— The CRISPR/Cas system is currently widely used for genome editing. The procedure of genome editing includes two necessary steps: (i) searching for the most effective guide RNA, and (ii)...

Over the years, our vision of the genome has changed from a linear molecule to that of a complex 3D structure that follows specific patterns and possesses a hierarchical organization. Currently, genomics is becoming “four-dimensional”: our attention is increasingly focused on the study of chromatin dynamics over time, in the fourth dimension. Recent methods for visualizing the movements of chromatin loci in living cells by targeting fluorescent proteins can be divided into two groups. The first group requires the insertion of a special sequence into the locus of interest, to which proteins that recognize the sequence are recruited (e.g., FROS and ParB-INT methods). In the methods of the second approach, “programmed” proteins are targeted to the locus of interest (i.e., systems based on CRISPR/Cas, TALE, and zinc finger proteins). In the present review, we discuss these approaches, examine their strengths and weaknesses, and identify the key scientific problems that can be studied using these methods.

Topoisomerase inhibitors are widely used in cancer chemotherapy. However, one of the potential long-term adverse effects of such therapy is acute leukemia. A key feature of such therapy-induced acute myeloid leukemia (t-AML) is recurrent chromosomal translocations involving AML1 (RUNX1) or MLL (KMT2A) genes. The formation of chromosomal translocation depends on the spatial proximity of translocation partners and the mobility of the DNA ends. It is unclear which of these two factors might be decisive for recurrent t-AML translocations. Here, we used fluorescence in situ hybridization (FISH) and chromosome conformation capture followed by sequencing (4C-seq) to investigate double-strand DNA break formation and the mobility of broken ends upon etoposide treatment, as well as contacts between translocation partner genes. We detected the separation of the parts of the broken AML1 gene, as well as the increased mobility of these separated parts. 4C-seq analysis showed no evident contacts of AML1 and MLL with loci, implicated in recurrent t-AML translocations, either before or after etoposide treatment. We suggest that separation of the break ends and their increased non-targeted mobility—but not spatial predisposition of the rearrangement partners—plays a major role in the formation of these translocations.

In recent years, various technologies have emerged for the imaging of chromatin loci in living cells via catalytically inactive Cas9 (dCas9). These technologies facilitate a deeper understanding of the mechanisms behind the chromatin dynamics and provide valuable kinetic data that could not have previously been obtained via FISH applied to fixed cells. However, such technologies are relatively complicated, as they involve the expression of several chimeric proteins as well as sgRNAs targeting the visualized loci, a process that entails many technical subtleties. Therefore, the effectiveness in visualizing a specific target locus may be quite low. In this study, we directly compared two versions of a previously published CRISPR-Sirius method based on the use of sgRNAs containing eight MS2 or PP7 stem loops and the expression of MCP or PCP fused to fluorescent proteins. We assessed the visualization efficiency for several unique genomic loci by comparing the two approaches in delivering sgRNA genes (transient transfection and lentiviral transduction), as well as two CRISPR-Sirius versions (with PCP and with MCP). The efficiency of visualization varied among the loci, and not all loci could be visualized. However, the MCP-sfGFP version provided more efficient visualization in terms of the number of cells with signals than PCP-sfGFP for all tested loci. We also showed that lentiviral transduction was more efficient in locus imaging than transient transfection for both CRISPR-Sirius systems. Most of the target loci in our study were located at the borders of topologically associating domains, and we defined a set of TAD borders that could be effectively visualized using the MCP-sfGFP version of the CRISPR-Sirius system. Altogether, our study validates the use of the CRISPR-Sirius technology for live-cell visualization and highlights various technical details that should be considered when using this method.

Cohesin organizes the genome into spatially segregated loops and topologically associated domains by loop extrusion. In addition, it ensures cohesion of sister chromatids after replication. Thus, cohesin is expected to limit chromatin dynamics by ensuring cohesion and compacting chromatin in the interphase. Nonetheless, loop extrusion is an example of chromatin dynamics; thus, cohesin could promote the dynamics of genomic loci at the scale of individual loops and contact domains. Moreover, given that the extruding activity of cohesin after replication is supplemented by its cohesive activity, the impact of cohesin on chromatin dynamics in different phases of the cell cycle may vary. Of particular interest is the cohesin’s role in the regulation of the dynamics of damaged chromatin, which remains insufficiently studied. Here, we visualized a genomic locus using the CRISPR-Sirius system in human cells with auxin-induced depletion of the cohesin subunit RAD21. Cohesin depletion increased the local spatial dynamics of the visualized locus on a time scale of fractions of a second to one minute. This effect was observed in both replicated and unreplicated chromatin. However, the increase in the mobility of the visualized locus upon cohesin depletion was more pronounced in the former. In addition, we showed that cohesin depletion did not affect the local mobility of double-strand break repair foci visualized using a fluorescent fragment of the repair factor 53BP1. Cohesin depletion did not affect the local mobility of repair foci in either replicated or unreplicated chromatin. The results indicate that cohesin constrains local spatial dynamics of genomic loci. At the same time, cohesive activity of cohesin is not indispensable for restricting chromatin dynamics, although it enhances the confinement effect. On the other hand, repair foci are less mobile structures than point chromatin loci, and cohesin does not affect their dynamics on the studied time scales.

CRISPR/Cas systems (Clustered regularly interspaced palindromic repeats / CRISPR-associated) are rapidly becoming a commonplace and popular tool for gene editing in research and clinical contexts. However, the quality of CRISPR/Cas experiments depends heavily on the guide RNA (gRNA) design; therefore, a reliable, easy, and rapid method for verifying gRNA cleavage efficacy is necessary. Engineered nuclease-induced translocations (ENIT) are an easy and cost-efficient method for the verification of gRNA efficacy, which involves tracking induced chromosomal mutations, using polymerase chain reaction (PCR). We have customized this method using both direct PCR and nested PCR approaches and have been able to reduce the sample preparation time. We present a simple and reliable gRNA testing approach that requires no specific enzymes or equipment.•The approach requires only routinely used enzymes and equipment.•Cost- and time-efficient, requiring approximately 30 min for PCR sample preparation, without requiring DNA purification.•High sensitivity, with induced translocation detected in 100 of 10,000 cells in the general population.