Biology subjects

Qi, L. S.

Publications and source records attributed to Qi, L. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Programmable Surface Receptor-Mediated Cell Targeting with Designer Antibody-Routed Nanoparticles (DARTs)

Precise in vivo delivery remains a massive barrier to therapeutic genome editing. Recently, major strides have been made towards cell type-specific delivery, with potential to improve the safety profiles of future gene therapies. However, programming such targeted tropism is limited by the minimal repertoire of scFv sequences, nanobodies, and viral fusogens that enable cell-type specificity, and the slow, iterative design-build-test cycle required to identify and optimize them. Here, we overcome this bottleneck by developing the designer antibody-routed (DART) platform: a modular, plug-and-play tool that decouples delivery vehicle production from tropism engineering. By presenting generic, bioorthogonal antibody-binding interfaces on the surfaces of enveloped particles, the DART system enables conjugation of those particles with off-the-shelf antibodies that direct cell entry without requiring genetic or chemical manipulation. Designer antibody-routed virus-like particles (DARTs) leverage this facile conjugation method to interrogate and identify transduction-competent surface antigens across cell types. They enable combinatorial targeting and the discovery of antigens that synergistically enhance transduction. Finally, DARTs function in the fetal murine nervous system, empirically identifying CD56 as a previously uncharacterized surface antigen enabling editing in hard-to-transduce neural populations. Overall, the DART platform provides a universal tool for rapidly profiling unique, targetable antigens and leveraging them for precision editing applications.

bioengineering↗

bioRxiv2026

PHAROS: turning single-cell perturbation models into target-directed drug-combination screens

Combination therapies are central to cancer treatment, but exhaustive screening is impractical. We introduce PHAROS, a framework that turns a pretrained single-cell perturbation model into a target-directed search engine for drug combinations. PHAROS predicts how a cell population changes under a drug, one drug at a time, then chains these predictions together to simulate drug combinations. It scores each simulated outcome against the desired target state and uses a search algorithm to find the most promising combinations, all without retraining the underlying model. Across two independent combinatorial perturbation datasets, PHAROS recovered exact or mechanism-matched two-drug responses in cell lines, both seen and unseen during model training. Its rankings were specific to the requested conversion and were not explained by single-drug effects, additive effects, or shared mechanism of action. In exploratory analyses of patient-derived metastatic HR+/HER2$-$ breast tumors and basal cell carcinoma (BCC), PHAROS prioritized FDA-approved regimens, distinguished combinations by their predicted tumor-versus-immune objective profiles, and nominated pathway-level hypotheses, while explicitly identifying both tumor cohorts as outside the model's supported distribution. PHAROS provides a modular route from pretrained virtual-cell models to inverse, single-cell combination-screening platforms.

bioinformatics↗

bioRxiv2025

CRISPR-Cas13d-mediated targeting of a context-specific essential gene enables selective elimination of uveal melanoma

Uveal melanoma, the most common eye cancer in adults, remains limited to surgical intervention and chemotherapy, with a dismal survival rate that has not improved in over 50 years. To address this therapeutic impasse, we systematically analyzed public gene expression, RNAi, and CRISPR knockout datasets and identified RASGRP3 as an essential gene specifically for uveal melanoma. RasGRP3 is uniquely overexpressed and essential for survival in uveal melanoma cells, but dispensable in healthy cells. RasGRP3 remains "undruggable" due to its intracellular localization and lack of targetable binding pockets. To overcome this, we developed a CRISPR-Cas13d RNA-targeting therapeutic that specifically knocks down RasGRP3 mRNA. This Cas13d-based therapeutic mediates selective uveal melanoma killing through two synergistic mechanisms: (i) direct silencing of the essential RasGRP3 transcript, and (ii) collateral RNA degradation triggered by the cleavage of overexpressed RasGRP3. When delivered via optimized lipid nanoparticles encoding Cas13d mRNA and guide RNA, this strategy eliminated >97% of uveal melanoma cells while sparing healthy cells, including retinal pigment epithelial cells. This approach outperformed conventional Cas9 and siRNA methods in potency without inducing permanent genomic alterations. Our findings establish a RNA-targeting therapeutic for uveal melanoma and a framework for Cas13d-based interventions against broad "undruggable" cancers.

synthetic biology↗

bioRxiv2025

A Super-Resolution Spatial Atlas of SARS-CoV-2 Infection in Human Cells

Coronaviral replication depends on double-membrane vesicles (DMVs), yet where polyprotein processing occurs and how replication organelles mature remain unresolved. We built a multi{-}color super-resolution atlas of SARS-CoV-2 RNA, non-structural and structural proteins in infected human cells, combining 3D single-molecule localization with radial and angular pair-correlation analysis. The atlas uncovers fundamental spatial principles. First, nsp5 (3CLpro) localizes within the DMV lumen near pores, with nsp7-nsp16 positioned interior to nsp4, supporting a protease-dependent maturation model in which nsp5 action permits membrane closure and subsequently completes intravesicular polyprotein processing to activate replication complexes. Second, we identify dsRNA connectors bridging DMVs and non-canonical, nsp3/4-lacking dsRNA granules decorated with replicase components, consistent with a condensate-mediated route for trafficking replication intermediates. Third, mapping structural proteins reveals M- and S-positive virion assembly intermediates along the secretory route, while premature intracellular S1 shedding captures the baseline instability of the original virus isolate prior to evolutionary adaptation. Finally, the antiviral nirmatrelvir induces multilayered bodies of uncleaved polyproteins (nsp4-5-10-16) that persist after washout and precede rapid rebound, suggesting a drug-induced reservoir state. Beyond mapping the viral architecture, this atlas resolves the spatial context of proteolysis and organelle remodeling, providing a framework for pan-coronaviral mechanisms and antiviral design.

microbiology↗

bioRxiv2025

Programmable macromolecule delivery via engineered trogocytosis

Trogocytosis, the transfer of plasma membrane fragments during cell-cell contact, offers potential for macromolecular delivery but is limited by uncertain fate of trogocytosed molecules, constraints to membrane cargo, and unclear generalizability. Here, we demonstrate that donor cells engineered with designed receptors specific to intrinsic ligands can transfer proteins to recipient cells through direct contact. We identified key principles for enhancing contact-mediated transfer and subsequent functionalization of transferred macromolecules, including receptor design, pH-responsive membrane fusion, inducible cargo localization, release, and subcellular translocation. Exploiting these findings, we developed TRANSFER, a versatile delivery system that integrates logic gate-based control to sense multiple ligand inputs and deliver diverse functional cargos for genome editing and targeted cell ablation across cell types. The study establishes trogocytosis as a novel, programmable framework for cell-based macromolecular delivery.

bioengineering↗

bioRxiv2024

PreciCE: Precision engineering of cell fates via data-driven multi-gene control of transcriptional networks

The directed differentiation of stem cells into specific cell types is critical for regenerative medicine and cell-based applications. However, current methods for cell fate control are inefficient, imprecise, and rely on laborious trial-and-error. To address these limitations, we present a method for data-driven multi-gene modulation of transcriptional networks. We develop bidirectional CRISPR-based tools based on dCas12a, Cas13d, and dCas9 for simultaneously activating and repressing many genes. Due to the vast combinatorial complexity of multi-gene regulation, we introduce a machine learning-based computational algorithm that uses single-cell RNA sequencing data to predict multi-gene perturbation sets for converting a starting cell type into a desired target cell type. By combining these technologies, we establish a unified workflow for data-driven cell fate engineering and demonstrate its efficacy in controlling early stem cell differentiation while suppressing alternative lineages through logic-based cell fate operations. This approach represents a significant advancement in the use of synthetic biology to engineer cell identity.

synthetic biology↗

bioRxiv2024

Self-organization of embryonic stem cells into a reproducible embryo model through epigenome editing

Embryonic stem cells (ESCs) can self-organize in vitro into developmental patterns with spatial organization and molecular similarity to that of early embryonic stages. This self-organization of ESCs requires transmission of signaling cues, via addition of small molecule chemicals or recombinant proteins, to induce distinct embryonic cellular fates and subsequent assembly into structures that can mimic aspects of early embryonic development. During natural embryonic development, different embryonic cell types co-develop together, where each cell type expresses specific fate-inducing transcription factors through activation of non-coding regulatory elements and interactions with neighboring cells. However, previous studies have not fully explored the possibility of engineering endogenous regulatory elements to shape self-organization of ESCs into spatially-ordered embryo models. Here, we hypothesized that cell-intrinsic activation of a minimum number of such endogenous regulatory elements is sufficient to self-organize ESCs into early embryonic models. Our results show that CRISPR-based activation (CRISPRa) of only two endogenous regulatory elements in the genome of pluripotent stem cells is sufficient to generate embryonic patterns that show spatial and molecular resemblance to that of pre-gastrulation mouse embryonic development. Quantitative single-cell live fluorescent imaging showed that the emergence of spatially-ordered embryonic patterns happens through the intrinsic induction of cell fate that leads to an orchestrated collective cellular motion. Based on these results, we propose a straightforward approach to efficiently form 3D embryo models through intrinsic CRISPRa-based epigenome editing and independent of external signaling cues. CRISPRa-Programmed Embryo Models (CPEMs) show highly consistent composition of major embryonic cell types that are spatially-organized, with nearly 80% of the structures forming an embryonic cavity. Single cell transcriptomics confirmed the presence of main embryonic cell types in CPEMs with transcriptional similarity to pre-gastrulation mouse embryos and revealed novel signaling communication links between different embryonic cell types. Our findings offer a programmable embryo model and demonstrate that minimum intrinsic epigenome editing is sufficient to self-organize ESCs into highly consistent pre-gastrulation embryo models

developmental biology↗

bioRxiv2024

Virus-derived Synthetic Riboswitches for Real-time Coronaviruses Sensing in Live Cells

Real-time sensing of viral infection in live cells are crucial for virology research and antiviral development. However, existing methods face challenges of low signal sensitivity and the necessity for viral manipulation and cell fixation. Here, we develop a viral riboswitch (vRibo) approach that employs the viral replicase to induce transgene expression upon viral infection. The vRibo is designed to detect viral real-time transcription and replication in live cells, which in response triggers the translation of reporter and therapeutic genes. By integrating a viral packaging sequence, vRibo can be transmitted to neighboring cells through progeny virions, effectively acting as a Trojan Horse. The negative-strand vRibo elements demonstrated effective detection of several coronaviruses, including 229E and OC43, due to the conservation of cis-acting RNA structures across coronaviruses. Notably, vRibo functions as a dual-purpose system, acting both as an infection detector and inducible antiviral system. vRibo has the potential for basic virology research applications and can be adopted in improving the inducible expression of mRNA medicines for future coronaviruses.

bioengineering↗

bioRxiv2023

Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles

The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelle where the replication of viral genomic RNA (vgRNA) occurs. To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain vgRNA clusters along with viral double-stranded RNA (dsRNA) clusters and the replication enzyme, encapsulated by membranes derived from the host endoplasmic reticulum (ER). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of ER labels and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are enclosed by DMVs at early infection stages which then merge into vesicle packets as infection progresses. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

microbiology↗

bioRxiv2023

Biodesign of Frugal CRISPR Kits for Equitable and Accessible Education

Equitable and accessible education on life sciences and bioengineering is crucial to training the next generation of scientists, fostering transparency in public decision-making, and ensuring biotechnology democratization that can benefit a wide-ranging population. As a ground-breaking technology for genome engineering, CRISPR has transformed research and therapeutics. However, hands-on exposure to this technology for educational purposes has remained restricted, primarily due to extensive resources required to execute CRISPR experiments. In this study, we develop an accessible and frugal CRISPR kit, tailored for K-12 education settings. Our CRISPR kit eliminates the need for equipment, prioritizes biosafety, and utilizes cost-effective reagents. By combining CRISPRi gene regulation, chromoproteins, cell-free transcription-translation systems, and smartphone-based quantification, our kit offers a user-friendly approach and a reliable assessment of CRISPR activity, eliminating the need for a traditional laboratory setup. Experiments conducted by high school students in real-world settings highlight the kits utility for conducting reliable CRISPR experiments. The frugal CRISPR kit provides a modular and expandable platform to offer hands-on experience in genome engineering, and will facilitate equitable and accessible education and technology democratization for communities of diverse socioeconomic and geographic backgrounds.

bioengineering↗

bioRxiv2023

casmini-tool: a comprehensive database for efficient and specific guide RNA design using dCasMINI

The dCasMINI protein is a hypercompact, nuclease-inactivated CRISPR-Cas system engineered for transcriptional modulation and epigenetic editing [Xu et al., 2021]. The small size of dCas-MINI (529 amino acids), less than half the size of comparable Cas9 molecules, makes it ideal for AAV-based therapies which are frequently limited by AAVs small cargo capacity. Unlike Cas9 or Cas12a, there are no available computational tools for designing dCasMINI guides. To facilitate and accelerate the development of dCasMINI-based applications, we synthesized knowledge regarding dCasMINI guide design and built a website to assist researchers in designing optimal guides for dCasMINI-based experiments for transcriptional inhibition (CRISPRi) and activation (CRISPRa); to ensure that our tool would be useful for therapeutic guide design, in which a guides off-target safety profile is of paramount importance, we specifically optimized alignment parameters for high-sensitivity to comprehensively report genome-wide off-targets. To investigate dCasMINIs full protospacer adjacent motif (PAM) profile, we engineered libraries of PAMs and exhaustively characterized dCasMINIs ability to activate a locus with different PAMs. We also experimentally investigated the importance of each nucleotide position on the guide RNAs ability to activate its target, and characterized a 6bp high-fidelity seed region at the 5 end of the protospacer sequence which we identified to be intolerant to mismatches and deletions, and thus critical for true binding events. Taken together, our tool offers CRISPRi/a guide design for every protein-coding gene in the human genome along with comprehensive off-target prediction, incorporating the most up-to-date information about dCasMINIs full PAM and protospacer design rules. The tool is freely available to use at www.casmini-tool.com.

bioengineering↗

bioRxiv2023

Robust nuclease-dead S. aureus dCas9-mediated alpha-synuclein knockdown in substantia nigra in a humanized mouse model of Parkinson's disease

Parkinsons disease (PD) is becoming increasingly prevalent due to an aging society, which places a substantial disease burden on patients and their families and an annual cost estimated at 52 billion dollars. However, no approved disease modulatory therapies that halt disease progression are available. Alpha-synuclein is a critical therapeutic target found in aggregated form in Lewy bodies which is the diagnostic hallmark of PD. Familial autosomal dominant forms of PD can present with causative exonic point mutations, copy number multiplications, and non-coding risk variants in the alpha-synuclein gene. The disease onset, severity, and progression depend on the gene expression levels of the alpha-synuclein. Here, we demonstrate that Streptococcus aureus dCas9 (sadCas9)-mediated CRISPR interference (CRISPRi) reduces alpha-synuclein mRNA and protein levels in a humanized mouse model. The mechanism of action is based on the principle that a complementary single guide RNA (sgRNA) recruits the sadCas9 protein to the promoter region of alpha-synuclein and modulates target gene transcription, leading to reduced gene expression. We show robust downregulation of alpha-synuclein in neurons after unilateral stereotactic injection into the substantia nigra of adult mice 1 and 6 months after surgery. This work shows proof of concept that viral-mediated sadCas9 CRISPR interference can be a promising therapeutic strategy to reduce alpha-synuclein in vivo. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/556425v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@4d9247org.highwire.dtl.DTLVardef@beb419org.highwire.dtl.DTLVardef@21e0f9org.highwire.dtl.DTLVardef@391d5a_HPS_FORMAT_FIGEXP M_FIG C_FIG eTOC synopsisThis study focuses on the development of an AAV9 nuclease-dead S. aureus CRISPR/Cas9 expression system designed to target the human SNCA promoter. The objective is to achieve downregulation of -synuclein (a-syn) expression. After performing surgery and introducing the expression system, the levels of a-syn were measured at 1 month and 6 months post-surgery. The results indicate a significant downregulation of a-syn at both time points. Furthermore, it was observed that the initial immune response to the system attenuated over time, reaching control levels at the 6-month mark. These findings suggest the potential of this expression system for long-term downregulation of a-syn and provide insights into the immune response dynamics associated with its use.

neuroscience↗

bioRxiv2023

Discovery and engineering of hypercompact epigenetic modulators for durable gene activation

Programmable epigenetic modulators provide a powerful toolkit for controlling gene expression in novel therapeutic applications, but recent discovery efforts have primarily selected for potency of effect rather than contextual robustness or durability thereof. Current CRISPR-based tools are further limited by large cargo sizes that impede clinical delivery and, in gene activation contexts, by brief activity windows that preclude transient, single-dose strategies such as lipid nanoparticle (LNP) delivery. To address these limitations, we perform high-throughput screening to discover novel classes of transcriptional modulators derived from thousands of human, viral, and archaeal proteomes. We identify high-potency activators capable of mitotically stable gene activation in a multitude of cellular contexts and leverage machine learning models to rationally engineer variants with improved activities. In liver and T-cells, novel hypercompact activators (64 to 98 amino acids) derived from vIRF2 core domain (vCD) achieve superior potency and durable activation lasting weeks beyond the current large activators ([~]five-fold larger). In a humanized mouse model, we target a human hypercholesterolemia susceptibility gene and achieve activation persisting five weeks after a single dose by LNP delivery. Our discovery pipeline provides a predictive rubric for the development of contextually robust, potent, and persistent activators of compact size, broadly advancing the therapeutic potential of epigenetic gene activation.

synthetic biology↗

bioRxiv2023

Development of compact transcriptional effectors using high-throughput measurements in diverse contexts

Human nuclear proteins contain >1000 transcriptional effector domains that can activate or repress transcription of target genes. We lack a systematic understanding of which effector domains regulate transcription robustly across genomic, cell-type, and DNA-binding domain (DBD) contexts. Here, we developed dCas9-mediated high-throughput recruitment (HT-recruit), a pooled screening method for quantifying effector function at endogenous targets, and tested effector function for a library containing 5092 nuclear protein Pfam domains across varied contexts. We find many effectors depend on target and DBD contexts, such as HLH domains that can act as either activators or repressors. We then confirm these findings and further map context dependencies of effectors drawn from unannotated protein regions using a larger library containing 114,288 sequences tiling chromatin regulators and transcription factors. To enable efficient perturbations, we select effectors that are potent in diverse contexts, and engineer (1) improved ZNF705 KRAB CRISPRi tools to silence promoters and enhancers, and (2) a compact human activator combination NFZ for better CRISPRa and inducible circuit delivery. Together, this effector-by-context functional map reveals context-dependence across human effectors and guides effector selection for robustly manipulating transcription.

systems biology↗

bioRxiv2023

Programmable Drug Control of Receptor Valency Modulates the Potency of Cell Therapeutics

Designer T cells offer a novel paradigm for treating diseases like cancer, yet they are often hindered by target recognition evasion and limited in vivo control. To overcome these challenges, we develop valency-controlled receptors (VCRs), a novel class of synthetic receptors engineered to enable precise modulation of immune cell activity. VCRs use custom-designed valency-control ligands (VCLs) to modulate T cell signaling via spatial molecular clustering. Using multivalent DNA origami as VCL, we first establish that valency is important for tuning the activity of CD3-mediated immune activation. We then generate multivalent formats of clinically relevant drugs as VCL and incorporate VCR into the architecture of chimeric antigen receptors (CARs). Our data demonstrate that VCL-mediated VCRs can significantly amplify CAR activities and improve suboptimal CARs. Finally, through medicinal chemistry, we synthesize programmable, bioavailable VCL drugs that potentiate targeted immune response against low-antigen tumors both in vitro and in vivo. Our findings establish receptor valency as a core mechanism for enhancing CAR functionality and offer a synthetic chemical biology platform for strengthening customizable, potent, and safer cell therapies.

synthetic biology↗

bioRxiv2022

SARS-CoV-2 escapes direct NK cell killing through Nsp1-mediated downregulation of ligands for NKG2D

SummaryNatural killer (NK) cells are cytotoxic effector cells that target and lyse virally-infected cells; many viruses therefore encode mechanisms to escape such NK cell killing. Here, we interrogated the ability of SARS-CoV-2 to modulate NK cell recognition and lysis of infected cells. We found that NK cells exhibit poor cytotoxic responses against SARS-CoV-2-infected targets, preferentially killing uninfected bystander cells. We demonstrate that this escape is driven by downregulation of ligands for the activating receptor NKG2D ("NKG2D-L"). Indeed, early in viral infection, prior to NKG2D-L downregulation, NK cells are able to target and kill infected cells; however, this ability is lost as viral proteins are expressed. Finally, we found that SARS-CoV-2 non-structural protein 1 (Nsp1) mediates downregulation of NKG2D-L and that Nsp1 alone is sufficient to confer resistance to NK cell killing. Collectively, our work reveals that SARS-CoV-2 evades NK cell cytotoxicity and describes a mechanism by which this occurs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/496341v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@bcffeeorg.highwire.dtl.DTLVardef@469b0eorg.highwire.dtl.DTLVardef@16dd205org.highwire.dtl.DTLVardef@f78070_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

Cas9-Mediated Knockout of Ndrg2 Enhances the Regenerative Potential of Dendritic Cells for Wound Healing

Chronic wounds impose a significant healthcare burden to a broad patient population. Cell based therapies, while having shown benefits for the treatment of chronic wounds, have not achieved widespread adoption into clinical practice. Here, we developed a novel CRISPR/Cas9 approach to precisely edit dendritic cells (DCs) to enhance their therapeutic potential for healing chronic wounds. Using single-cell RNA sequencing (scRNA-seq) of tolerogenic DCs, we discover N-myc downregulated gene 2 (Ndrg2), which marks a specific population of DC progenitors, as a promising target for CRISPR knockout (KO). Ndrg2-KO alters the transcriptomic profile of DCs and preserves an immature cell state with a strong, pro-angiogenic and regenerative capacity. We then incorporated our CRISPR-based cell engineering within a hydrogel technology for in vivo cell delivery and developed a highly effective translational approach for DC based immunotherapy that accelerated healing of full-thickness wounds in both non-diabetic and diabetic mouse models. These findings could open the door to future clinical trials using safe gene editing in DCs for treating various types of chronic wounds.

cell biology↗

bioRxiv2021

Targeted Reactivation of X-linked Endogenous FOXP3 Gene from X-chromosome Inactivation in Human Female Breast Cancer Cells

Unlike autosomal tumor suppressors, which must be inactivated by a two-hit Knudson mechanism, X-linked tumor suppressors can be inactivated by a single hit due to X-chromosome inactivation (XCI). Here, we argue that targeted reactivation of the non-mutated allele from XCI offers a potential therapy for female breast cancers. Towards this goal, we developed a dual CRISPR interference and activation (CRISPRi/a) approach for simultaneously silencing and reactivating multiple X-linked genes using two orthogonal, nuclease-deficient CRISPR/Cas9 (dCas9) proteins. We verified the efficacy for use of Streptococcus pyogenes (Sp) dCas9-KRAB for silencing XIST and Staphylococcus aureus (Sa) dCas9-VPR for activating FOXP3 in various cell lines of human female breast cancers. We confirmed CRISPR reactivation of the non-mutated copy of FOXP3 from XCI in human breast cancer CRL2316 cells, which express a synonymous heterozygous mutation (p.L266L; c.798G>C) in the coding region of FOXP3. Further, simultaneous silencing of XIST from XCI led to enhanced and prolonged FOXP3 reactivation. We optimized CRISPRa by fusing SadCas9 to the demethylase TET1 and observed enhanced FOXP3 activation. Analysis of the conserved CpG-rich region of FOXP3 intron 1 confirmed that CRISPRi/a-mediated simultaneous FOXP3 activation and XIST silencing were accompanied by elevated H4 acetylation, including H4K5ac, H4K8ac, and H4K16ac, and H3K4me3 and lower DNA methylation. This indicates that CRISPRi/a targeting to XIST and FOXP3 loci alters their transcription and their nearby epigenetic modifications. The simultaneous activation and repression of the X-linked, endogenous FOXP3 and XIST from XCI offers a useful research tool and a potential therapeutic for female breast cancers. This approach also provides new routes of targeted therapy for other X-chromosome-linked genetic disorders.

cancer biology↗