptomic profiles and cell types associated with MCAP, revealing molecular intricacies of this genetic syndrome.

DOI: https://doi.org/10.1038/s41598-024-72167-8
Cell. 2024 Oct 31. pii: S0092-8674(24)01144-9. [Epub ahead of print]187(22): 6393-6410.e16

Method of moments framework for differential expression analysis of single-cell RNA sequencing data.

Min Cheol Kim, Rachel Gate, David S Lee, Andrew Tolopko, Andrew Lu, Erin Gordon, Eric Shifrut, Pablo E Garcia-Nieto, Alexander Marson, Vasilis Ntranos, Chun Jimmie Ye.

  Differential expression analysis of single-cell RNA sequencing (scRNA-seq) data is central for characterizing how experimental factors affect the distribution of gene expression. However, distinguishing between biological and technical sources of cell-cell variability and assessing the statistical significance of quantitative comparisons between cell groups remain challenging. We introduce Memento, a tool for robust and efficient differential analysis of mean expression, variability, and gene correlation from scRNA-seq data, scalable to millions of cells and thousands of samples. We applied Memento to 70,000 tracheal epithelial cells to identify interferon-responsive genes, 160,000 CRISPR-Cas9 perturbed T cells to reconstruct gene-regulatory networks, 1.2 million peripheral blood mononuclear cells (PBMCs) to map cell-type-specific quantitative trait loci (QTLs), and the 50-million-cell CELLxGENE Discover corpus to compare arbitrary cell groups. In all cases, Memento identified more significant and reproducible differences in mean expression compared with existing methods. It also identified differences in variability and gene correlation that suggest distinct transcriptional regulation mechanisms imparted by perturbations.

Keywords:  Bootstrap method; CD4 T cells; CELLxGENE Discover; Differential expression; Gene expression variance; Gene-regulatory networks; Memento; Single-cell transcriptomics; Spatial genomics; scRNA-seq analysis

DOI:  https://doi.org/10.1016/j.cell.2024.09.044
Mol Cell. 2024 Oct 24. pii: S1097-2765(24)00826-8. [Epub ahead of print]

Senescence suppresses the integrated stress response and activates a stress-remodeled secretory phenotype.

Matthew J Payea, Showkat A Dar, Carlos Anerillas, Jennifer L Martindale, Cedric Belair, Rachel Munk, Sulochan Malla, Jinshui Fan, Yulan Piao, Xiaoling Yang, Abid Rehman, Nirad Banskota, Kotb Abdelmohsen, Myriam Gorospe, Manolis Maragkakis.

  Senescence is a state of indefinite cell-cycle arrest associated with aging, cancer, and age-related diseases. Here, we find that translational deregulation, together with a corresponding maladaptive integrated stress response (ISR), is a hallmark of senescence that desensitizes senescent cells to stress. We present evidence that senescent cells maintain high levels of eIF2α phosphorylation, typical of ISR activation, but translationally repress production of the stress response activating transcription factor 4 (ATF4) by ineffective bypass of the inhibitory upstream open reading frames (uORFs). Surprisingly, ATF4 translation remains inhibited even after acute proteotoxic and amino acid starvation stressors, resulting in a highly diminished stress response. We also find that stress augments the senescence-associated secretory phenotype with sustained remodeling of inflammatory factors expression that is suppressed by non-uORF carrying ATF4 mRNA expression. Our results thus show that senescent cells possess a unique response to stress, which entails an increase in their inflammatory profile.

Keywords:  ATF4; ER stress; ISR; SASP; integrated stress response; nanopore direct RNA sequencing; proteomics; ribosome sequencing; senescence; senescence-associated secretory phenotype; translation

DOI:  https://doi.org/10.1016/j.molcel.2024.10.003
N Engl J Med. 2024 Oct 31. 391(17): 1584-1596

Inavolisib-Based Therapy in PIK3CA-Mutated Advanced Breast Cancer.

Nicholas C Turner, Seock-Ah Im, Cristina Saura, Dejan Juric, Sibylle Loibl, Kevin Kalinsky, Peter Schmid, Sherene Loi, Patrapim Sunpaweravong, Antonino Musolino, Huiping Li, Qingyuan Zhang, Zbigniew Nowecki, Roland Leung, Eirini Thanopoulou, Noopur Shankar, Guiyuan Lei, Thomas J Stout, Katherine E Hutchinson, Jennifer L Schutzman, Chunyan Song, Komal L Jhaveri.

BACKGROUND: Inavolisib is a highly potent and selective inhibitor of the alpha isoform of the p110 catalytic subunit of the phosphatidylinositol 3-kinase complex (encoded by PIK3CA) that also promotes the degradation of mutated p110α. Inavolisib plus palbociclib-fulvestrant has shown synergistic activity in preclinical models and promising antitumor activity in early-phase trials.METHODS: In a phase 3, double-blind, randomized trial, we compared first-line inavolisib (at an oral dose of 9 mg once daily) plus palbociclib-fulvestrant (inavolisib group) with placebo plus palbociclib-fulvestrant (placebo group) in patients with PIK3CA-mutated, hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative locally advanced or metastatic breast cancer who had had relapse during or within 12 months after the completion of adjuvant endocrine therapy. The primary end point was progression-free survival as assessed by the investigator.
RESULTS: A total of 161 patients were assigned to the inavolisib group and 164 to the placebo group; the median follow-up was 21.3 months and 21.5 months, respectively. The median progression-free survival was 15.0 months (95% confidence interval [CI], 11.3 to 20.5) in the inavolisib group and 7.3 months (95% CI, 5.6 to 9.3) in the placebo group (hazard ratio for disease progression or death, 0.43; 95% CI, 0.32 to 0.59; P<0.001). An objective response occurred in 58.4% of the patients in the inavolisib group and in 25.0% of those in the placebo group. The incidence of grade 3 or 4 neutropenia was 80.2% in the inavolisib group and 78.4% in the placebo group; grade 3 or 4 hyperglycemia, 5.6% and 0%, respectively; grade 3 or 4 stomatitis or mucosal inflammation, 5.6% and 0%; and grade 3 or 4 diarrhea, 3.7% and 0%. No grade 3 or 4 rash was observed. Discontinuation of any trial agent because of adverse events occurred in 6.8% of the patients in the inavolisib group and in 0.6% of those in the placebo group.
CONCLUSIONS: In patients with PIK3CA-mutated, hormone receptor-positive, HER2-negative locally advanced or metastatic breast cancer, inavolisib plus palbociclib-fulvestrant led to significantly longer progression-free survival than placebo plus palbociclib-fulvestrant, with a greater incidence of toxic effects. The percentage of patients who discontinued any trial agent because of adverse events was low. (Funded by F. Hoffmann-La Roche; INAVO120 ClinicalTrials.gov number, NCT04191499.).

DOI: https://doi.org/10.1056/NEJMoa2404625
J Biol Chem. 2024 Oct 23. pii: S0021-9258(24)02432-3. [Epub ahead of print] 107930

Downregulation of PIK3IP1/TrIP on T cells is controlled by TCR signal strength, PKC and metalloprotease-mediated cleavage.

Benjamin M Murter, Sean C Robinson, Hridesh Banerjee, Louis Lau, Uzodinma U Uche, Andrea L Szymczak-Workman, Lawrence P Kane.

  The protein known as PI3K-interacting protein (PIK3IP1), or transmembrane inhibitor of PI3K (TrIP), is highly expressed by T cells and can modulate PI3K activity in these cells. Several studies have also revealed that TrIP is rapidly downregulated following T cell activation. However, it is unclear as to how this downregulation is controlled. Using a novel monoclonal antibody that robustly stains cell-surface TrIP, we demonstrate that TrIP is lost from the surface of activated T cells in a manner dependent on the strength of signaling through the T cell receptor (TCR) and specific downstream signaling pathways, in particular classical PKC isoforms. TrIP expression returns by 24 hours after stimulation, suggesting that it may play a role in resetting TCR signaling at later time points. We also provide evidence that ADAM family proteases are required for both constitutive and stimulation-induced downregulation of TrIP in T cells. Finally, by expressing truncated forms of TrIP in cells, we identify the region in the extracellular stalk domain of TrIP that is targeted for proteolytic cleavage.

Keywords:  ADAM; PIK3IP1; T-cell receptor (TCR); TrIP; T‐cell; cell signaling; cellular immune response; matrix metalloproteinase (MMP); phosphatidylinositide 3‐kinase (PI 3‐kinase)

DOI:  https://doi.org/10.1016/j.jbc.2024.107930
Cell Death Discov. 2024 Oct 25. 10(1): 452

The oncogenic axis YAP/MYC/EZH2 impairs PTEN tumor suppression activity enhancing lung tumorigenicity.

Federica Lo Sardo, Chiara Turco, Beatrice Messina, Andrea Sacconi, Francesca Romana Auciello, Claudio Pulito, Sabrina Strano, Sima Lev, Giovanni Blandino.

The tumor suppressor PTEN (phosphatase and tensin homolog deleted in chromosome 10) is genetically deleted or downregulated in many cancer types. Loss of PTEN protein expression is frequently found in lung cancer while genetic alterations are less abundant. PTEN expression is regulated at multiple genetic and epigenetic levels and even partial reduction of its expression increases cancer occurrence. We show that YAP and TAZ cooperate with EZH2, and MYC to transcriptionally repress onco-suppressor genes, including PTEN, in non-small cell lung cancer (NSCLC) cells. YAP/TAZ-EZH2-MYC transcriptional regulators form a nuclear complex that represses PTEN transcription, while their combinatorial targeting restores PTEN expression, attenuates NSCLC cell growth, and prevents compensatory responses induced by single treatments. Datasets analysis of NSCLC patients revealed that PTEN expression is negatively correlated to YAP/TAZ, EZH2 and MYC and that low expression of PTEN is predictive of poor prognosis, especially at earlier stages of the disease. These findings highlight the repressive role of the YAP/TAZ-EZH2-MYC axis on tumor-suppressor genes and offer a potential therapeutic strategy for lung cancer patients with low PTEN levels.

DOI: https://doi.org/10.1038/s41420-024-02216-8
bioRxiv. 2024 Oct 19. pii: 2024.10.19.619045. [Epub ahead of print]

CRISPR associated enzymes are mislocalized to the cytoplasm in iPSC-derived neurons resulting in KRAB-specific degradation.

Gregory Cajka, Matthew Liu, Ophir Shalem.

The use of CRISPR-associated enzymes in iPSC-derived neurons for precise gene targeting and high-throughput gene perturbation screens offers great potential but presents unique challenges compared to dividing cell lines. CRISPRi screens in iPSC-derived neurons and glia have already been successful in relating gene function to neurological phenotypes; however, loss of dCas9-KRAB expression after differentiation has been observed by many labs and has been largely ascribed to transgene silencing after differentiation. Here, we investigated the expression levels of different CRISPR enzymes in iPSC and Ngn2-derived neurons using piggybac delivery. We found that the commonly used dCas9-KRAB (using the KOX1 domain) displayed dramatic reduction in protein expression levels following neuronal differentiation, yet surprisingly, nCas9 constructs retained comparable protein expression between iPSCs and neurons. We further found that CRISPR constructs, primarily relying on the SV40 Nuclear Localization Signal (NLS), fail to efficiently localize to the nuclei of neurons, despite having robust nuclear levels in iPSCs, leading to KRAB-specific cytoplasmic degradation. By adding a neuronal-specific NLS, we were able to correct neuronal nuclear localization and protein expression, confirming the contribution of mislocalization to the instability of dCas9-KRAB in neurons. As the lack of nuclear localization can have a profound impact on editing and gene perturbation efficiency, we suggest further investigation across both cultured and in-vivo postmitotic cell models.

DOI: https://doi.org/10.1101/2024.10.19.619045