Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids

Prime editing is a recently reported genome editing tool using a nickase-cas9 fused to a reverse transcriptase that directly synthesizes the desired edit at the target site. Here, we explore the use of prime editing in human organoids. Common TP53 mutations can be correctly modeled in human adult stem cell–derived colonic organoids with efficiencies up to 25% and up to 97% in hepatocyte organoids. Next, we functionally repaired the cystic fibrosis CFTR-F508del mutation and compared prime editing to CRISPR/Cas9–mediated homology-directed repair and adenine base editing on the CFTR-R785* mutation. Whole-genome sequencing of prime editing–repaired organoids revealed no detectable off-target effects. Despite encountering varying editing efficiencies and undesired mutations at the target site, these results underline the broad applicability of prime editing for modeling oncogenic mutations and showcase the potential clinical application of this technique, pending further optimization.

In Vitro 3D Cultures to Model the Tumor Microenvironment

It is now well established that the tumor microenvironment plays a key role in determining cancer growth, metastasis and drug resistance. Thus, it is fundamental to understand how cancer cells interact and communicate with their stroma and how this crosstalk regulates disease initiation and progression. In this setting, 3D cell cultures have gained a lot of interest in the last two decades, due to their ability to better recapitulate the complexity of tumor microenvironment and therefore to bridge the gap between 2D monolayers and animal models. Herein, we present an overview of the 3D systems commonly used for studying tumor–stroma interactions, with a focus on recent advances in cancer modeling and drug discovery and testing.

Spontaneous and Induced Tumors in Germ-Free Animals: A General Review

Cancer, bacteria, and immunity relationships are much-debated topics in the last decade. Microbiome′s importance for metabolic and immunologic modulation of the organism adaptation and responses has become progressively evident, and models to study these relationships, especially about carcinogenesis, have acquired primary importance. The availability of germ-free (GF) animals, i.e., animals born and maintained under completely sterile conditions avoiding the microbiome development offers a unique tool to investigate the role that bacteria can have in carcinogenesis and tumor development. The comparison between GF animals with the conventional (CV) counterpart with microbiome can help to evidence conditions and mechanisms directly involving bacterial activities in the modulation of carcinogenesis processes. Here, we review the literature about spontaneous cancer and cancer modeling in GF animals since the early studies, trying to offer a practical overview on the argument.

Mini-review: advances in 3D bioprinting of vascularized constructs

3D in vitro constructs have gained more and more relevance in tissue engineering and in cancer-modeling. In recent years, with the development of thicker and more physiologically relevant tissue patches, the integration of a vascular network has become pivotal, both for sustaining the construct in vitro and to help the integration with the host tissue once implanted. Since 3D bioprinting is rising to be one of the most versatile methods to create vascularized constructs, we here briefly review the most promising advances in bioprinting techniques.