scholarly journals Integrated computational and experimental identification of p53, KRAS and VHL mutant selection associated with CRISPR-Cas9 editing

2018 ◽  
Author(s):  
Sanju Sinha ◽  
Karina Barbosa Guerra ◽  
Kuoyuan Cheng ◽  
Mark DM Leiserson ◽  
David M Wilson ◽  
...  
Keyword(s):  
Gene Editing ◽  
Mutant Cell ◽  
Cell Types ◽  
Driver Mutations ◽  
Mutant Selection ◽  
Cancer Driver ◽  
Genome Wide ◽  
A Genome ◽  
Induced Changes ◽  
Different Cell Types

AbstractRecent studies have reported that CRISPR-Cas9 gene editing induces a p53-dependent DNA damage response in primary cells, which may select for cells with oncogenic p53 mutations11,12. It is unclear whether these CRISPR-induced changes are applicable to different cell types, and whether CRISPR gene editing may select for other oncogenic mutations. Addressing these questions, we analyzed genome-wide CRISPR and RNAi screens to systematically chart the mutation selection potential of CRISPR knockouts across the whole exome. Our analysis suggests that CRISPR gene editing can select for mutants of KRAS and VHL, at a level comparable to that reported for p53. These predictions were further validated in a genome-wide manner by analyzing independent CRISPR screens and patients’ tumor data. Finally, we performed a new set of pooled and arrayed CRISPR screens to evaluate the competition between CRISPR-edited isogenic p53 WT and mutant cell lines, which further validated our predictions. In summary, our study systematically charts and points to the potential selection of specific cancer driver mutations during CRISPR-Cas9 gene editing.

scholarly journals A systematic genome-wide mapping of oncogenic mutation selection during CRISPR-Cas9 genome editing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sanju Sinha ◽  
Karina Barbosa ◽  
Kuoyuan Cheng ◽  
Mark D. M. Leiserson ◽  
Prashant Jain ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cell Types ◽  
Selective Advantage ◽  
Driver Mutations ◽  
Cancer Driver ◽  
Oncogenic Mutation ◽  
Genome Wide ◽  
Selection For ◽  
Mutant Cells ◽  
Selection Of

AbstractRecent studies have reported that genome editing by CRISPR–Cas9 induces a DNA damage response mediated by p53 in primary cells hampering their growth. This could lead to a selection of cells with pre-existing p53 mutations. In this study, employing an integrated computational and experimental framework, we systematically investigated the possibility of selection of additional cancer driver mutations during CRISPR-Cas9 gene editing. We first confirm the previous findings of the selection for pre-existing p53 mutations by CRISPR-Cas9. We next demonstrate that similar to p53, wildtype KRAS may also hamper the growth of Cas9-edited cells, potentially conferring a selective advantage to pre-existing KRAS-mutant cells. These selective effects are widespread, extending across cell-types and methods of CRISPR-Cas9 delivery and the strength of selection depends on the sgRNA sequence and the gene being edited. The selection for pre-existing p53 or KRAS mutations may confound CRISPR-Cas9 screens in cancer cells and more importantly, calls for monitoring patients undergoing CRISPR-Cas9-based editing for clinical therapeutics for pre-existing p53 and KRAS mutations.

scholarly journals DeeReCT-TSS: A novel meta-learning-based method annotates TSS in multiple cell types based on DNA sequences and RNA-seq data

2021 ◽  
Author(s):  
Juexiao Zhou ◽  
Bin Zhang ◽  
Haoyang Li ◽  
Longxi Zhou ◽  
Zhongxiao Li ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Cell Types ◽  
Rna Seq ◽  
Sources Of Information ◽  
Genome Wide ◽  
A Genome ◽  
Meta Learning ◽  
Multiple Cell ◽  
Different Cell Types ◽  
Genome Scale

The accurate annotation of TSSs and their usage is critical for the mechanistic understanding of gene regulation under different biological contexts. To fulfill this, specific high-throughput experimental technologies have been developed to capture TSSs in a genome-wide manner. Various computational tools have also been developed for in silico prediction of TSSs solely based on genomic sequences. Most of these tools have drastic false positive predictions when applied on the genome-scale. Here, we present DeeReCT-TSS, a deep-learning-based method that is capable of TSSs identification across the whole genome based on DNA sequences and conventional RNA-seq data. We show that by effectively incorporating these two sources of information, DeeReCT-TSS significantly outperforms other solely sequence-based methods on the precise annotation of TSSs used in different cell types. Furthermore, we develop a meta-learning-based extension for simultaneous transcription start site (TSS) annotation on 10 cell types, which enables the identification of cell-type-specific TSS. Finally, we demonstrate the high precision of DeeReCT-TSS on two independent datasets from the ENCODE project by correlating our predicted TSSs with experimentally defined TSS chromatin states.

scholarly journals A systematic genome-wide mapping of oncogenic mutation selection during CRISPR-Cas9 genome editing

2021 ◽  
Author(s):  
Sanju Sinha ◽  
Karina Guerra ◽  
Kuoyuan Cheng ◽  
Mark Leiserson ◽  
Prashant Jain ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cell Types ◽  
Selective Advantage ◽  
Driver Mutations ◽  
Cancer Driver ◽  
Oncogenic Mutation ◽  
Genome Wide ◽  
Selection For ◽  
Mutant Cells ◽  
Selection Of

Abstract Recent studies have reported that genome editing by CRISPR–Cas9 induces a DNA damage response mediated by p53 in primary cells hampering their growth. This could lead to a selection of cells with pre-existing p53 mutations. In this study, employing an integrated computational and experimental framework, we systematically investigated the possibility of selection of additional cancer driver mutations during CRISPR-Cas9 gene editing. We first confirm the previous findings of the selection for pre-existing p53 mutations by CRISPR-Cas9. We next demonstrate that similar to p53, wildtype KRAS may also hamper the growth of Cas9-edited cells, potentially conferring a selective advantage to pre-existing KRAS-mutant cells. These selective effects are widespread, extending across cell-types and methods of CRISPR-Cas9 delivery and the strength of selection depends on the sgRNA sequence and the gene being edited. The selection for pre-existing p53 or KRAS mutations may confound CRISPR-Cas9 screens in cancer cells and more importantly, calls for monitoring patients undergoing CRISPR-Cas9-based editing for clinical therapeutics for pre-existing p53 and KRAS mutations.

scholarly journals Delivering genes across the blood-brain barrier: LY6A, a novel cellular receptor for AAV-PHP.B capsids

2019 ◽  
Author(s):  
Qin Huang ◽  
Ken Y. Chan ◽  
Isabelle G. Tobey ◽  
Yujia Alina Chan ◽  
Tim Poterba ◽  
...  
Keyword(s):  
Genome Wide Association Study ◽  
Neurological Diseases ◽  
Cell Types ◽  
Mouse Strains ◽  
Adeno Associated Virus ◽  
Genome Wide ◽  
A Genome ◽  
Gene Therapy Vectors ◽  
Different Cell Types ◽  
Mouse Central Nervous System

The engineered AAV-PHP.B family of adeno-associated virus efficiently delivers genes throughout the mouse central nervous system. To guide their application across disease models, and to inspire the development of translational gene therapy vectors useful for targeting neurological diseases in humans, we sought to elucidate the host factors responsible for the CNS tropism of AAV-PHP.B vectors. Leveraging CNS tropism differences across mouse strains, we conducted a genome-wide association study, and rapidly identified and verified LY6A as an essential receptor for the AAV-PHP.B vectors in brain endothelial cells. Importantly, this newly discovered mode of AAV binding and transduction is independent of other known AAV receptors and can be imported into different cell types to confer enhanced transduction by the AAV-PHP.B vectors.

scholarly journals Development of a CRISPR-SaCas9 system for projection- and function-specific gene editing in the rat brain

2020 ◽  
Vol 6 (12) ◽  
pp. eaay6687 ◽  
Author(s):  
Haojie Sun ◽  
Su Fu ◽  
Shuang Cui ◽  
Xiangsha Yin ◽  
Xiaoyan Sun ◽  
...  
Keyword(s):  
Rat Brain ◽  
Gene Editing ◽  
High Efficiency ◽  
Protein A ◽  
Cell Types ◽  
Cell Labeling ◽  
Specific Gene ◽  
Creb Binding Protein ◽  
A Genome ◽  
And Function

A genome editing technique based on the clustered regularly interspaced short palindromic repeats (CRISPR)–associated endonuclease Cas9 enables efficient modification of genes in various cell types, including neurons. However, neuronal ensembles even in the same brain region are not anatomically or functionally uniform but divide into distinct subpopulations. Such heterogeneity requires gene editing in specific neuronal populations. We developed a CRISPR-SaCas9 system–based technique, and its combined application with anterograde/retrograde AAV vectors and activity-dependent cell-labeling techniques achieved projection- and function-specific gene editing in the rat brain. As a proof-of-principle application, we knocked down the cbp (CREB-binding protein), a sample target gene, in specific neuronal subpopulations in the medial prefrontal cortex, and demonstrated the significance of the projection- and function-specific CRISPR-SaCas9 system in revealing neuronal and circuit basis of memory. The high efficiency and specificity of our projection- and function-specific CRISPR-SaCas9 system could be widely applied in neural circuitry studies.

scholarly journals PDTM-26. NOVEL SHARED AND DISTINCT EPIGENOMIC MECHANISMS OF G34R AND K27M MUTATIONS IN CHILDHOOD GLIOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi192-vi193
Author(s):  
Michael Chen ◽  
Kelly Bush ◽  
Nichole Lewis ◽  
Vanessa Cervantes ◽  
Paul Knoepfler
Keyword(s):  
Gene Editing ◽  
Gene Knockout ◽  
Point Mutations ◽  
Glioma Cells ◽  
Driver Mutations ◽  
High Grade ◽  
Shared Key ◽  
Induced Changes ◽  
Open Question

Abstract Alterations in histone H3.3 are common driver mutations in high-grade pediatric gliomas, but the central oncogenic mechanisms remain an open question. To identify important mutant H3.3 effectors, we used CRISPR-Cas9 to precisely introduce H3.3 K27M and G34R mutations into previously H3.3-wildtype human astrocyte and glioma cells, while in parallel reverting mutations in glioma cells back to wildtype. K27M and G34R mutations invoked some strikingly similar epigenomic effects supporting a new model in which some major aspects of their oncogenic functions are shared. For instance, both K27M and G34R induced changes at many of the same genomic loci in specific histone marks, with the largest changes in H3K27me3 including in particular within super-enhancers, which also exhibited perturbed transcriptional function. K27M and G34R mutations induced some gene expression changes that were unique to each mutation, but both mutations changed similar functional ontological clusters and ASCL1 is a shared key putative effector. H3.3 mutant glioma cells are sensitive to ASCL1 knockdown or overexpression, resulting in cell viability that is reduced or increased, respectively. Comparison of our panel of glioma cells gene-edited with precise point mutations to edited glioma cells in other studies that performed gene knockout or overexpression reveals striking differences in the resulting phenotypes. We also determined that certain drugs exhibited specificity to H3.3 mutation-bearing cells including DAPT, JQ1, and ONC201. In vivo, we found that reversion of K27M to WT in glioma cells significantly reduced tumorigenicity in mouse xenograft assays and introduction of G34R mutations in previously WT glioma cells increased tumor growth. Overall, gene editing of gliomas and comparison of otherwise isogenic sets of cells defines both distinct and shared gliomagenesis mechanisms that can be targeted for development of oncohistone-based therapeutics.

scholarly journals The Antisense Transcriptomes of Human Cells

Science ◽  
2008 ◽  
Vol 322 (5909) ◽  
pp. 1855-1857 ◽  
Author(s):  
Yiping He ◽  
Bert Vogelstein ◽  
Victor E. Velculescu ◽  
Nickolas Papadopoulos ◽  
Kenneth W. Kinzler
Keyword(s):  
Mammalian Cells ◽  
Cell Types ◽  
Human Cells ◽  
Antisense Transcripts ◽  
Genome Wide ◽  
Human Genes ◽  
A Genome ◽  
Dna Strand ◽  
The Relationship ◽  
Rna Transcript

Transcription in mammalian cells can be assessed at a genome-wide level, but it has been difficult to reliably determine whether individual transcripts are derived from the plus or minus strands of chromosomes. This distinction can be critical for understanding the relationship between known transcripts (sense) and the complementary antisense transcripts that may regulate them. Here, we describe a technique that can be used to (i) identify the DNA strand of origin for any particular RNA transcript, and (ii) quantify the number of sense and antisense transcripts from expressed genes at a global level. We examined five different human cell types and in each case found evidence for antisense transcripts in 2900 to 6400 human genes. The distribution of antisense transcripts was distinct from that of sense transcripts, was nonrandom across the genome, and differed among cell types. Antisense transcripts thus appear to be a pervasive feature of human cells, which suggests that they are a fundamental component of gene regulation.

scholarly journals Replication timing maintains the global epigenetic state in human cells

2019 ◽  
Author(s):  
Kyle N. Klein ◽  
Peiyao A. Zhao ◽  
Xiaowen Lyu ◽  
Daniel A. Bartlett ◽  
Amar Singh ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Cancer Cell Line ◽  
Replication Timing ◽  
Cell Types ◽  
Control Factor ◽  
Epigenetic State ◽  
Genome Wide ◽  
A Genome ◽  
A Cell ◽  
Early Replication

AbstractDNA is replicated in a defined temporal order termed the replication timing (RT) program. RT is spatially segregated in the nucleus with early/late replication corresponding to Hi-C A/B chromatin compartments, respectively. Early replication is also associated with active histone modifications and transcriptional permissiveness. However, the mechanistic interplay between RT, chromatin state, and genome compartmentalization is largely unknown. Here we report that RT is central to epigenome maintenance and compartmentalization in both human embryonic stem cells (hESCs) and cancer cell line HCT116. Knockout (KO) of the conserved RT control factor RIF1, rather than causing discrete RT switches as previously suspected, lead to dramatically increased cell to cell heterogeneity of RT genome wide, despite RIF1’s enrichment in late replicating chromatin. RIF1 KO hESCs have a nearly random RT program, unlike all prior RIF1 KO cells, including HCT116, which show localized alterations. Regions that retain RT, which are prevalent in HCT116 but rare in hESCs, consist of large H3K9me3 domains revealing two independent mechanisms of RT regulation that are used to different extents in different cell types. RIF1 KO results in a striking genome wide downregulation of H3K27ac peaks and enrichment of H3K9me3 at large domains that remain late replicating, while H3K27me3 and H3K4me3 are re-distributed genome wide in a cell type specific manner. These histone modification changes coincided with global reorganization of genome compartments, transcription changes and a genome wide strengthening of TAD structures. Inducible degradation of RIF1 revealed that disruption of RT is upstream of genome compartmentalization changes. Our findings demonstrate that disruption of RT leads to widespread epigenetic mis-regulation, supporting previously speculative models in which the timing of chromatin assembly at the replication fork plays a key role in maintaining the global epigenetic state, which in turn drives genome architecture.

scholarly journals The epigenetic landscape in purified myonuclei from fast and slow muscles

2021 ◽  
Author(s):  
Mads Bengtsen ◽  
Ivan Myhre Winje ◽  
Einar Eftestøl ◽  
Johannes Landskron ◽  
Chengyi Sun ◽  
...  
Keyword(s):  
Binding Sites ◽  
Association Studies ◽  
Cell Types ◽  
Specific Marker ◽  
Fiber Types ◽  
Epigenetic Landscape ◽  
Super Enhancer ◽  
Genome Wide ◽  
A Genome ◽  
Pericentriolar Material

AbstractMuscle cells have different phenotypes adapted to different usage and can be grossly divided into fast/glycolytic and slow/oxidative types. While most muscles contain a mixture of such fiber types, we aimed at providing a genome-wide analysis of chromatin environment by ChIP-Seq in two muscle extremes, the almost completely fast/glycolytic extensor digitorum longus (EDL) and slow/oxidative soleus muscles. Muscle is a heterogeneous tissue where less than 60% of the nuclei are inside muscle fibers. Since cellular homogeneity is critical in epigenome-wide association studies we devised a new method for purifying skeletal muscle nuclei from whole tissue based on the nuclear envelope protein Pericentriolar material 1 (PCM1) being a specific marker for myonuclei. Using antibody labeling and a magnetic-assisted sorting approach we were able to sort out myonuclei with 95% purity. The sorting eliminated influence from other cell types in the tissue and improved the myo-specific signal. A genome-wide comparison of the epigenetic landscape in EDL and soleus reflected the functional properties of the two muscles each with a distinct regulatory program involving distal enhancers, including a glycolytic super-enhancer in the EDL. The two muscles are also regulated by different sets of transcription factors; e.g. in soleus binding sites for MEF2C, NFATC2 and PPARA were enriched, while in EDL MYOD1 and SOX1 binding sites were found to be overrepresented. In addition, novel factors for muscle regulation such as MAF, ZFX and ZBTB14 were identified.

scholarly journals Integrative analysis of epigenetics data identifies gene-specific regulatory elements

2019 ◽  
Author(s):  
Florian Schmidt ◽  
Alexander Marx ◽  
Marie Hebel ◽  
Martin Wegner ◽  
Nina Baumgarten ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Cell Types ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Genome Wide ◽  
A Genome ◽  
Gene Level ◽  
Regulatory Landscape ◽  
Regulatory Sites ◽  
Validation Experiments

AbstractUnderstanding the complexity of transcriptional regulation is a major goal of computational biology. Because experimental linkage of regulatory sites to genes is challenging, computational methods considering epigenomics data have been proposed to create tissue-specific regulatory maps. However, we showed that these approaches are not well suited to account for the variations of the regulatory landscape between cell-types. To overcome these drawbacks, we developed a new method called STITCHIT, that identifies and links putative regulatory sites to genes. Within STITCHIT, we consider the chromatin accessibility signal of all samples jointly to identify regions exhibiting a signal variation related to the expression of a distinct gene. STITCHIToutperforms previous approaches in various validation experiments and was used with a genome-wide CRISPR-Cas9 screen to prioritize novel doxorubicin-resistance genes and their associated non-coding regulatory regions. We believe that our work paves the way for a more refined understanding of transcriptional regulation at the gene-level.

Sign in / Sign up

Export Citation Format

Share Document