Deconstructing p53 pathways in tumor suppression.
AMER ASSOC CANCER RESEARCH. 2018: 17
View details for Web of Science ID 000432307300006
CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity.
Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases.
View details for DOI 10.1038/s41588-018-0070-7
View details for PubMedID 29507424
A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies
2018; Epub ahead of print: 460?71
Primary cilia organize Hedgehog signaling and shape embryonic development, and their dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen can robustly identify factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovered novel components of several ciliary structures, including a protein complex that contains ?-tubulin and ?-tubulin and is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and showed that many congenital heart disorders are caused by loss of ciliary signaling. Collectively, our study enables a systematic analysis of ciliary function and of ciliopathies, and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.
View details for DOI 10.1038/s41588-018-0054-7
View details for PubMedCentralID PMC5862771
CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity
2017; 549 (7670): 101?5
Cancer cells exploit the expression of the programmed death-1 (PD-1) ligand 1 (PD-L1) to subvert T-cell-mediated immunosurveillance. The success of therapies that disrupt PD-L1-mediated tumour tolerance has highlighted the need to understand the molecular regulation of PD-L1 expression. Here we identify the uncharacterized protein CMTM6 as a critical regulator of PD-L1 in a broad range of cancer cells, by using a genome-wide CRISPR-Cas9 screen. CMTM6 is a ubiquitously expressed protein that binds PD-L1 and maintains its cell surface expression. CMTM6 is not required for PD-L1 maturation but co-localizes with PD-L1 at the plasma membrane and in recycling endosomes, where it prevents PD-L1 from being targeted for lysosome-mediated degradation. Using a quantitative approach to profile the entire plasma membrane proteome, we find that CMTM6 displays specificity for PD-L1. Notably, CMTM6 depletion decreases PD-L1 without compromising cell surface expression of MHC class I. CMTM6 depletion, via the reduction of PD-L1, significantly alleviates the suppression of tumour-specific T cell activity in vitro and in vivo. These findings provide insights into the biology of PD-L1 regulation, identify a previously unrecognized master regulator of this critical immune checkpoint and highlight a potential therapeutic target to overcome immune evasion by tumour cells.
View details for DOI 10.1038/nature23643
View details for Web of Science ID 000409388700041
View details for PubMedID 28813417
View details for PubMedCentralID PMC5706633
Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens
CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens.
View details for DOI 10.1038/ncomms15178
View details for Web of Science ID 000400851300001
View details for PubMedID 28474669
Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions
2017; 35 (5): 463-?
Identification of effective combination therapies is critical to address the emergence of drug-resistant cancers, but direct screening of all possible drug combinations is infeasible. Here we introduce a CRISPR-based double knockout (CDKO) system that improves the efficiency of combinatorial genetic screening using an effective strategy for cloning and sequencing paired single guide RNA (sgRNA) libraries and a robust statistical scoring method for calculating genetic interactions (GIs) from CRISPR-deleted gene pairs. We applied CDKO to generate a large-scale human GI map, comprising 490,000 double-sgRNAs directed against 21,321 pairs of drug targets in K562 leukemia cells and identified synthetic lethal drug target pairs for which corresponding drugs exhibit synergistic killing. These included the BCL2L1 and MCL1 combination, which was also effective in imatinib-resistant cells. We further validated this system by identifying known and previously unidentified GIs between modifiers of ricin toxicity. This work provides an effective strategy to screen synergistic drug combinations in high-throughput and a CRISPR-based tool to dissect functional GI networks.
View details for DOI 10.1038/nbt.3834
View details for Web of Science ID 000400809800021
View details for PubMedID 28319085
Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes
2016; 34 (6): 634-636
We compared the ability of short hairpin RNA (shRNA) and CRISPR/Cas9 screens to identify essential genes in the human chronic myelogenous leukemia cell line K562. We found that the precision of the two libraries in detecting essential genes was similar and that combining data from both screens improved performance. Notably, results from the two screens showed little correlation, which can be partially explained by the identification of distinct essential biological processes with each technology.
View details for DOI 10.1038/nbt.3567
View details for Web of Science ID 000377846400030
View details for PubMedID 27159373
View details for PubMedCentralID PMC4900911
Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification
NATURE CHEMICAL BIOLOGY
2016; 12 (5): 361-?
Broad-spectrum antiviral drugs targeting host processes could potentially treat a wide range of viruses while reducing the likelihood of emergent resistance. Despite great promise as therapeutics, such drugs remain largely elusive. Here we used parallel genome-wide high-coverage short hairpin RNA (shRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screens to identify the cellular target and mechanism of action of GSK983, a potent broad-spectrum antiviral with unexplained cytotoxicity. We found that GSK983 blocked cell proliferation and dengue virus replication by inhibiting the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). Guided by mechanistic insights from both genomic screens, we found that exogenous deoxycytidine markedly reduced GSK983 cytotoxicity but not antiviral activity, providing an attractive new approach to improve the therapeutic window of DHODH inhibitors against RNA viruses. Our results highlight the distinct advantages and limitations of each screening method for identifying drug targets, and demonstrate the utility of parallel knockdown and knockout screens for comprehensive probing of drug activity.
View details for DOI 10.1038/NCHEMBIO.2050
View details for Web of Science ID 000374322800011
View details for PubMedID 27018887
View details for PubMedCentralID PMC4836973
Novel Population Genetics in Ciliates due to Life Cycle and Nuclear Dimorphism.
Molecular biology and evolution
2014; 31 (8): 2084-2093
Our understanding of population genetics comes primarily from studies of organisms with canonical life cycles and nuclear organization, either haploid or diploid, sexual, or asexual. Although this template yields satisfactory results for the study of animals and plants, the wide variety of genomic organizations and life cycles of unicellular eukaryotes can make these organisms behave differently in response to mutation, selection, and drift than predicted by traditional population genetic models. In this study, we show how each of these unique features of ciliates affects their evolutionary parameters in mutation-selection, selection-drift, and mutation-selection-drift situations. In general, ciliates are less efficient in eliminating deleterious mutations-these mutations linger longer and at higher frequencies in ciliate populations than in sexual populations--and more efficient in selecting beneficial mutations. Approaching this problem via analytical techniques and simulation allows us to make specific predictions about the nature of ciliate evolution, and we discuss the implications of these results with respect to the high levels of polymorphism and high rate of protein evolution reported for ciliates.
View details for DOI 10.1093/molbev/msu150
View details for PubMedID 24784136
The Protein Invasion: A Broad Review on the Origin of the Translational System
JOURNAL OF MOLECULAR EVOLUTION
2013; 77 (4): 185-196
Translation, coded peptide synthesis, arguably exists at the heart of modern cellular life. By orchestrating an incredibly complex interaction between tRNAs, mRNAs, aaRSs, the ribosome, and numerous other small molecules, the translational system allows the interpretation of data in the form of DNA to create massively complex proteins which control and enact almost every cellular function. A natural question then, is how did this system evolve? Here we present a broad review of the existing theories of the last two decades on the origin of the translational system. We attempt to synthesize the wide variety of ideas as well as organize them into modular components, addressing the evolution of the peptide-RNA interaction, tRNA, mRNA, the ribosome, and the first proteins separately. We hope to provide both a comprehensive overview of the literature as well as a framework for future discussions and novel theories.
View details for DOI 10.1007/s00239-013-9592-x
View details for Web of Science ID 000326456600005
View details for PubMedID 24145863
Ambushing the ambush hypothesis: predicting and evaluating off-frame codon frequencies in Prokaryotic Genomes
In this paper, we address the evidence for the Ambush Hypothesis. Proposed by Seligmann and Pollock, this hypothesis posits that there exists a selection for off-frame stop codons (OSCs) to counteract the possible deleterious effects of translational frameshifts, including the waste of resources and potential cytotoxicity. Two main types of study have been used to support the hypothesis. Some studies analyzed codon usage and showed that codons with more potential to create OSCs seem to be favored over codons with lower potential; they used this finding to support the Ambush Hypothesis. Another study used 342 bacterial genomes to evaluate the hypothesis directly, finding significant excesses of OSCs in these genomes.We repeated both analyses with newer datasets and searched for other factors that could explain the observed trends. In the first case, the relative frequency of codons with the potential to create OSCs is directly correlated with the GC content of organisms, as stop codons are GC-poor. When evaluating the frequency of OSCs directly in 1,976 bacterial genomes we also detected a significant excess. However, when comparing the excess of OSCs with similarly obtained results for the frequency of out-of-frame sense codons, some sense codons have a more significant excess than stop codons.Two avenues of study have been used to support the Ambush Hypothesis. Using the same methods as these previous studies, we demonstrate that the evidence in support of the Ambush Hypothesis does not hold up against more rigorous testing.
View details for DOI 10.1186/1471-2164-14-418
View details for Web of Science ID 000321389800002
View details for PubMedID 23799949
View details for PubMedCentralID PMC3700767
Model for the Evolution of Extremely Fragmented Macronuclei in Ciliates
2013; 8 (5)
While all ciliates possess nuclear dimorphism, several ciliates - like those in the classes Phyllopharyngea, Spirotrichea, and Armophorea - have an extreme macronuclear organization. Their extensively fragmented macronuclei contain upwards of 20,000 chromosomes, each with upwards of thousands of copies. These features have evolved independently on multiple occasions throughout ciliate evolutionary history, and currently no models explain these structures in an evolutionary context. In this paper, we propose that competition between two forces - the limitation and avoidance of chromosomal imbalances as a ciliate undergoes successive asexual divisions, and the costs of replicating massive genomes - is sufficient to explain this particular nuclear structure. We present a simulation of ciliate cell evolution under control of these forces, allowing certain features of the population to change over time. Over a wide range of parameters, we observe the repeated emergence of this unusual genomic organization found in nature. Although much remains to be understood about the evolution of macronuclear genome organization, our results show that the proposed model is a plausible explanation for the emergence of these extremely fragmented, highly polyploid genomes.
View details for DOI 10.1371/journal.pone.0064997
View details for Web of Science ID 000319330200178
View details for PubMedID 23705024
View details for PubMedCentralID PMC3660376
An Alternative Look at Code Evolution: Using Non-canonical Codes to Evaluate Adaptive and Historic Models for the Origin of the Genetic Code
JOURNAL OF MOLECULAR EVOLUTION
2013; 76 (1-2): 71-80
The canonical code has been shown many times to be highly robust against point mutations; that is, mutations that change a single nucleotide tend to result in similar amino acids more often than expected by chance. There are two major types of models for the origin of the code, which explain how this sophisticated structure evolved. Adaptive models state that the primitive code was specifically selected for error minimization, while historic models hypothesize that the robustness of the code is an artifact or by-product of the mechanism of code evolution. In this paper, we evaluated the levels of robustness in existing non-canonical codes as well as codes that differ in only one codon assignment from the standard code. We found that the level of robustness of many of these codes is comparable or better than that of the standard code. Although these results do not preclude an adaptive origin of the genetic code, they suggest that the code was not selected for minimizing the effects of point mutations.
View details for DOI 10.1007/s00239-013-9542-7
View details for Web of Science ID 000315578300007
View details for PubMedID 23344715