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
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