Direct haplotype-resolved 5-base HiFi sequencing for genome-wide profiling of hypermethylation outliers in a rare disease cohort.

Long-read HiFi genome sequencing allows for accurate detection and direct phasing of single nucleotide variants, indels, and structural variants. Recent algorithmic development enables simultaneous detection of CpG methylation for analysis of regulatory element activity directly in HiFi reads. We present a comprehensive haplotype resolved 5-base HiFi genome sequencing dataset from a rare disease cohort of 276 samples in 152 families to identify rare (~0.

Massively parallel identification of functionally consequential noncoding genetic variants in undiagnosed rare disease patients.

Clinical whole genome sequencing has enabled the discovery of potentially pathogenic noncoding variants in the genomes of rare disease patients with a prior history of negative genetic testing. However, interpreting the functional consequences of noncoding variants and distinguishing those that contribute to disease etiology remains a challenge. Here we address this challenge by experimentally profiling the functional consequences of rare noncoding variants detected in a cohort of undiagnosed rare disease patients at scale using a massively parallel reporter assay.

An CRISPR Screening Platform for Prioritizing Therapeutic Targets in AML.

CRISPR-Cas9-based genetic screens have successfully identified cell type-dependent liabilities in cancer, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed using established cell lines, evaluating the physiologic relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to validate and prioritize AML-enriched dependencies , including in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing.

SMAD4 represses FOSL1 expression and pancreatic cancer metastatic colonization.

Metastasis is a complex and poorly understood process. In pancreatic cancer, loss of the transforming growth factor (TGF)-β/BMP effector SMAD4 is correlated with changes in altered histopathological transitions, metastatic disease, and poor prognosis. In this study, we use isogenic cancer cell lines to identify SMAD4 regulated genes that contribute to the development of metastatic colonization.

Autoregulates Its Expression in the Pulmonary Endothelium After Endotoxin Stimulation in a Histone Acetylation-Dependent Manner.

The innate immune response of pulmonary endothelial cells (EC) to lipopolysaccharide (LPS) induces Forkhead box protein C2 (FOXC2) activation through Toll Like Receptor 4 (TLR4). The mechanisms by which FOXC expression is regulated in lung EC under LPS stimulation remain unclear. We postulated that FOXC2 regulates its own expression in sepsis, and its transcriptional autoregulation directs lymphatic EC cell-fate decision.

LKB1/ Is a Tumor Suppressor in the Progression of Myeloproliferative Neoplasms.

The myeloproliferative neoplasms (MPN) frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/ led to enhanced self-renewal of murine MPN cells. Deletion of in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis, and an accumulation of immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells .

Identification of functional regulatory elements in the human genome using pooled CRISPR screens.

Background: Genome-scale pooled CRISPR screens are powerful tools for identifying genetic dependencies across varied cellular processes. The vast majority of CRISPR screens reported to date have focused exclusively on the perturbation of protein-coding gene function. However, protein-coding genes comprise < 2% of the sequence space in the human genome leaving a substantial portion of the genome uninterrogated.

Agreement between two large pan-cancer CRISPR-Cas9 gene dependency data sets.

Genome-scale CRISPR-Cas9 viability screens performed in cancer cell lines provide a systematic approach to identify cancer dependencies and new therapeutic targets. As multiple large-scale screens become available, a formal assessment of the reproducibility of these experiments becomes necessary. We analyze data from recently published pan-cancer CRISPR-Cas9 screens performed at the Broad and Sanger Institutes.

Deubiquitinases Maintain Protein Homeostasis and Survival of Cancer Cells upon Glutathione Depletion.

Cells are subjected to oxidative stress during the initiation and progression of tumors, and this imposes selective pressure for cancer cells to adapt mechanisms to tolerate these conditions. Here, we examined the dependency of cancer cells on glutathione (GSH), the most abundant cellular antioxidant. While cancer cell lines displayed a broad range of sensitivities to inhibition of GSH synthesis, the majority were resistant to GSH depletion.

Cells Lacking the Tumor Suppressor Gene Are Hyperdependent on Aurora B Kinase for Survival.

Small cell lung cancer (SCLC) accounts for 15% of lung cancers and is almost always linked to inactivating and mutations. SCLC frequently responds, albeit briefly, to chemotherapy. The canonical function of the gene product RB1 is to repress the E2F transcription factor family.

Pooled Lentiviral-Delivery Genetic Screens.

Pooled cell-based screens of mammalian genetic perturbations enable systematic large-scale, even genome-scale, evaluation of gene function. Pooled screens introduce genetic perturbations into a cell population through viral transduction such that each cell integrates into its DNA a single or small number of library perturbations with barcodes identifying the perturbations. One then selects and physically isolates the subset of cells that exhibit the phenotype of interest.

Orthologous CRISPR-Cas9 enzymes for combinatorial genetic screens.

Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes.

p53 regulates enhancer accessibility and activity in response to DNA damage.

The tumor suppressor p53 is a well-characterized transcription factor that can bind gene promoters and regulate target gene transcription in response to DNA damage. Recent studies, however, have revealed that p53 binding events occur predominantly within regulatory enhancer elements. The effect of p53 binding on enhancer function has not been systematically evaluated.

Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display.

Noncoding RNAs play diverse roles throughout biology and exhibit broad functional capacity. To investigate and harness these capabilities, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-Display (CRISP-Disp), a targeted localization method that uses Cas9 to deploy large RNA cargos to DNA loci. We demonstrate that functional RNA domains up to at least 4.

Integrative genomic analysis reveals widespread enhancer regulation by p53 in response to DNA damage.

The tumor suppressor p53 has been studied extensively as a direct transcriptional activator of protein-coding genes. Recent studies, however, have shed light on novel regulatory functions of p53 within noncoding regions of the genome. Here, we use a systematic approach that integrates transcriptome-wide expression analysis, genome-wide p53 binding profiles and chromatin state maps to characterize the global regulatory roles of p53 in response to DNA damage.

Identification and validation of miRNA target sites within nontraditional miRNA targets.

miRNAs are endogenous small RNAs that regulate gene expression through recognition of complementary RNA sequences. While miRNAs have generally been understood to repress gene expression posttranscriptionally through recognition of 3'-untranslated regions (3'-UTRs) of mRNA transcripts, they have the potential to target additional classes of RNAs. Understanding the expanding pool of potential miRNA targets has been hindered by the lack of tools for predicting target sites within these RNAs.

'Lnc'-ing enhancers to MYC regulation.

Long noncoding RNAs (lncRNAs) are emerging as important functional components in the establishment of long-range chromosomal interactions. In a recent paper published in Cell Research, Xiang et al. provide mechanistic insight into this phenomenon by characterizing the role of CCAT1-L, a colorectal cancer-specific lncRNA, in intra-chromosome looping between the MYC gene promoter and distal upstream enhancer elements that regulate MYC transcription.

Transcriptional regulation by miRNA mimics that target sequences downstream of gene termini.

Transcriptome studies have revealed that protein-coding loci within the human genome are overlapped at their 3'-termini by noncoding RNA (ncRNA) transcripts. Small duplex RNAs designed to be fully complementary to these 3' ncRNAs can modulate transcription of the upstream gene. Robust regulation by designed RNAs suggests that endogenous small RNAs might also recognize 3' ncRNAs and regulate gene expression.

Transcriptional gene silencing in mammalian cells by miRNA mimics that target gene promoters.

Synthetic small duplex RNAs that are fully complementary to gene promoters can silence transcription in mammalian cells. microRNAs (miRNAs) are endogenous small regulatory RNAs that sequence specifically regulate gene expression. We have developed a computational method to identify potential miRNA target sites within gene promoters.

Involvement of argonaute proteins in gene silencing and activation by RNAs complementary to a non-coding transcript at the progesterone receptor promoter.

Double-stranded RNAs that are complementary to non-coding transcripts at gene promoters can activate or inhibit gene expression in mammalian cells. Understanding the mechanism for modulating gene expression by promoter-targeted antigene RNAs (agRNAs) will require identification of the proteins involved in recognition. Previous reports have implicated argonaute (AGO) proteins, but identifications have differed with involvement of AGO1, AGO2, or both AGO1 and AGO2 being reported by different studies.

Transcriptional regulation by small RNAs at sequences downstream from 3' gene termini.

Transcriptome studies reveal many noncoding transcripts overlapping 3' gene termini. The function of these transcripts is unknown. Here we have characterized transcription at the progesterone receptor (PR) locus and identified noncoding transcripts that overlap the 3' end of the gene.