CRISPRi enables isoform-specific loss-of-function screens and identification of gastric cancer-specific isoform dependencies

Davies, Rebecca; Liu, Ling; Tong, S. Y.; Tuano, Natasha; Chaturvedi, Richa; Huang, Kie Kyon; Itman, Catherine; Mandoli, Amit; Qamra, Aditi; Hu, Changyuan; Powell, David R.; Daly, Roger J.; Tan, Patrick; Rosenbluh, Joseph

doi:10.1186/s13059-021-02266-6

Cited by 18 publications

(16 citation statements)

References 46 publications

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“…2I). We have previously reported that bidirectional promoters can result in off-target effects in CRISPRi screens (Davies et al, 2021;Rosenbluh et al, 2017), and in some cases we found inconsistencies that are likely due to off-target effects. For example, ATXN7 scored as a strong tumorsuppressor gene using CRISPRko in both 2D and 3D assays but did not score with CRISPRi (Supplementary Fig.…”

Section: Bc Associated Risk Genes That Upon Suppression or Overexpression Induce Cell Proliferation Phenotype In 2d And 3d Culturesmentioning

confidence: 62%

“…High-throughput sequencing library was generated using one-step PCR to amplify the integrated sequence within the construct and the addition of a barcode as previously described (Davies et al, 2021;Rosenbluh et al, 2016;Rosenbluh et al, 2017). PCR products were then purified using AMPure beads and samples sequenced using HiSeq (Illumina).…”

Section: Library Preparation Sequencing and Analysismentioning

confidence: 99%

“…For each gene we chose top scoring 5 sgRNAs (based on CRISPick scores). Libraries were prepared as previously described (Davies et al, 2021;Rosenbluh et al, 2016;Rosenbluh et al, 2017…”

Section: Generation Of Pooled Sgrna Librarymentioning

confidence: 99%

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CRISPR screens identify gene targets and drug repositioning opportunities at breast cancer risk loci

Tuano

Beesley

Manning

et al. 2021

Preprint

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Genome-wide association studies (GWAS) have identified >200 loci associated with breast cancer (BC) risk. The majority of candidate causal variants (CCVs) are in non-coding regions and are likely to modulate cancer risk by regulating gene expression. We recently developed a scoring system, INQUISIT, to predict candidate risk genes at BC-risk loci. Here, we used pooled CRISPR activation and suppression screens to validate INQUISIT predictions, and to define the cancer phenotypes they mediate. We measured proliferation in 2D, 3D, and in immune-deficient mice, as well as the effect on the DNA damage response. We performed 60 CRISPR screens and identified 21 high-confidence INQUISIT predictions that mediate a cancer phenotype. We validated the direct regulation of a subset of genes by BC-risk variants using HiCHIP and CRISPRqtl. Furthermore, we show the utility of expression profiling for drug repurposing against these targets. We provide a platform for identifying gene targets of risk variants, and lay a blueprint of interventions for BC risk reduction and treatment.

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Section: Bc Associated Risk Genes That Upon Suppression or Overexpression Induce Cell Proliferation Phenotype In 2d And 3d Culturesmentioning

confidence: 62%

Section: Library Preparation Sequencing and Analysismentioning

confidence: 99%

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CRISPR screens identify gene targets and drug repositioning opportunities at breast cancer risk loci

Tuano

Beesley

Manning

et al. 2021

Preprint

Self Cite

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show abstract

“…For each gene we chose top scoring 5 sgRNAs (based on CRISPick scores). Libraries were prepared as previously described 22,23,67 . Briefly, oligonucleotide pools (CustomArray) contained the sgRNA sequence appended to BsmBI cutting sites and overhang sequences for PCR amplification.…”

Section: Discussionmentioning

confidence: 99%

“…4A). The inconsistencies detected are likely due to CRISPRi bidirectional promoter off-target effects 22,23 . For example, ATXN7 scored as a strong tumor-suppressor using CRISPRko in 2D and 3D assays but did not score with CRISPRi (Extended Data Fig.…”

Section: Bc Risk Genes That Induce a Proliferation Phenotype In 2d And 3d Culturesmentioning

confidence: 99%

CRISPR screens identify gene targets and drug repositioning opportunities at breast cancer risk loci

Rosenbluh

Tuano

Milne

et al. 2022

Preprint

Self Cite

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Genome-wide association studies (GWAS) have identified >200 loci associated with breast cancer (BC) risk. The majority of candidate causal variants (CCVs) are in non-coding regions and likely modulate cancer risk by regulating gene expression. However, pinpointing the exact target of the association and identifying the phenotype it mediates is a major challenge in the interpretation and translation of GWAS. Here, we used pooled CRISPR activation and suppression screens to evaluate predicted GWAS target genes, and to define the cancer phenotypes they mediate. We measured proliferation in 2D, 3D, and in immune-deficient mice, as well as the effect on DNA repair. We performed 60 CRISPR screens and identified 21 genes predicted with high confidence to be GWAS targets that drive a cancer phenotype by driving a proliferation or DNA damage response in breast cells. We validated the regulation of a subset of these genes by BC-risk variants, and show the utility of expression profiling for drug repurposing. We provide a platform for identifying gene targets of risk variants, and present a blueprint of interventions for BC risk reduction and treatment.

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Epigenomic insights into common human disease pathology

Bell

2024

Cell. Mol. Life Sci.

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The epigenome—the chemical modifications and chromatin-related packaging of the genome—enables the same genetic template to be activated or repressed in different cellular settings. This multi-layered mechanism facilitates cell-type specific function by setting the local sequence and 3D interactive activity level. Gene transcription is further modulated through the interplay with transcription factors and co-regulators. The human body requires this epigenomic apparatus to be precisely installed throughout development and then adequately maintained during the lifespan. The causal role of the epigenome in human pathology, beyond imprinting disorders and specific tumour suppressor genes, was further brought into the spotlight by large-scale sequencing projects identifying that mutations in epigenomic machinery genes could be critical drivers in both cancer and developmental disorders. Abrogation of this cellular mechanism is providing new molecular insights into pathogenesis. However, deciphering the full breadth and implications of these epigenomic changes remains challenging. Knowledge is accruing regarding disease mechanisms and clinical biomarkers, through pathogenically relevant and surrogate tissue analyses, respectively. Advances include consortia generated cell-type specific reference epigenomes, high-throughput DNA methylome association studies, as well as insights into ageing-related diseases from biological ‘clocks’ constructed by machine learning algorithms. Also, 3rd-generation sequencing is beginning to disentangle the complexity of genetic and DNA modification haplotypes. Cell-free DNA methylation as a cancer biomarker has clear clinical utility and further potential to assess organ damage across many disorders. Finally, molecular understanding of disease aetiology brings with it the opportunity for exact therapeutic alteration of the epigenome through CRISPR-activation or inhibition.

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CRISPRi enables isoform-specific loss-of-function screens and identification of gastric cancer-specific isoform dependencies

Cited by 18 publications

References 46 publications

CRISPR screens identify gene targets and drug repositioning opportunities at breast cancer risk loci

CRISPR screens identify gene targets and drug repositioning opportunities at breast cancer risk loci

CRISPR screens identify gene targets and drug repositioning opportunities at breast cancer risk loci

Epigenomic insights into common human disease pathology

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