Universal toxin-based selection for precise genome engineering in human cells.

Prokaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. However, in many primary cell types, the efficiency of genome editing remains low, impeding the development of gene- and cell-based therapeutic applications. A safe strategy for robust and efficient enrichment of precisely genetically engineered cells is urgently required.

In vivo genome and base editing of a human PCSK9 knock-in hypercholesterolemic mouse model.

Background: Plasma concentration of low-density lipoprotein (LDL) cholesterol is a well-established risk factor for cardiovascular disease. Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which regulates cholesterol homeostasis, has recently emerged as an approach to reduce cholesterol levels. The development of humanized animal models is an important step to validate and study human drug targets, and use of genome and base editing has been proposed as a mean to target disease alleles.

BE-FLARE: a fluorescent reporter of base editing activity reveals editing characteristics of APOBEC3A and APOBEC3B.

Background: Base Editing is a precise genome editing method that uses a deaminase-Cas9 fusion protein to mutate cytidine to thymidine in target DNA in situ without the generation of a double-strand break. However, the efficient enrichment of genetically modified cells using this technique is limited by the ability to detect such events.

Results: We have developed a Base Editing FLuorescent Activity REporter (BE-FLARE), which allows for the enrichment of cells that have undergone editing of target loci based on a fluorescence shift from BFP to GFP.

Functional abnormalities in induced Pluripotent Stem Cell-derived cardiomyocytes generated from titin-mutated patients with dilated cardiomyopathy.

Aims: Dilated cardiomyopathy (DCM), a myocardial disorder that can result in progressive heart failure and arrhythmias, is defined by ventricular chamber enlargement and dilatation, and systolic dysfunction. Despite extensive research, the pathological mechanisms of DCM are unclear mainly due to numerous mutations in different gene families resulting in the same outcome-decreased ventricular function. Titin (TTN)-a giant protein, expressed in cardiac and skeletal muscles, is an important part of the sarcomere, and thus TTN mutations are the most common cause of adult DCM.

Tbx1 represses gene expression and is correlated with histone 3 deacetylation of the anterior heart field enhancer.

The gene is haploinsufficient in 22q11.2 deletion syndrome (22q11.2DS), and genetic evidence from human patients and mouse models points to a major role of this gene in the pathogenesis of this syndrome.

Subtype-specific promoter-driven action potential imaging for precise disease modelling and drug testing in hiPSC-derived cardiomyocytes.

Aims: Cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) are increasingly used in disease modelling and drug evaluation. However, they are typically a heterogeneous mix of ventricular-, atrial-, and nodal-like cells based on action potentials (APs) and gene expression. This heterogeneity and the paucity of methods for high-throughput functional phenotyping hinder the full exploitation of their potential.

Elucidating arrhythmogenic mechanisms of long-QT syndrome CALM1-F142L mutation in patient-specific induced pluripotent stem cell-derived cardiomyocytes.

Aims: Calmodulin (CaM) is a small protein, encoded by three genes (CALM1-3), exerting multiple Ca2+-dependent modulatory roles. A mutation (F142L) affecting only one of the six CALM alleles is associated with long QT syndrome (LQTS) characterized by recurrent cardiac arrests. This phenotypic severity is unexpected from the predicted allelic balance.

Embryonic heart progenitors and cardiogenesis.

The mammalian heart is a highly specialized organ, comprised of many different cell types arising from distinct embryonic progenitor populations during cardiogenesis. Three precursor populations have been identified to contribute to different myocytic and nonmyocytic cell lineages of the heart: cardiogenic mesoderm cells (CMC), the proepicardium (PE), and cardiac neural crest cells (CNCCs). This review will focus on molecular cues necessary for proper induction, expansion, and lineage-specific differentiation of these progenitor populations during cardiac development in vivo.

Tbx1 is a negative modulator of Mef2c.

The developmental role of the T-box transcription factor Tbx1 is exquisitely dosage-sensitive. In this study, we performed a microarray-based transcriptome analysis of E9.5 embryo tissues across a previously generated Tbx1 mouse allelic series.

dSTAM expression pattern during wild type and mutant egg chamber development in D. melanogaster.

STAM (signal-transducing adaptor molecule) is a protein highly conserved from yeast to mammals. In Drosophila melanogaster the basic molecular architecture of the protein is comprised of a N-terminal VHS domain, an ubiquitin-interacting motif and a central Src homology-3 domain. In this paper we examine the expression pattern of the stam gene and the localisation of the STAM protein during D.