Raw Data for: Rapid generation of homozygous fluorescent knock-in human cells using CRISPR/Cas9 genome editing and validation by automated imaging and digital PCR screening. Authors: Andrea Callegari, Moritz Kueblbeck, Beatriz Serrano-Solano, Natalia Rosalia Morero and Jan Ellenberg.

Raw Data for: Rapid generation of homozygous fluorescent knock-in human cells using CRISPR/Cas9 genome editing and validation by automated imaging and digital PCR screening. Authors: Andrea Callegari, Moritz Kueblbeck, Beatriz Serrano-Solano, Natalia Rosalia Morero and Jan Ellenberg.</p

EGF-induced centrosome separation promotes mitotic progression and cell survival

Timely and accurate assembly of the mitotic spindle is critical for the faithful segregation of chromosomes, and centrosome separation is a key step in this process. The timing of centrosome separation varies dramatically between cell types; however, the mechanisms responsible for these differences and its significance are unclear. Here, we show that activation of epidermal growth factor receptor (EGFR) signaling determines the timing of centrosome separation. Premature separation of centrosomes decreases the requirement for the major mitotic kinesin Eg5 for spindle assembly, accelerates mitosis, and decreases the rate of chromosome missegregation. Importantly, EGF stimulation impacts upon centrosome separation and mitotic progression to different degrees in different cell lines. Cells with high EGFR levels fail to arrest in mitosis upon Eg5 inhibition. This has important implications for cancer therapy because cells with high centrosomal response to EGF are more susceptible to combinatorial inhibition of EGFR and Eg5

ChromoTrace: Computational reconstruction of 3D chromosome configurations for super-resolution microscopy

<div><p>The 3D structure of chromatin plays a key role in genome function, including gene expression, DNA replication, chromosome segregation, and DNA repair. Furthermore the location of genomic loci within the nucleus, especially relative to each other and nuclear structures such as the nuclear envelope and nuclear bodies strongly correlates with aspects of function such as gene expression. Therefore, determining the 3D position of the 6 billion DNA base pairs in each of the 23 chromosomes inside the nucleus of a human cell is a central challenge of biology. Recent advances of super-resolution microscopy in principle enable the mapping of specific molecular features with nanometer precision inside cells. Combined with highly specific, sensitive and multiplexed fluorescence labeling of DNA sequences this opens up the possibility of mapping the 3D path of the genome sequence in situ. Here we develop computational methodologies to reconstruct the sequence configuration of all human chromosomes in the nucleus from a super-resolution image of a set of fluorescent in situ probes hybridized to the genome in a cell. To test our approach, we develop a method for the simulation of DNA in an idealized human nucleus. Our reconstruction method, ChromoTrace, uses suffix trees to assign a known linear ordering of in situ probes on the genome to an unknown set of 3D in-situ probe positions in the nucleus from super-resolved images using the known genomic probe spacing as a set of physical distance constraints between probes. We find that ChromoTrace can assign the 3D positions of the majority of loci with high accuracy and reasonable sensitivity to specific genome sequences. By simulating appropriate spatial resolution, label multiplexing and noise scenarios we assess our algorithms performance. Our study shows that it is feasible to achieve genome-wide reconstruction of the 3D DNA path based on super-resolution microscopy images.</p></div

Distribution of path lengths.

<p>The relationship between the number of colors and the length of the paths found by ChromoTrace is shown in this plot. A violin plot is shown for each number of colors and the relation to the logarithm (log base 10) of the path length. More colors lead to longer paths and after 10 colors the path length does not increase as recall becomes greater than 0.99. Within the violin plot the first and third quartiles are shown.</p

Segmented simulated LE profiles.

<p>(A) The reconstruction performance, recall versus precision when running ChromoTrace for whole genome and individual chromosomes. (B) The percent of missing probes across all 100 simulations for all of the polymer chains and a single chain. (C) The percent of LE’s that were clustered into the wrong locus for the whole genome and chromosome 20. (D) the percent of clusters that contained LE’s from multiple starting loci.</p

Reconstruction performance for the main simulations.

<p>The reconstruction algorithms performance is shown in terms of the relationship between precision and recall given the number of colors in the probe design. (A) Recall against precision genome wide (triangles) and for chromosome 20 (circles). Precision is good for both genome and chromosome scale regions for all the different probe designs whereas recall is much more dependent on the number of available colors and improves as the number of colors is increased. (B) Total number of contacts in 100 kb windows against the area under the precision-recall curve given the number of colors in the probe design.</p

Suffix tree of the sequence <i>BGRY</i> <i>BGRY</i> <i>BGRY</i>.

<p>Every subsequence of the sequence is spelled out on edges from the root, at the top of the tree, to a leaf node, at the bottom of the tree.</p

Differences in simulation packing densities.

<p>Reconstruction performance when decreasing the packing density of the simulations. (A-B) For all positions across the simulations, the proportion of directly adjacent spaces that are occupied for the new (blue) and original (red) simulations respectively. The distribution is left shifted for the new simulations compared to the original and the median number of occupied spaces is reduced reflecting a decrease in density. (C) Genome wide performance of the reconstruction algorithm for the new (triangles) and original (circles) simulations in terms of precision and recall given the number of colors in the probe design.</p

Robustness to missing and mislabeled probes.

<p>Relationship between amount of error for two different modes (missing and mislabeled probes) and the overall reconstruction performance given the number of colors in the probe design is displayed in panels A through D. The number of colors in the probe design is indicated using different shades of black-blue. Panels A and C show the proportion of error against precision for mislabeled and missing probe errors respectively and panels B and D show the proportion of error against recall.</p

Illustration of the ChromoTrace algorithm.

<p>(A) The 3D coordinates that would be obtained from super-resolution microscope imaging are converted into an distance graph. Given the pre-specified linear labeling sequence of green-red-blue-blue-green a trivial path is detected. Note that in the microscopy image the connection between points is unknown and only the colors remain. (B) Diagram of the extension algorithm exploring the ambiguous extension phase.</p