Biology subjects

Brinkman, E. K.

Publications and source records attributed to Brinkman, E. K..

5 recordsLinked to original sources

bioRxiv2023

CRISPR-Cas9-mediated genome engineering exaggerates genomic deletion at 10q23.31 including the PTEN gene locus mimicking cancer profiles

The CRISPR-Cas9 system is a powerful tool for studying gene functions and has tremendous potential for disease treatment. However, precise genome editing requires thorough assessments to minimize unintended on- and off-target effects. Here, we report an unexpected deletion of a 287 kb region on Chromosome 10 (10q23.31) in chronic myelogenous leukemia HAP1 cells, which are frequently used in CRISPR screens. The deleted region encodes regulatory genes, including PAPSS2, ATAD1, KLLN, and PTEN. We found that this deletion was not a direct consequence of CRISPR-Cas9 off-targeting but rather occurred frequently by the process of generating CRISPR-Cas9-modifed cells. The deletion was associated with global changes in histone acetylation and gene expression, affecting fundamental cellular processes such as cell cycle and DNA replication. We detected this deletion in cancer patient genomes. As in HAP1 cells, the deletion contributed to similar gene expression patterns among cancer patients despite interindividual differences. Overall, our findings suggest that the unintended deletion of 10q23.31 can confound CRISPR-Cas9 studies, highlights the importance of assessing unintended genomic changes in CRISPR-Cas9-modified cells and may have clinical significance in cancer research. HighlightsO_LICRISPR-Cas9-modified HAP1 cells carry an unexpected large genomic deletion at 10q23.31 encompassing four protein-coding genes frequently expressed across various cell types. C_LIO_LIThe 10q23.31 deletion is accompanied by global changes in histone modification and transcriptomes. C_LIO_LIThe generation of CRISPR-Cas9-modified cells rather than Cas9 activity increases the frequencies of the deletion at 10q23.31. C_LIO_LIThe 10q23.31 deletion identified in HAP1 cells resembles a commonly occurring deletion pattern in cancer patients. C_LI

cancer biology↗

bioRxiv2020

Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance

DNA double-strand break (DSB) repair is mediated by multiple pathways, including classical non-homologous end-joining pathway (NHEJ) and several homology-driven repair pathways. This is particularly important for Cas9-mediated genome editing, where the outcome critically depends on the pathway that repairs the break. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a newly developed multiplexed reporter assay in combination with Cas9 cutting, we systematically measured the relative activities of three DSB repair pathways as function of chromatin context in >1,000 genomic locations. This revealed that NHEJ is broadly biased towards euchromatin, while microhomology-mediated end-joining (MMEJ) is more efficient in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 shifts the balance towards NHEJ. Single-strand templated repair (SSTR), often used for precise CRISPR editing, competes with MMEJ, and this competition is weakly associated with chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance, and guidance for the design of Cas9-mediated genome editing experiments.

molecular biology↗

bioRxiv2018

TSA-Seq Mapping of Nuclear Genome Organization

While nuclear compartmentalization is an essential feature of three-dimensional genome organization, no genomic method exists for measuring chromosome distances to defined nuclear structures. Here we describe TSA-Seq, a new mapping method able to estimate mean chromosomal distances from nuclear speckles genome-wide and predict several Mbp chromosome trajectories between nuclear compartments without sophisticated computational modeling. Ensemble-averaged results reveal a clear nuclear lamina to speckle axis correlated with a striking spatial gradient in genome activity. This gradient represents a convolution of multiple, spatially separated nuclear domains, including two types of transcription \"hot-zones\". Transcription hot-zones protruding furthest into the nuclear interior and positioning deterministically very close to nuclear speckles have higher numbers of total genes, the most highly expressed genes, house-keeping genes, genes with low transcriptional pausing, and super-enhancers. Our results demonstrate the capability of TSA-Seq for genome-wide mapping of nuclear structure and suggest a new model for nuclear spatial organization of transcription.

genomics↗

bioRxiv2017

Easy quantification of template-directed CRISPR/Cas9 editing

Template-directed CRISPR/Cas9 editing is a powerful tool for introducing subtle mutations in genomes. However, the success rate of incorporation of the desired mutations at the target site is difficult to predict and therefore must be empirically determined. Here, we adapted the widely used TIDE method for quantification of templated editing events, including point mutations. The resulting TIDER method is a rapid, cheap and accessible tool for testing and optimization of template-directed genome editing strategies.

genetics↗

bioRxiv2017

Quantitative Analysis Shows That Repair Of Cas9-Induced Double-Strand DNA Breaks Is Slow And Error-Prone

The RNA-guided DNA endonuclease Cas9 is a powerful tool for genome editing. Little is known about the kinetics and fidelity of the double-strand break (DSB) repair process that follows a Cas9 cutting event in living cells. Here, we developed a strategy to measure the kinetics of DSB repair for single loci in human cells. Quantitative modeling of repaired DNA in time series after Cas9 activation reveals a relatively slow repair rate (~6h). Furthermore, the double strand break is predominantly repaired in an error-prone fashion (at least 70%). Both classical and microhomology-mediated end-joining pathways are active and contribute to the repair in a stochastic manner. However, the balance between these two pathways changes over time and can be altered by chemical inhibition of DNAPKcs or additional ionizing radiation. Our strategy is generally applicable to study DSB repair kinetics and fidelity in single loci, and demonstrates that Cas9-induced DSBs are repaired in an unusual manner.

molecular biology↗