Inducible, split base editors for in vivo cancer functional genomics

Nature Biotechnology · 2026-04-15

Cancer functional genomics using CRISPR base editors (BEs) holds great promise for molecular characterization and new target discovery. However, traditional BEs, using intact DNA deaminases as mutators, are often constrained by limited control and nonspecific toxicities. Here we developed a small-molecule-controllable system using split-engineered BEs (seBEs). By placing deaminase activity under small-molecule control, seBEs significantly reduced cellular toxicity and enabled robust and inducible in vivo functional genomics screens. High-density seBE genetic screens using ~11,000 single guide RNAs in vitro and ~3,700 single guide RNAs in vivo reveal known and previously unknown loss-of-function and dominant-negative mutations in cancer therapeutic targets. A deeper tiling seBE screen against Adar1, a key mediator in cancer immunotherapy, reveals critical residues within functional domains that show no phenotype in vitro but distinctively elicit non-cell-autonomous cancer dependencies in vivo. Overall, our seBE system offers a generalizable, controllable and highly efficient method to systematically identify key residues in cancer functional genomics.

Independent genetic strategies define the scope and limits of CDKL5 deficiency disorder reversal

Cell Reports Medicine · 2025-01-23 · 4 citations

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a neurodevelopmental syndrome caused by mutations in the X-linked CDKL5 gene. The early onset of CDD suggests that CDKL5 is essential during development, but post-developmental re-expression rescues multiple CDD-related phenotypes in hemizygous male mice. Since most patients are heterozygous females, studies in clinically relevant female models are essential. Here, we systematically compare phenotype reversal across age and sex using two independent mouse models of CDD. We find that early re-activation of endogenous Cdkl5 in heterozygous females reverses most phenotypes, except working memory. Later re-expression improves several traits but has limited effects on cognitive function. Seizure prevention is more effective with early intervention in heterozygous females but becomes limited after seizure onset. These findings demonstrate the robust potential of CDKL5 re-expression to reverse CDD-related phenotypes in both sexes while underscoring the critical impact of age and disease stage in designing clinical trials. • Restoring CDKL5 expression in heterozygous STOP females reverses CDD-related deficits • Genetic rescue in heterozygous FLEX females replicates CDD-related phenotype reversal • Aging limits the reversal of CDD-related phenotypes in male and female mice • Early but not late CDKL5 restoration in female mice prevents age-related epilepsy CDKL5 deficiency disorder (CDD) is a severe neurodevelopmental syndrome. Here, we report early restoration of CDKL5 expression reverses multiple symptoms in male and female mouse models. Outcomes vary by age and sex, underscoring the need for early intervention and careful consideration of these factors in designing clinical trials.

Allelic contribution of Nrxn1α to autism-relevant behavioral phenotypes in mice

PLoS Genetics · 2023-02-27 · 22 citations

Copy number variations (CNVs) in the Neurexin 1 (NRXN1) gene, which encodes a presynaptic protein involved in neurotransmitter release, are some of the most frequently observed single-gene variants associated with autism spectrum disorder (ASD). To address the functional contribution of NRXN1 CNVs to behavioral phenotypes relevant to ASD, we carried out systematic behavioral phenotyping of an allelic series of Nrxn1 mouse models: one carrying promoter and exon 1 deletion abolishing Nrxn1α transcription, one carrying exon 9 deletion disrupting Nrxn1α protein translation, and one carrying an intronic deletion with no observable effect on Nrxn1α expression. We found that homozygous loss of Nrxn1α resulted in enhanced aggression in males, reduced affiliative social behaviors in females, and significantly altered circadian activities in both sexes. Heterozygous or homozygous loss of Nrxn1α affected the preference for social novelty in male mice, and notably, enhanced repetitive motor skills and motor coordination in both sexes. In contrast, mice bearing an intronic deletion of Nrxn1 did not display alterations in any of the behaviors assessed. These findings demonstrate the importance of Nrxn1α gene dosage in regulating social, circadian, and motor functions, and the variables of sex and genomic positioning of CNVs in the expression of autism-related phenotypes. Importantly, mice with heterozygous loss of Nrxn1, as found in numerous autistic individuals, show an elevated propensity to manifest autism-related phenotypes, supporting the use of models with this genomic architecture to study ASD etiology and assess additional genetic variants associated with autism.

Temporal manipulation of Cdkl5 reveals essential postdevelopmental functions and reversible CDKL5 deficiency disorder–related deficits

Journal of Clinical Investigation · 2021-10-14 · 60 citations

CDKL5 deficiency disorder (CDD) is an early onset, neurodevelopmental syndrome associated with pathogenic variants in the X-linked gene encoding cyclin-dependent kinase-like 5 (CDKL5). CDKL5 has been implicated in neuronal synapse maturation, yet its postdevelopmental necessity and the reversibility of CDD-associated impairments remain unknown. We temporally manipulated endogenous Cdkl5 expression in male mice and found that postdevelopmental loss of CDKL5 disrupts numerous behavioral domains, hippocampal circuit communication, and dendritic spine morphology, demonstrating an indispensable role for CDKL5 in the adult brain. Accordingly, restoration of Cdkl5 after the early stages of brain development using a conditional rescue mouse model ameliorated CDD-related behavioral impairments and aberrant NMDA receptor signaling. These findings highlight the requirement of CDKL5 beyond early development, underscore the potential for disease reversal in CDD, and suggest that a broad therapeutic time window exists for potential treatment of CDD-related deficits.

The Transcription Factor NR4A2 Plays an Essential Role in Driving Prolactin Expression in Female Pituitary Lactotropes

Endocrinology · 2020-03-19 · 8 citations

Differentiation of the hormone-producing cells of the pituitary represents an informative model of cell fate determination. The generation and maintenance of 2 pituitary lineages, the growth hormone (GH)- producing somatotropes and the prolactin (PRL)- producing lactotropes, are dependent on the pituitary-specific transcription factor, POU1F1. While POU1F1 is expressed in both cell types, and plays a role in activation of both the Gh and Prl genes, expression of Gh and Prl is restricted to somatotropes and lactotropes, respectively. These observations imply the existence of additional factors that contribute to the somatotrope and lactotrope identities and their hormone expressions. Prior transcriptome analysis of primary somatotropes and lactotropes isolated from the mouse pituitary identified enrichment of a transcription factor, Nr4a2, in the lactotropes. Nr4a2 was shown in a cell culture model to bind the Prl promoter at a position adjacent to Pou1f1 and to synergize with Pou1f1 in driving Prl transcription. Here we demonstrate in vivo the role of Nr4a2 as an enhancer of Prl expression by conditional gene inactivation of the Nr4a2 gene in mouse lactotropes. We demonstrate that nuclear orphan receptor transcription factor (NR4A2) binding at the Prl promoter is dependent on actions of POU1F1; while POU1F1 is essential to loading polymerase (Pol) II on the Prl promoter, Nr4a2 plays a role in enhancing Pol II release into the Prl gene body. These studies establish an in vivo role of Nr4a2 in enhancing Prl expression in mouse lactotropes, explore its mechanism of action, and establish a system for further study of the lactotrope lineage in the pituitary.

Single-cell transcriptomic analysis of adult mouse pituitary reveals sexual dimorphism and physiologic demand-induced cellular plasticity

Protein & Cell · 2020 · 89 citations

• Biology
• Cell biology
• Endocrinology

The anterior pituitary gland drives highly conserved physiologic processes in mammalian species. These hormonally controlled processes are central to somatic growth, pubertal transformation, fertility, lactation, and metabolism. Current cellular models of mammalian anteiror pituitary, largely built on candidate gene based immuno-histochemical and mRNA analyses, suggest that each of the seven hormones synthesized by the pituitary is produced by a specific and exclusive cell lineage. However, emerging evidence suggests more complex relationship between hormone specificity and cell plasticity. Here we have applied massively parallel single-cell RNA sequencing (scRNA-seq), in conjunction with complementary imaging-based single-cell analyses of mRNAs and proteins, to systematically map both cell-type diversity and functional state heterogeneity in adult male and female mouse pituitaries at single-cell resolution and in the context of major physiologic demands. These quantitative single-cell analyses reveal sex-specific cell-type composition under normal pituitary homeostasis, identify an array of cells associated with complex complements of hormone-enrichment, and undercover non-hormone producing interstitial and supporting cell-types. Interestingly, we also identified a Pou1f1-expressing cell population that is characterized by a unique multi-hormone gene expression profile. In response to two well-defined physiologic stresses, dynamic shifts in cellular diversity and transcriptome profiles were observed for major hormone producing and the putative multi-hormone cells. These studies reveal unanticipated cellular complexity and plasticity in adult pituitary, and provide a rich resource for further validating and expanding our molecular understanding of pituitary gene expression programs and hormone production.

Transcriptome analyses of female somatotropes and lactotropes reveal novel regulators of cell identity in the pituitary

Endocrinology · 2018-09-21 · 17 citations

The differentiation of the hormone-producing cell lineages of the anterior pituitary represents an informative model of mammalian cell fate determination. The generation and maintenance of two of these lineages, the GH-producing somatotropes and prolactin (PRL)-producing lactotropes, are dependent on the pituitary-specific transcription factor POU1F1. Whereas POU1F1 is expressed in both cell types, and plays a direct role in the activation of both the Gh and Prl genes, GH expression is restricted to somatotropes and PRL expression is restricted to lactotropes. These observations imply the existence of additional, cell type-enriched factors that contribute to the somatotrope and lactotrope cell identities. In this study, we use transgenic mouse models to facilitate sorting of somatotrope and lactotrope populations based on the expression of fluorescent markers expressed under Gh and Prl gene transcriptional controls. The transcriptomic analyses reveal a concordance of gene expression profiles in the two populations. The limited number of divergent mRNAs between the two populations includes a set of transcription factors that may have roles in pituitary lineage divergence and/or in regulating expression of cell type-specific genes after differentiation. Four of these factors were validated for lineage enrichment at the level of protein expression, two somatotrope enriched and two lactotrope enriched. Three of these four factors were shown to have corresponding activities in appropriate enhancement or repression of landmark genes in a cell culture model system. These studies identify novel regulators of the somatotropes and lactotropes, and they establish a useful database for further study of these lineages in the anterior pituitary.

Single cell transcriptomic analysis of the adult mouse pituitary reveals a novel multi-hormone cell cluster and physiologic demand-induced lineage plasticity

bioRxiv (Cold Spring Harbor Laboratory) · 2018-11-22 · 8 citations

Abstract The anterior pituitary gland drives a set of highly conserved physiologic processes in mammalian species. These hormonally-controlled processes are central to somatic growth, pubertal transformation, fertility, lactation, and metabolism. Current models, largely built upon candidate gene based immuno-histochemical and mRNA analyses, suggest that each of the seven hormones synthesized by the pituitary is produced by a specific and exclusive cell lineage. However, emerging evidence suggests more complex models of hormone specificity and cell plasticity. Here we have applied massively parallel single-cell RNA sequencing (scRNA-seq), in conjunction with a set of orthogonal mRNA and protein imaging studies, to systematically map the cellular composition of adult male and female mouse pituitaries at single-cell resolution and in the setting of major physiologic demands. These analyses reveal sex-specific cellular diversity associated with normal pituitary homeostasis, and identify an array of cells with complex complements of hormone-enrichment as well as a series of non-hormone producing interstitial and supporting cell lineages. These scRNA-seq studies identify a major cell population that is characterized by a unique multi-hormone gene expression profile. The detection of dynamic shifts in cellular representations and transcriptome profiles in response to two well-defined physiologic stresses suggests corresponding roles of a number of these clusters in cellular plasticity within the adult pituitary. These studies point to an unanticipated complexity and plasticity in pituitary cellular composition that expands upon current models and concepts of pituitary gene expression and hormone production.

PRDM16 binds MED1 and controls chromatin architecture to determine a brown fat transcriptional program

Genes & Development · 2015-02-01 · 148 citations

PR (PRD1-BF1-RIZ1 homologous) domain-containing 16 (PRDM16) drives a brown fat differentiation program, but the mechanisms by which PRDM16 activates brown fat-selective genes have been unclear. Through chromatin immunoprecipitation (ChIP) followed by deep sequencing (ChIP-seq) analyses in brown adipose tissue (BAT), we reveal that PRDM16 binding is highly enriched at a broad set of brown fat-selective genes. Importantly, we found that PRDM16 physically binds to MED1, a component of the Mediator complex, and recruits it to superenhancers at brown fat-selective genes. PRDM16 deficiency in BAT reduces MED1 binding at PRDM16 target sites and causes a fundamental change in chromatin architecture at key brown fat-selective genes. Together, these data indicate that PRDM16 controls chromatin architecture and superenhancer activity in BAT.

An Autoregulatory Pathway Establishes the Definitive Chromatin Conformation at the <i>Pit-1</i> Locus

Molecular and Cellular Biology · 2015-02-18 · 10 citations

The transcription factor Pit-1 (POU1-F1) plays a dominant role in cell lineage expansion and differentiation in the anterior pituitary. Prior studies of the mouse Pit-1 (mPit-1) gene revealed that this master regulatory locus is activated at embryonic day 13.5 (E13.5) by an early enhancer (EE), whereas its subsequent expression throughout adult life is maintained by a more distal definitive enhancer (DE). Here, we demonstrate that the sequential actions of these two enhancers are linked to corresponding shifts in their proximities to the Pit-1 promoter. We further demonstrate that the looping of the definitive enhancer to the mPit-1 promoter is critically dependent on a self-sustaining autoregulatory mechanism mediated by the Pit-1 protein. These Pit-1-dependent actions are accompanied by localized recruitment of CBP and enrichment for H3K27 acetylation within the Pit-1 locus. These data support a model in which the sequential actions of two developmentally activated enhancers are linked to a corresponding shift in higher-order chromatin structures. This shift establishes an autoregulatory circuit that maintains durable expression of Pit-1 throughout adult life.