A CRISPR-based approach for proteomic analysis of a single genomic locus (original) (raw)
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Role of the CRISPR system in controlling gene transcription and monitoring cell fate (Review)
Molecular Medicine Reports
Even though the accrual of transcripts is implicated in distinct disease states, our knowledge regarding their functional role remains obscure. The CRISPR system has surged at the forefront of genome engineering tools in the field of RNA modulation. In the present review, we discuss some exciting applications of the CRISPR system, including the manipulation of RNA sequences, the visualization of chromosomal loci in living cells and the modulation of transcription. The CRISPR system has been documented to be very reliable and specific in altering gene expression, via leveraging inactive catalytically dead CRISPR-associated protein 9 (Cas9). In the present review, the CRISPR system is presented as an eminent tool for the meticulous analysis of gene regulation, loci mapping and complex pathways.
The influence of eukaryotic chromatin state on CRISPR–Cas9 editing efficiencies
Current Opinion in Biotechnology
CRISPR/Cas technologies have rapidly become routine in many laboratories. Despite this, the efficiency of CRISPR/Cas9 functioning cannot entirely be predicted, and it is not fully understood which factors contribute to this variability. Recent studies indicate that heterochromatin can negatively affect Cas9 binding and functioning. Investigating chromatin factors indicates that 5-cytosine methylation does not directly block Cas9 binding. Nucleosomes, however, can completely block Cas9 access to DNA in cell-free assays and present a substantial hurdle in vivo. In addition to being associated with an open chromatin state, active transcription can directly stimulate DNA cleavage by influencing Cas9 release rates in a strand-specific manner. With these insights and a better understanding of genome-wide chromatin and transcription states, CRISPR/Cas9 effectiveness and reliability can be improved.
Generation of CRISPR-cas9 construct for knockout of genes encoding chromatin-associated proteins
Bulletin of the National Research Centre, 2019
Background: Eukaryotic genomes have a multiscale three-dimensional organization varying from nucleosomes, loops, topologically associating domains, and chromosome territories. Chromatin, DNA wrapped around histone proteins, helps in packaging long DNA within tiny nuclear spaces. We used CRISPR-cas9, which is a system of single-protein and single-guide RNAs for genome engineering and also is simple and target specific. Method: Two major protein families involved in maintaining and regulating structure and dynamics of chromatin are trithorax group (TrxG) and polycomb group (PcG) proteins. This study was undertaken to generate knockout cell lines of some TrxG and PcG proteins using the CRISPR-based approach in order to study their role in higher order chromatin organization. Results: From TrxG, ISWI and Acf were selected, and from PcG, Pc and Psc were selected. Three pAc-sgRNA-Cas9puro-vector constructs for ISWI gene, one pAc-sgRNA-cas9-puro-vector construct for Pc, gene and two pAc-sgRNA-cas9-puro-vector constructs for each of the Acf and Psc gene were generated. These constructs were confirmed by PCR and sequencing. Conclusion: In the future, these constructs will be used to study the role of their respective target genes in chromatin organization.
CRISPR-Mediated Epigenome Editing
The Yale journal of biology and medicine, 2016
Mounting evidence has called into question our understanding of the role that the central dogma of molecular biology plays in human pathology. The conventional view that elucidating the mechanisms for translating genes into proteins can account for a panoply of diseases has proven incomplete. Landmark studies point to epigenetics as a missing piece of the puzzle. However, technological limitations have hindered the study of specific roles for histone post-translational modifications, DNA modifications, and non-coding RNAs in regulation of the epigenome and chromatin structure. This feature highlights CRISPR systems, including CRISPR-Cas9, as novel tools for targeted epigenome editing. It summarizes recent developments in the field, including integration of optogenetic and functional genomic approaches to explore new therapeutic opportunities, and underscores the importance of mitigating current limitations in the field. This comprehensive, analytical assessment identifies current re...
Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex
Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene signature based on the top screening hits correlated with a gene expression signature of BRAF inhibitor resistance in cell lines and patientderived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.
bioRxiv (Cold Spring Harbor Laboratory), 2022
CRISPR-Cas9-mediated genome editing has been widely adopted for basic and applied biological research in eukaryotic systems. While many studies consider DNA sequences of CRISPR target sites as the primary determinant for CRISPR mutagenesis efficiency and mutation profiles, increasing evidence reveals the substantial role of chromatin context. Nonetheless, most prior studies are limited by the lack of sufficient epigenetic resources and/or by only transiently expressing CRISPR-Cas9 in a short time window. In this study, we leveraged the wealth of high-resolution epigenomic resources in Arabidopsis (Arabidopsis thaliana) to address the impact of chromatin features on CRISPR-Cas9 mutagenesis using stable transgenic plants. Our results indicated that DNA methylation and chromatin features could lead to substantial variations in mutagenesis efficiency by up to 250-fold. Low mutagenesis efficiencies were mostly associated with repressive heterochromatic features. This repressive effect appeared to persist through cell divisions but could be alleviated through substantial reduction of DNA methylation at CRISPR target sites. Moreover, specific chromatin features, such as H3K4me1, H3.3, and H3.1, appear to be associated with significant variation in CRISPR-Cas9 mutation profiles mediated by the non-homologous end joining repair pathway. Our findings provide strong evidence that specific chromatin features could have substantial and lasting impacts on both CRISPR-Cas9 mutagenesis efficiency and DNA double-strand break repair outcomes. in eukaryotes (Jinek et al., 2012). The key steps in CRISPR-Cas9-mediated genome editing involve searching, binding and then cleaving a 20-nucleotide target site directed by a guide RNA (gRNA). The resulting cleavage product with double-strand breaks (DSBs) can then be repaired by either
The role of CRISPR/Cas9 technology in genomics edit
International Journal of Advanced Academic Studies
Many genoms editing tools have been used successfully to revise and control the genomes of various animals, but endonucleases under the guidance of RNA are known by the title of CRISPR / CAS9; and are naturally changing the genomic engineering domain. Since the introduction of CRISP / Cas9 is much simpler than ZFN or TALEN, which is originated from the simple construction of CRISPR / Cas9 vectors that target a specific place in the host genome. This technology has been changed importantly, quickly and only within a few years as a standard tool for targeted editing the Gene. In this article, I will discuss about that how this technology is formed and how it will be developed in the coming years. Editing genemic strategies by CRISPR / Cas9 will accelerate functional genomic studies in the coming years. CRISPR / Cas9 will not only change genetic engineering, but also various fields of research in biological studies.
Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression
Cell, 2013
Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale.
Interrogation of Enhancer Function by Enhanced CRISPR Epigenetic Editing
2019
ABSTRACTTissue-specific gene expression requires coordinated control of gene-proximal and -distalcis-regulatory elements (CREs), yet functional analysis of gene-distal CREs such as enhancers remains challenging. Here we describe enhanced CRISPR/dCas9-based epigenetic editing systems, enCRISPRa and enCRISPRi, for multiplexed analysis of enhancer functionin situandin vivo. Using dual effectors capable of re-writing enhancer-associated chromatin modifications, we show that enCRISPRa and enCRISPRi modulate gene transcription by remodeling local epigenetic landscapes at sgRNA-targeted enhancers and associated genes. Comparing with existing methods, the new systems display more robust perturbation of enhancer activity and gene transcription with minimal off-targets. Allele-specific targeting of enCRISPRa to oncogenicTAL1super-enhancer modulatesTAL1expression and cancer progression in xenotransplants. Multiplexed perturbations of lineage-specific enhancers using an enCRISPRi knock-in mouse...