Tengfei Xiao

Sequence determinants of improved CRISPR sgRNA design

The CRISPR/Cas9 system has revolutionized mammalian somatic cell genetics. Genome-wide functional screens using CRISPR/Cas9-mediated knockout or dCas9 fusion-mediated inhibition/activation (CRISPRi/a) are powerful techniques for discovering phenotype-associated gene function. We systematically assessed the DNA sequence features that contribute to single guide RNA (sgRNA) efficiency in CRISPR-based screens. Leveraging the information from multiple designs, we derived a new sequence model for predicting sgRNA efficiency in CRISPR/Cas9 knockout experiments. Our model confirmed known features and suggested new features including a preference for cytosine at the cleavage site. The model was experimentally validated for sgRNA-mediated mutation rate and protein knockout efficiency. Tested on independent data sets, the model achieved significant results in both positive and negative selection conditions and outperformed existing models. We also found that the sequence preference for CRISPRi/a is substantially different from that for CRISPR/Cas9 knockout and propose a new model for predicting sgRNA efficiency in CRISPRi/a experiments. These results facilitate the genome-wide design of improved sgRNA for both knockout and CRISPRi/a studies.

MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens.

We propose the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) method for prioritizing single-guide RNAs, genes and pathways in genome-scale CRISPR/Cas9 knockout screens. MAGeCK demonstrates better performance compared with existing methods, identifies both positively and negatively selected genes simultaneously, and reports robust results across different experimental conditions. Using public datasets, MAGeCK identified novel essential genes and pathways, including EGFR in vemurafenib-treated A375 cells harboring a BRAF mutation. MAGeCK also detected cell type-specific essential genes, including BCR and ABL1, in KBM7 cells bearing a BCR-ABL fusion, and IGF1R in HL-60 cells, which depends on the insulin signaling pathway for proliferation.

Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR-Cas9 library

CRISPR–Cas9 screens have been widely adopted to analyze coding-gene functions, but high-throughput screening of non-coding elements using this method is more challenging because indels caused by a single cut in non-coding regions are unlikely to produce a functional knockout. A high-throughput method to produce deletions of non-coding DNA is needed. We report a high-throughput genomic deletion strategy to screen for functional long non-coding RNAs (lncRNAs) that is based on a lentiviral paired-guide RNA (pgRNA) library. Applying our screening method, we identified 51 lncRNAs that can positively or negatively regulate human cancer cell growth. We validated 9 of 51 lncRNA hits using CRISPR–Cas9-mediated genomic deletion, functional rescue, CRISPR activation or inhibition and gene-expression profiling. Our high-throughput pgRNA genome deletion method will enable rapid identification of functional mammalian non-coding elements.

Long Noncoding RNA ADINR Regulates Adipogenesis by Transcriptionally Activating C/EBPα

Summary C/EBPα is a critical transcriptional regulator of adipogenesis. How C/EBPα transcription is itself regulated is poorly understood, however, and remains a key question that needs to be addressed for a complete understanding of adipogenic development. Here, we identify a lncRNA, ADINR (adipogenic differentiation induced noncoding RNA), transcribed from a position ∼450 bp upstream of the C/EBPα gene, that orchestrates C/EBPα transcription in vivo. Depletion of ADINR leads to a severe adipogenic defect that is rescued by overexpression of C/EBPα. Moreover, we reveal that ADINR RNA specifically binds to PA1 and recruits MLL3/4 histone methyl-transferase complexes so as to increase H3K4me3 and decrease H3K27me3 histone modification in the C/EBPα locus during adipogenesis. These results show that ADINR plays important roles in regulating the differentiation of human mesenchymal stem cells into adipocytes by modulating C/EBPα in cis.

Quality control, modeling, and visualization of CRISPR screens with MAGeCK-VISPR

High-throughput CRISPR screens have shown great promise in functional genomics. We present MAGeCK-VISPR, a comprehensive quality control (QC), analysis, and visualization workflow for CRISPR screens. MAGeCK-VISPR defines a set of QC measures to assess the quality of an experiment, and includes a maximum-likelihood algorithm to call essential genes simultaneously under multiple conditions. The algorithm uses a generalized linear model to deconvolute different effects, and employs expectation-maximization to iteratively estimate sgRNA knockout efficiency and gene essentiality. MAGeCK-VISPR also includes VISPR, a framework for the interactive visualization and exploration of QC and analysis results. MAGeCK-VISPR is freely available at http://bitbucket.org/liulab/mageck-vispr.

Transcriptional profiling analysis and functional prediction of long noncoding RNAs in cancer.

Long noncoding RNAs (lncRNAs), which are noncoding RNAs (ncRNAs) with length more than 200 nucleotides (nt), have been demonstrated to be involved in various types of cancer. Consequently, it has been frequently discussed that lncRNAs with aberrant expression in cancer serve as potential diagnostic biomarkers and therapeutic targets. However, one major challenge of developing cancer biomarkers is tumor heterogeneity which means that tumor cells show different cellular morphology, metastatic potential as well as gene expression. In this study, a custom designed microarray platform covering both mRNAs and lncRNAs was applied to tumor tissues of gastric, colon, liver and lung. 316 and 157 differentially expressed (DE-) protein coding genes and lncRNAs common to these four types of cancer were identified respectively. Besides, the functional roles of common DE-lncRNAs were inferred based on their expression and genomic position correlation with mRNAs. Moreover, mRNAs and lncRNAs with tissue specificity were also identified, suggesting their particular roles with regard to specific biogenesis and functions of different organs. Based on the large-scale survey of mRNAs and lncRNAs in four types of cancer, this study may offer new biomarkers common or specific for various types of cancer.

Inactivation of the PBRM1 tumor suppressor gene amplifies the HIF-response in VHL−/− clear cell renal carcinoma

Significance Mutational inactivation of the VHL tumor suppressor gene is the signature lesion in the most common form of kidney cancer and causes inappropriate accumulation of the HIF transcription factor, which activates genes that normally facilitate adaptation to hypoxia but, in the context of kidney cancer, also promote tumorigenesis. Additional mutational events are needed, in conjunction with VHL loss, to cause kidney cancer. The most common of these are inactivating mutations of the PBRM1 tumor suppressor gene, which encodes a component [BRG1-associated factor 180 (BAF180)] of a multiprotein complex [Polybromo- and BRG1-associated factors-containing complex (PBAF)] that regulates the positions of nucleosomes throughout the genome. We describe here kidney cancer cell-based models for monitoring BAF180 function and show that loss of BAF180 accentuates the transcriptional response to HIF. Most clear cell renal carcinomas (ccRCCs) are initiated by somatic inactivation of the VHL tumor suppressor gene. The VHL gene product, pVHL, is the substrate recognition unit of an ubiquitin ligase that targets the HIF transcription factor for proteasomal degradation; inappropriate expression of HIF target genes drives renal carcinogenesis. Loss of pVHL is not sufficient, however, to cause ccRCC. Additional cooperating genetic events, including intragenic mutations and copy number alterations, are required. Common examples of the former are loss-of-function mutations of the PBRM1 and BAP1 tumor suppressor genes, which occur in a mutually exclusive manner in ccRCC and define biologically distinct subsets of ccRCC. PBRM1 encodes the Polybromo- and BRG1-associated factors-containing complex (PBAF) chromatin remodeling complex component BRG1-associated factor 180 (BAF180). Here we identified ccRCC lines whose ability to proliferate in vitro and in vivo is sensitive to wild-type BAF180, but not a tumor-associated BAF180 mutant. Biochemical and functional studies linked growth suppression by BAF180 to its ability to form a canonical PBAF complex containing BRG1 that dampens the HIF transcriptional signature.

High-dimensional genomic data bias correction and data integration using MANCIE

High-dimensional genomic data analysis is challenging due to noises and biases in high-throughput experiments. We present a computational method matrix analysis and normalization by concordant information enhancement (MANCIE) for bias correction and data integration of distinct genomic profiles on the same samples. MANCIE uses a Bayesian-supported principal component analysis-based approach to adjust the data so as to achieve better consistency between sample-wise distances in the different profiles. MANCIE can improve tissue-specific clustering in ENCODE data, prognostic prediction in Molecular Taxonomy of Breast Cancer International Consortium and The Cancer Genome Atlas data, copy number and expression agreement in Cancer Cell Line Encyclopedia data, and has broad applications in cross-platform, high-dimensional data integration.

The Caenorhabditis elegans intermediate-size transcriptome shows high degree of stage-specific expression

Earlier studies have revealed a substantial amount of transcriptional activity occurring outside annotated protein-coding genes of the Caenorhabditis elegans genome. One important fraction of this transcriptional activity relates to intermediate-size (70–500 nt) transcripts (is-ncRNAs) of mostly unknown function. Profiling the expression of this segment of the transcriptome on a tiling array through the C. elegans life cycle identified 5866 hitherto unannotated transcripts. The novel loci were distributed across intronic and intergenic space, with some enrichment toward protein-coding gene termini. The majority of the putative is-ncRNAs showed either stage-specific expression, or distinct developmental variation in their expression levels. More than 200 loci showed male-specific expression, and conserved loci were significantly enriched on the X chromosome, both observations strongly suggesting involvement of is-ncRNAs in sex-specific functions. Half of the novel loci were conserved in other nematodes, and numerous loci showed significant conservational correlations to nearby coding genes. Assuming functional roles for most of the novel loci, the data imply a nematode is-ncRNA tool kit of considerable size and variety.

Genome-wide CRISPR screen identifies HNRNPL as a prostate cancer dependency regulating RNA splicing

Significance Alternative RNA splicing and the spliceosome machinery have been implicated in cancer progression. A genome-wide CRISPR screen identified the RNA processing factor heterogeneous nuclear ribonucleoprotein L (HNRNPL) as required for prostate cancer growth by regulating alternative RNA splicing and circular RNA formation. HNRNPL and its RNA clients are overexpressed during prostate cancer progression, supporting their potential role as therapeutic targets. Alternative RNA splicing plays an important role in cancer. To determine which factors involved in RNA processing are essential in prostate cancer, we performed a genome-wide CRISPR/Cas9 knockout screen to identify the genes that are required for prostate cancer growth. Functional annotation defined a set of essential spliceosome and RNA binding protein (RBP) genes, including most notably heterogeneous nuclear ribonucleoprotein L (HNRNPL). We defined the HNRNPL-bound RNA landscape by RNA immunoprecipitation coupled with next-generation sequencing and linked these RBP–RNA interactions to changes in RNA processing. HNRNPL directly regulates the alternative splicing of a set of RNAs, including those encoding the androgen receptor, the key lineage-specific prostate cancer oncogene. HNRNPL also regulates circular RNA formation via back splicing. Importantly, both HNRNPL and its RNA targets are aberrantly expressed in human prostate tumors, supporting their clinical relevance. Collectively, our data reveal HNRNPL and its RNA clients as players in prostate cancer growth and potential therapeutic targets.