Understanding the impact of guide RNA (gRNA) and genomic locus on CRISPR-Cas9 activity is crucial to design effective gene editing assays. However, it is challenging to profile Cas9 activity in the endogenous cellular environment. Here we leverage our TRIP technology to integrate ~ 1k barcoded reporter genes in the genomes of mouse embryonic stem cells. We target the integrated reporters (IRs) using RNA-guided Cas9 and characterize induced mutations by sequencing. We report that gRNA-sequence and IR locus explain most variation in mutation efficiency. Predominant insertions of a gRNA-specific nucleotide are consistent with template-dependent repair of staggered DNA ends with 1-bp 5′ overhangs. We confirm that such staggered ends are induced by Cas9 in mouse pre-B cells. To explain observed insertions, we propose a model generating primarily blunt and occasionally staggered DNA ends. Mutation patterns indicate that gRNA-sequence controls the fraction of staggered ends, which could be used to optimize Cas9-based insertion efficiency.

The spatio-temporal regulation of gene expression determines the fate and function of various cells and tissues and, as a consequence, the correct development and functioning of complex organisms. Certain mechanisms of gene activity regulation provide adequate cell responses to changes in environmental factors. Aside from gene expression disorders that lead to various pathologies, alterations of expression of particular genes were shown to significantly decrease or increase the lifespan in a wide range of organisms from yeast to human. Drosophila fruit fly is an ideal model system to explore mechanisms of longevity and aging due to low cost, easy handling and maintenance, large number of progeny per adult, short life cycle and lifespan, relatively low number of paralogous genes, high evolutionary conservation of epigenetic mechanisms and signalling pathways, and availability of a wide range of tools to modulate gene expression in vivo. Here, we focus on the organization of the evolutionarily conserved signaling pathways whose components significantly influence the aging process and on the interconnections of these pathways with gene expression regulation.

Drosophila melanogaster Suppressor of Under-Replication (SuUR) gene encodes a protein that modulates replicative properties of heterochromatin in endocycles of polytene cells. The SuUR mutation abolishes underreplication of intercalary heterochromatin and results in partial underreplication of pericentric heterochromatin. We performed a genome-wide mapping of SUUR target genes in non-polytenic Drosophila Kc cells by using the DamID approach. We show that SUUR preferentially binds genes that are transcriptionally silent and late-replicated. Distinct subsets of SUUR targets are associated with PcG proteins (Pc and Esc; Polycomb and Extra sexcombs), heterochromatic proteins [HP1 and SU(VAR)3-9] and B-type lamin. The SUUR binding profile negatively correlates with the DNA polytenization levels of salivary gland polytene chromosomes. Finally, SUUR target genes are repressed in Drosophila embryos and gradually activated later in development. Together these results suggest that SUUR is a ubiquitous marker of heterochromatin in different cell types.

Dam identification (DamID) is a powerful technique to generate genome-wide maps of chromatin protein binding. Due to its high sensitivity, it is particularly suited to study the genome interactions of chromatin proteins in small tissue samples in model organisms such as Drosophila. Here, we report an intein-based approach to tune the expression level of Dam and Dam-fusion proteins in Drosophila by addition of a ligand to fly food. This helps to suppress possible toxic effects of Dam. In addition, we describe a strategy for genetically controlled expression of Dam in a specific cell type in complex tissues. We demonstrate the utility of the latter by generating a glia-specific map of Polycomb in small samples of brain tissue. These new DamID tools will be valuable for the mapping of binding patterns of chromatin proteins in Drosophila tissues and especially in cell lineages.