Protocol for efficient CRISPR-Cas9-mediated fluorescent tag knockin in hard-to-transfect erythroid cell lines.

Precise insertion of fluorescent tags by CRISPR-Cas9-mediated homologous recombination (HR) in mammalian genes is a powerful tool allowing to study gene function and protein gene products. Here, we present a protocol for efficient HR-mediated targeted insertion of fluorescent markers in the genome of hard-to-transfect erythroid cell lines MEL (mouse erythroleukemic) and MEDEP (mouse ES cell-derived erythroid progenitor line). We describe steps for plasmid construction, electroporation, amplification, and verification of genome editing.

Efficient genome editing in erythroid cells unveils novel MYB target genes and regulatory functions.

Targeted genome editing holds great promise in biology. However, efficient genome modification, including gene knock-in (KI), remains an unattained goal in multiple cell types and loci due to poor transfection efficiencies and low target genes expression, impeding the positive selection of recombined cells. Here, we describe a genome editing approach to achieve efficient gene targeting using hard to transfect erythroid cell lines.

DeSUMOylation of chromatin-bound proteins limits the rapid transcriptional reprogramming induced by daunorubicin in acute myeloid leukemias.

Genotoxicants have been used for decades as front-line therapies against cancer on the basis of their DNA-damaging actions. However, some of their non-DNA-damaging effects are also instrumental for killing dividing cells. We report here that the anthracycline Daunorubicin (DNR), one of the main drugs used to treat Acute Myeloid Leukemia (AML), induces rapid (3 h) and broad transcriptional changes in AML cells.

High-throughput methods for the analysis of transcription factors and chromatin modifications: Low input, single cell and spatial genomic technologies.

Genome-wide analysis of transcription factors and epigenomic features is instrumental to shed light on DNA-templated regulatory processes such as transcription, cellular differentiation or to monitor cellular responses to environmental cues. Two decades of technological developments have led to a rich set of approaches progressively pushing the limits of epigenetic profiling towards single cells. More recently, disruptive technologies using innovative biochemistry came into play.

Erythroid Cell Research: 3D Chromatin, Transcription Factors and Beyond.

Studies of the regulatory networks and signals controlling erythropoiesis have brought important insights in several research fields of biology and have been a rich source of discoveries with far-reaching implications beyond erythroid cells biology. The aim of this review is to highlight key recent discoveries and show how studies of erythroid cells bring forward novel concepts and refine current models related to genome and 3D chromatin organization, signaling and disease, with broad interest in life sciences.