Advancing gastric cancer precision medicine with novel genomic screens†.

A recent study published in The Journal of Pathology used an shRNA library targeting all known human genes involved in metabolism to identify genes important for gastric cancer. The screen identified aspartyl-tRNA synthetase (DARS) as a potential drug target, and patients whose tumors had high DARS levels had a worse prognosis, particularly among diffuse-type gastric cancer. These findings identify a potential therapeutic target for precision medicine of gastric cancer patients, and may be useful for further investigations to discover additional interacting targets. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Diversifying preclinical research tools: expanding patient-derived models to address cancer health disparities.

Cancer health disparities define a critical healthcare issue for racial/ethnic minorities in the USA. Key findings have led to cancer treatment improvements tailored to minority patients, but such successes have been rare. Here, we highlight how the use of patient-derived xenograft (PDX) and organoid models could resolve current blocks toward precision cancer health equity.

Imaging Unique DNA Sequences in Individual Cells Using a CRISPR-Cas9-Based, Split Luciferase Biosensor.

An extensive arsenal of biosensing tools has been developed based on the clustered regularly interspaced short palindromic repeat (CRISPR) platform, including those that detect specific DNA sequences both in vitro and in live cells. To date, DNA imaging approaches have traditionally used full fluorescent reporter-based fusion probes. Such "always-on" probes differentiate poorly between bound and unbound probe and are unable to sensitively detect unique copies of a target sequence in individual cells. Herein we describe a DNA biosensor that provides a sensitive readout for such low-copy DNA sequences through proximity-mediated reassembly of two independently optimized fragments of NanoLuc luciferase (NLuc), a small, bright luminescent reporter. Applying this "turn-on" probe in live cells, we demonstrate an application not easily achieved by fluorescent reporter-based probes, detection of individual endogenous genomic loci using standard epifluorescence microscopy. This approach could enable detection of gene edits during ex vivo editing procedures and should be a useful platform for many other live cell DNA biosensing applications.

Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing.

One important application of clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas 9 is the development of knock-out cell lines, specifically to study the function of new genes/proteins associated with a disease, identified during the genetic diagnosis. For the development of such cell lines, two major issues have to be untangled: insertion of the CRISPR tools (the Cas9 and the guide RNA) with high efficiency into the chosen cells, and restriction of the Cas9 activity to the specific deletion of the chosen gene. The protocol described here is dedicated to the insertion of the CRISPR tools in difficult to transfect cells, such as muscle cells. This protocol is based on the use of lentiviruses, produced with plasmids publicly available, for which all the cloning steps are described to target a gene of interest. The control of Cas9 activity has been performed using an adaptation of a previously described system called KamiCas9, in which the transduction of the cells with a lentivirus encoding a guide RNA targeting the Cas9 allows the progressive abolition of Cas9 expression. This protocol has been applied to the development of a RYR1-knock out human muscle cell line, which has been further characterized at the protein and functional level, to confirm the knockout of this important calcium channel involved in muscle intracellular calcium release and in excitation-contraction coupling. The procedure described here can easily be applied to other genes in muscle cells or in other difficult to transfect cells and produce valuable tools to study these genes in human cells.