Single-cell transcriptome analysis of epithelial, immune, and stromal signatures and interactions in human ovarian cancer.

A comprehensive investigation of ovarian cancer (OC) progression at the single-cell level is crucial for enhancing our understanding of the disease, as well as for the development of better diagnoses and treatments. Here, over half a million single-cell transcriptome data were collected from 84 OC patients across all clinical stages. Through integrative analysis, we identified heterogeneous epithelial-immune-stromal cellular compartments and their interactions in the OC microenvironment. The epithelial cells displayed clinical subtype features with functional variance. A significant increase in distinct T cell subtypes was identified including Tregs and CD8+ exhausted T cells from stage IC2. Additionally, we discovered antigen-presenting cancer-associated fibroblasts (CAFs), with myofibroblastic CAFs (myCAFs) exhibiting enriched extracellular matrix (ECM) functionality linked to tumor progression at stage IC2. Furthermore, the NECTIN2-TIGIT ligand-receptor pair was identified to mediate T cells communicating with epithelial, fibroblast, endothelial, and other cell types. Knock-out of NECTIN2 using CRISPR/Cas9 inhibited ovarian cancer cell (SKOV3) proliferation, and increased T cell proliferation when co-cultured. These findings shed light on the cellular compartments and functional aspects of OC, providing insights into the molecular mechanisms underlying stage IC2 and potential therapeutic strategies for OC.

Enrichment strategies to enhance genome editing.

Genome editing technologies hold great promise for numerous applications including the understanding of cellular and disease mechanisms and the development of gene and cellular therapies. Achieving high editing frequencies is critical to these research areas and to achieve the overall goal of being able to manipulate any target with any desired genetic outcome. However, gene editing technologies sometimes suffer from low editing efficiencies due to several challenges. This is often the case for emerging gene editing technologies, which require assistance for translation into broader applications. Enrichment strategies can support this goal by selecting gene edited cells from non-edited cells. In this review, we elucidate the different enrichment strategies, their many applications in non-clinical and clinical settings, and the remaining need for novel strategies to further improve genome research and gene and cellular therapy studies.

Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene.

CRISPR-Cas-mediated site-specific integration of transgenes by homology-directed repair (HDR) is challenging, especially in primary cells, where inferior editing efficiency may impede the development of gene- and cellular therapies. Various strategies for enrichment of cells with transgene integrations have been developed, but most strategies either generate unwanted genomic scars or rely on permanent integration and expression of a reporter gene used for selection. However, stable expression of a reporter gene may perturb cell homeostasis and function. Here we develop a broadly applicable and versatile enrichment strategy by harnessing the capability of CRISPR activation (CRISPRa) to transiently induce expression of a therapeutically relevant reporter gene used for immunomagnetic enrichment. This strategy is readily adaptable to primary human T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs), where enrichment of 1.8- to 3.3-fold and 3.2- to 3.6-fold was achieved, respectively. Furthermore, chimeric antigen receptor (CAR) T cells were enriched 2.5-fold and demonstrated improved cytotoxicity over non-enriched CAR T cells. Analysis of HDR integrations showed a proportion of cells harboring deletions of the transgene cassette arising either from impartial HDR or truncated adeno-associated virus (AAV) vector genomes. Nonetheless, this novel enrichment strategy expands the possibility to enrich for transgene integrations in research settings and in gene and cellular therapies.

A truncated reverse transcriptase enhances prime editing by split AAV vectors.

Prime editing is a new CRISPR-based, genome-editing technology that relies on the prime editor (PE), a fusion protein of Cas9-nickase and M-MLV reverse transcriptase (RT), and a prime editing guide RNA (pegRNA) that serves both to target PE to the desired genomic locus and to carry the edit to be introduced. Here, we make advancements to the RT moiety to improve prime editing efficiencies and truncations to mitigate issues with adeno-associated virus (AAV) viral vector size limitations, which currently do not support efficient delivery of the large prime editing components. These efforts include RT variant screening, codon optimization, and PE truncation by removal of the RNase H domain and further trimming. This led to a codon-optimized and size-minimized PE that has an expression advantage (1.4-fold) and size advantage (621 bp shorter). In addition, we optimize the split intein PE system and identify Rma-based Cas9 split sites (573-574 and 673-674) that combined with the truncated PE delivered by dual AAVs result in superior AAV titer and prime editing efficiency. We also show that this minimized PE gives rise to superior lentiviral vector titers (46-fold) over the regular PE in an all-in-one PE lentiviral vector. We finally deliver the minimized PE to mouse liver by dual AAV8 vectors and show up to 6% precise editing of the PCSK9 gene, thereby demonstrating the value of this truncated split PE system for in vivo applications.

Targeted regulation of transcription in primary cells using CRISPRa and CRISPRi.

Targeted transcriptional activation or interference can be induced with the CRISPR-Cas9 system (CRISPRa/CRISPRi) using nuclease-deactivated Cas9 fused to transcriptional effector molecules. These technologies have been used in cancer cell lines, particularly for genome-wide functional genetic screens using lentiviral vectors. However, CRISPRa and CRISPRi have not yet been widely applied to ex vivo cultured primary cells with therapeutic relevance owing to a lack of effective and nontoxic delivery modalities. Here we develop CRISPRa and CRISPRi platforms based on RNA or ribonucleoprotein (RNP) delivery by electroporation and show transient, programmable gene regulation in primary cells, including human CD34+ hematopoietic stem and progenitor cells (HSPCs) and human CD3+ T cells. We show multiplex and orthogonal gene modulation using multiple sgRNAs and CRISPR systems from different bacterial species, and we show that CRISPRa can be applied to manipulate differentiation trajectories of HSPCs. These platforms constitute simple and effective means to transiently control transcription and are easily adopted and reprogrammed to new target genes by synthetic sgRNAs. We believe these technologies will find wide use in engineering the transcriptome for studies of stem cell biology and gene function, and we foresee that they will be implemented to develop and enhance cellular therapeutics.