CRISPR-Cas9 screening develops an epigenetic and transcriptional gene signature for risk stratification and target prediction in neuroblastoma
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ABSTRACT:
SUBMITTER: Zhang L
PROVIDER: S-EPMC11338898 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen, we further describe strategies for confirming the screening phenotype, as well as genetic perturbation, through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9-15 weeks, followed by 4-5 weeks of validation.
| S-EPMC5526071 | biostudies-literature
Project description:Current genome-editing systems generally rely on inducing DNA double-strand breaks (DSBs). This may limit their utility in clinical therapies, as unwanted mutations caused by DSBs can have deleterious effects. CRISPR/Cas9 system has recently been repurposed to enable target gene activation, allowing regulation of endogenous gene expression without creating DSBs. However, in vivo implementation of this gain-of-function system has proven difficult. Here, we report a robust system for in vivo activation of endogenous target genes through trans-epigenetic remodeling. The system relies on recruitment of Cas9 and transcriptional activation complexes to target loci by modified single guide RNAs. As proof-of-concept, we used this technology to treat mouse models of diabetes, muscular dystrophy, and acute kidney disease. Results demonstrate that CRISPR/Cas9-mediated target gene activation can be achieved in vivo, leading to measurable phenotypes and amelioration of disease symptoms. This establishes new avenues for developing targeted epigenetic therapies against human diseases. VIDEO ABSTRACT.
| S-EPMC5732045 | biostudies-literature
Project description:BackgroundA common issue in CRISPR-Cas9 genome editing is off-target activity, which prevents the widespread use of CRISPR-Cas9 in medical applications. Among other factors, primary chromatin structure and epigenetics may influence off-target activity.MethodsIn this work, we utilize crisprSQL, an off-target database, to analyze the effect of 19 epigenetic descriptors on CRISPR-Cas9 off-target activity. Termed as 19 epigenetic features/scores, they consist of 6 experimental epigenetic and 13 computed nucleosome organization-related features. In terms of novel features, 15 of the epigenetic scores are newly considered. The 15 newly considered scores consist of 13 freshly computed nucleosome occupancy/positioning scores and 2 experimental features (MNase and DRIP). The other 4 existing scores are experimental features (CTCF, DNase I, H3K4me3, RRBS) commonly used in deep learning models for off-target activity prediction. For data curation, MNase was aggregated from existing experimental nucleosome occupancy data. Based on the sequence context information available in crisprSQL, we also computed nucleosome occupancy/positioning scores for off-target sites.ResultsTo investigate the relationship between the 19 epigenetic features and off-target activity, we first conducted Spearman and Pearson correlation analysis. Such analysis shows that some computed scores derived from training-based models and training-free algorithms outperform all experimental epigenetic features. Next, we evaluated the contribution of all epigenetic features in two successful machine/deep learning models which predict off-target activity. We found that some computed scores, unlike all 6 experimental features, significantly contribute to the predictions of both models. As a practical research contribution, we make the off-target dataset containing all 19 epigenetic features available to the research community.ConclusionsOur comprehensive computational analysis helps the CRISPR-Cas9 community better understand the relationship between epigenetic features and CRISPR-Cas9 off-target activity.
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Project description:DNA methylation is an epigenetic process that controls DNA accessibility and serves as a transcriptomic switch when deposited at regulatory regions. The adequate functioning of this process is indispensable for tissue homeostasis and cell fate determination. Conversely, altered DNA methylation patterns result in abnormal gene transcription profiles that contribute to tumor initiation and progression. However, whether the consequence of DNA methylation on gene expression and cell fate is uniform regardless of the cell type or state could so far not been tested due to the lack of technologies to target DNA methylation in-situ. Here, we have taken advantage of CRISPR/dCas9 technology adapted for epigenetic editing through site-specific targeting of DNA methylation to characterize the transcriptional changes of the candidate gene and the functional effects on cell fate in different tumor settings. As a proof-of-concept, we were able to induce de-novo site-specific methylation of the gene promoter of IGFBP2 up to 90% with long-term and bona-fide inheritance by daughter cells. Strikingly, this modification led to opposing expression profiles of the target gene in different cancer cell models and affected the expression of mesenchymal genes CDH1, VIM1, TGFB1 and apoptotic marker BCL2. Moreover, methylation-induced changes in expression profiles was also accompanied by a phenotypic switch in cell migration and cell morphology. We conclude that in different cell types the consequence of DNA methylation on gene expression and cell fate can be completely different.
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Project description:CRISPR/Cas9 is a versatile genome-editing technology that is widely used for studying the functionality of genetic elements, creating genetically modified organisms as well as preclinical research of genetic disorders. However, the high frequency of off-target activity (≥50%)-RGEN (RNA-guided endonuclease)-induced mutations at sites other than the intended on-target site-is one major concern, especially for therapeutic and clinical applications. Here, we review the basic mechanisms underlying off-target cutting in the CRISPR/Cas9 system, methods for detecting off-target mutations, and strategies for minimizing off-target cleavage. The improvement off-target specificity in the CRISPR/Cas9 system will provide solid genotype-phenotype correlations, and thus enable faithful interpretation of genome-editing data, which will certainly facilitate the basic and clinical application of this technology.
| S-EPMC4877446 | biostudies-other
Project description:Dysregulation of the epigenome due to alterations in chromatin modifier proteins commonly contribute to malignant transformation. To interrogate the roles of epigenetic modifiers in cancer cells, we generated an epigenome-wide CRISPR-Cas9 knockout library (EPIKOL) that targets a wide-range of epigenetic modifiers and their cofactors. We conducted eight screens in two different cancer types and showed that EPIKOL performs with high efficiency in terms of sgRNA distribution and depletion of essential genes. We discovered novel epigenetic modifiers that regulate triple-negative breast cancer (TNBC) and prostate cancer cell fitness. We confirmed the growth-regulatory functions of individual candidates, including SS18L2 and members of the NSL complex (KANSL2, KANSL3, KAT8) in TNBC cells. Overall, we show that EPIKOL, a focused sgRNA library targeting ~800 genes, can reveal epigenetic modifiers that are essential for cancer cell fitness under in vitro and in vivo conditions and enable the identification of novel anti-cancer targets. Due to its comprehensive epigenome-wide targets and relatively high number of sgRNAs per gene, EPIKOL will facilitate studies examining functional roles of epigenetic modifiers in a wide range of contexts, such as screens in primary cells, patient-derived xenografts as well as in vivo models.
| S-EPMC9381743 | biostudies-literature
Project description:Neuroblastoma is the most common extracranial solid tumor in children. Patients with neuroblastoma have a poor prognosis. The development of therapy targets and the ability to predict prognosis will be enhanced through further exploration of the genetically related genes of neuroblastoma. The present investigation utilized CRISPR-Cas9 genome-wide screening based on the DepMap database to determine essential genes for neuroblastoma cells' continued survival. WGCNA analysis was used to determine the progression-related genes, and a prognostic signature was constructed. The signature gene, NCAPG, was downregulated in neuroblastoma cells to explore its impact on various cellular processes. This research used DepMap and WGCNA to pinpoint 45 progression-related essential genes for neuroblastoma. A risk signature comprising NCAPG and MAD2L1 was established. The suppression of NCAPG prevented neuroblastoma cells from proliferating, migrating, and invading. The results of flow cytometric analysis demonstrated that NCAPG inhibition caused cell cycle arrest during the G2 and S phases and the activation of apoptosis. Additionally, NCAPG downregulation activated the p53-mediated apoptotic pathway, inducing cell apoptosis. The present work showed that NCAPG knockdown reduced neuroblastoma cell progression and may serve as a basis for further investigation into diagnostic indicators and therapy targets for neuroblastoma.
| S-EPMC10573393 | biostudies-literature
Project description:Metformin is a classic type II diabetes drug which possesses anti-tumor properties for various cancers. However, different cancers do not respond to metformin with the same effectiveness or acquire resistance. Thus, searching for vulnerabilities of metformin-resistant prostate cancer is a promising strategy to improve the therapeutic efficiency of the drug. A genome-scale CRISPR-Cas9 activation library search targeting 23,430 genes was conducted to identify the genes that confer resistance to metformin in prostate cancer cells. Candidate genes were selected by total reads of sgRNA and sgRNA diversity, and then a CCK8 assay was used to verify their resistance to metformin. Interestingly, we discovered that the activation of ECE1, ABCA12, BPY2, EEF1A1, RAD9A, and NIPSNAP1 contributed to in vitro resistance to metformin in DU145 and PC3 cell lines. Notably, a high level of RAD9A, with poor prognosis in PCa, was the most significant gene in the CCK8 assay. Furthermore, we discerned the tumor immune microenvironment with RAD9A expression by CIBERSORT. These results suggested that a high level of RAD9A may upregulate regulatory T cells to counterbalance metformin in the tumor immune microenvironment.
| S-EPMC7870801 | biostudies-literature
Project description:The CRISPR-Cas9 system, naturally a defense mechanism in prokaryotes, has been repurposed as an RNA-guided DNA targeting platform. It has been widely used for genome editing and transcriptome modulation, and has shown great promise in correcting mutations in human genetic diseases. Off-target effects are a critical issue for all of these applications. Here we review the current status on the target specificity of the CRISPR-Cas9 system.
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Project description:Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation-based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator-like (TALs) effectors. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.
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