RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas proteins have recently emerged as versatile tools to investigate and engineer the genome. The programmability of CRISPR-Cas has proven especially useful for probing genomic function in high-throughput. Facile single-guide RNA library synthesis allows CRISPR-Cas screening to rapidly investigate the functional consequences of genomic, transcriptomic, and epigenomic perturbations. Furthermore, by combining CRISPR-Cas perturbations with downstream single-cell analyses (flow cytometry, expression profiling, etc.), forward screens can generate robust data sets linking genotypes to complex cellular phenotypes. In the following review, we highlight recent advances in CRISPR-Cas genomic screening while outlining protocols and pitfalls associated with screen implementation. Finally, we describe current challenges limiting the utility of CRISPR-Cas screening as well as future research needed to resolve these impediments. As CRISPR-Cas technologies develop, so too will their clinical applications. Looking ahead, patient centric functional screening in primary cells will likely play a greater role in disease management and therapeutic development.

Deconstructing tissue-specific effects of genes and variants on proliferation is critical to understanding cellular transformation and systematically selecting cancer therapeutics. This requires scalable methods for multiplexed genetic screens tracking fitness across time, across lineages, and in a suitable niche, since physiological cues influence functional differences. Towards this, we present an approach, coupling single-cell cancer driver screens in teratomas with hit enrichment by serial teratoma reinjection, to simultaneously screen drivers across multiple lineages in vivo. Using this system, we analyzed population shifts and lineage-specific enrichment for 51 cancer associated genes and variants, profiling over 100,000 cells spanning over 20 lineages, across two rounds of serial reinjection. We confirmed that c-MYC alone or combined with myristoylated AKT1 potently drives proliferation in progenitor neural lineages, demonstrating signatures of malignancy. Additionally, mutant MEK1 ^S218D/S222D provides a proliferative advantage in mesenchymal lineages like fibroblasts. Our method provides a powerful platform for multi-lineage longitudinal study of oncogenesis.

Pluripotent stem cell-derived organoids have transformed our ability to recreate complex three-dimensional models of human tissue. However, the directed differentiation methods used to create them do not afford the ability to introduce cross-germ-layer cell types. Here, we present a bottom-up engineering approach to building vascularized human tissue by combining genetic reprogramming with chemically directed organoid differentiation. As a proof of concept, we created neuro-vascular and myo-vascular organoids via transcription factor overexpression in vascular organoids. We comprehensively characterized neuro-vascular organoids in terms of marker gene expression and composition, and demonstrated that the organoids maintain neural and vascular function for at least 45 days in culture. Finally, we demonstrated chronic electrical stimulation of myo-vascular organoid aggregates as a potential path toward engineering mature and large-scale vascularized skeletal muscle tissue from organoids. Our approach offers a roadmap to build diverse vascularized tissues of any type derived entirely from pluripotent stem cells.

Objective(s)

To quantify the effect of laryngeal prominence size on socially perceived attributes relating to gender expression. Chondrolaryngoplasty ("tracheal shave") is a common procedure performed for transgender women to feminize neck appearance. The extent of thyroid cartilage resection needed to convey socially-perceived feminine gender expression without destabilizing the voice is incompletely understood.

Methods

Cross-sectional evaluation of a randomized allocation of images of varying laryngeal prominence to a non-repeated, random sample from November 2021 to December 2021. Photos of laryngeal prominence were isolated against a constant neck baseline with lateral, oblique, and frontal views. The images were embedded into a web-based survey with visual analog scales to capture perceived scaled gender expression (masculinity, femininity) and social traits (e.g., attractiveness, friendliness, leadership). We performed bivariate and multivariate analyses relating the laryngeal prominence to perceived gender expression and social traits.

Results

The analytic sample included 1,026 respondents. Laryngeal grades similar to the demonstrated "grade M" in this study and smaller demonstrated similar perceptions of increased femininity and decreased masculinity. Grades larger than M demonstrate significantly increased perceived masculinity and significantly decreased perceived femininity. The lateral and oblique views of the neck appear to be the most gender-informative.

Conclusion

This crowd-sourced analysis of external laryngeal anatomy by a large population of observers provides clear, reproducible insights into social perceptions of gender identity and specifically femininity. These data will meaningfully inform patient counseling and surgical planning for gender-affirming interventions by establishing normative data representing the general public's perceptions.

Level of evidence

NA Laryngoscope, 133:2292-2300, 2023.

Genetic screens are powerful tools for both resolving biological function and identifying potential therapeutic targets, but require physiologically accurate systems to glean biologically useful information. Here, we enable genetic screens in physiologically relevant ex vivo cancer tissue models by integrating CRISPR-Cas-based genome engineering and biofabrication technologies. We first present a novel method for generating perfusable tissue constructs, and validate its functionality by using it to generate three-dimensional perfusable dense cultures of cancer cell lines and sustain otherwise ex vivo unculturable patient-derived xenografts. Using this system we enable large-scale CRISPR screens in perfused tissue cultures, as well as emulate a novel point-of-care diagnostics scenario of a clinically actionable CRISPR knockout (CRISPRko) screen of genes with FDA-approved drug treatments in ex vivo PDX cell cultures. Our results reveal differences across in vitro and in vivo cancer model systems, and highlight the utility of programmable tissue engineered models for screening therapeutically relevant cancer vulnerabilities.

Adenosine deaminases acting on RNA (ADARs) impact diverse cellular processes and pathological conditions, but their functions in early cell-fate specification remain less understood. To gain insights here, we began by charting time-course RNA editing profiles in human organs from fetal to adult stages. Next, we utilized hPSC differentiation to experimentally probe ADARs, harnessing brain organoids as neural specific, and teratomas as pan-tissue developmental models. We show that time-series teratomas faithfully recapitulate fetal developmental trends, and motivated by this, conducted pan-tissue, single-cell CRISPR-KO screens of ADARs in teratomas. Knocking out ADAR leads to a global decrease in RNA editing across all germ-layers. Intriguingly, knocking out ADAR leads to an enrichment of adipogenic cells, revealing a role for ADAR in human adipogenesis. Collectively, we present a multi-pronged framework charting time-resolved RNA editing profiles and coupled ADAR perturbations in developmental models, thereby shedding light on the role of ADARs in cell-fate specification.

Current treatments for chronic pain rely largely on opioids despite their substantial side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing. In particular, a hereditary loss-of-function mutation in NaV1.7, a sodium channel protein associated with signaling in nociceptive sensory afferents, leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence and structural similarity between NaV subtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of NaV1.7 in primary afferents via epigenome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and zinc finger proteins at the spinal level as a potential treatment for chronic pain. Toward this end, we first optimized the efficiency of NaV1.7 repression in vitro in Neuro2A cells and then, by the lumbar intrathecal route, delivered both epigenome engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain, and BzATP-induced pain. Our results show effective repression of NaV1.7 in lumbar dorsal root ganglia, reduced thermal hyperalgesia in the inflammatory state, decreased tactile allodynia in the neuropathic state, and no changes in normal motor function in mice. We anticipate that this long-lasting analgesia via targeted in vivo epigenetic repression of NaV1.7 methodology we dub pain LATER, might have therapeutic potential in management of persistent pain states.