Unexpected CRISPR off-target mutation pattern in vivo are not typically germline-like [preprint]

A computationally evolutionary investigation was performed to re-analyze the WGS data of the two studies published in Nature Methods (2015, 2017) with opposite conclusions on CRISPR off-target mutations. Our analysis concluded that the so-called unexpected SNVs pattern obtained by the study of Schaefer et al. are not typically germline-like. Some of unusual and unidentified mutations may arise, but the real reasons remain to be explored. Based on the available data and a direct comparison of the two studies, we presented two possible reasons and future re-analysis directions that may contribute to such different conclusions. To characterize the authentic CRISPR-mediated mutations, we are required to have appropriate controls to rule out other sources of mutations, which will be needed for benchmarking of targeting safety of CRISPR-based gene therapy

Evolution of the class C GPCR Venus flytrap modules involved positive selected functional divergence

<p>Abstract</p> <p>Background</p> <p>Class C G protein-coupled receptors (GPCRs) represent a distinct group of the GPCR family, which structurally possess a characteristically distinct extracellular domain inclusive of the Venus flytrap module (VFTM). The VFTMs of the class C GPCRs is responsible for ligand recognition and binding, and share sequence similarity with bacterial periplasmic amino acid binding proteins (PBPs). An extensive phylogenetic investigation of the VFTMs was conducted by analyzing for functional divergence and testing for positive selection for five typical groups of the class C GPCRs. The altered selective constraints were determined to identify the sites that had undergone functional divergence via positive selection. In order to structurally demonstrate the pattern changes during the evolutionary process, three-dimensional (3D) structures of the GPCR VFTMs were modelled and reconstructed from ancestral VFTMs.</p> <p>Results</p> <p>Our results show that the altered selective constraints in the VFTMs of class C GPCRs are statistically significant. This implies that functional divergence played a key role in characterizing the functions of the VFTMs after gene duplication events. Meanwhile, positive selection is involved in the evolutionary process and drove the functional divergence of the VFTMs. Our results also reveal that three continuous duplication events occurred in order to shape the evolutionary topology of class C GPCRs. The five groups of the class C GPCRs have essentially different sites involved in functional divergence, which would have shaped the specific structures and functions of the VFTMs.</p> <p>Conclusion</p> <p>Taken together, our results show that functional divergence involved positive selection and is partially responsible for the evolutionary patterns of the class C GPCR VFTMs. The sites involved in functional divergence will provide more clues and candidates for further research on structural-function relationships of these modules as well as shedding light on the activation mechanism of the class C GPCRs.</p

The Genomes of Oryza sativa: A History of Duplications

We report improved whole-genome shotgun sequences for the genomes of indica and japonica rice, both with multimegabase contiguity, or almost 1,000-fold improvement over the drafts of 2002. Tested against a nonredundant collection of 19,079 full-length cDNAs, 97.7% of the genes are aligned, without fragmentation, to the mapped super-scaffolds of one or the other genome. We introduce a gene identification procedure for plants that does not rely on similarity to known genes to remove erroneous predictions resulting from transposable elements. Using the available EST data to adjust for residual errors in the predictions, the estimated gene count is at least 38,000–40,000. Only 2%–3% of the genes are unique to any one subspecies, comparable to the amount of sequence that might still be missing. Despite this lack of variation in gene content, there is enormous variation in the intergenic regions. At least a quarter of the two sequences could not be aligned, and where they could be aligned, single nucleotide polymorphism (SNP) rates varied from as little as 3.0 SNP/kb in the coding regions to 27.6 SNP/kb in the transposable elements. A more inclusive new approach for analyzing duplication history is introduced here. It reveals an ancient whole-genome duplication, a recent segmental duplication on Chromosomes 11 and 12, and massive ongoing individual gene duplications. We find 18 distinct pairs of duplicated segments that cover 65.7% of the genome; 17 of these pairs date back to a common time before the divergence of the grasses. More important, ongoing individual gene duplications provide a never-ending source of raw material for gene genesis and are major contributors to the differences between members of the grass family

Effect of Duplicate Genes on Mouse Genetic Robustness: An Update

In contrast to S. cerevisiae and C. elegans, analyses based on the current knockout (KO) mouse phenotypes led to the conclusion that duplicate genes had almost no role in mouse genetic robustness. It has been suggested that the bias of mouse KO database toward ancient duplicates may possibly cause this knockout duplicate puzzle, that is, a very similar proportion of essential genes (PE) between duplicate genes and singletons. In this paper, we conducted an extensive and careful analysis for the mouse KO phenotype data and corroborated a strong effect of duplicate genes on mouse genetics robustness. Moreover, the effect of duplicate genes on mouse genetic robustness is duplication-age dependent, which holds after ruling out the potential confounding effect from coding-sequence conservation, protein-protein connectivity, functional bias, or the bias of duplicates generated by whole genome duplication (WGD). Our findings suggest that two factors, the sampling bias toward ancient duplicates and very ancient duplicates with a proportion of essential genes higher than that of singletons, have caused the mouse knockout duplicate puzzle; meanwhile, the effect of genetic buffering may be correlated with sequence conservation as well as protein-protein interactivity

Functional complementation between transcriptional methylation regulation and post-transcriptional microRNA regulation in the human genome

Abstract Background DNA methylation in the 5' promoter regions of genes and microRNA (miRNA) regulation at the 3' untranslated regions (UTRs) are two major epigenetic regulation mechanisms in most eukaryotes. Both DNA methylation and miRNA regulation can suppress gene expression and their corresponding protein product; thus, they play critical roles in cellular processes. Although there have been numerous investigations of gene regulation by methylation changes and miRNAs, there is no systematic genome-wide examination of their coordinated effects in any organism. Results In this study, we investigated the relationship between promoter methylation at the transcription level and miRNA regulation at the post-transcription level by taking advantage of recently released human methylome data and high quality miRNA and other gene annotation data. We found methylation level in the promoter regions and expression level was negatively correlated. Then, we showed that miRNAs tended to target the genes with a low DNA methylation level in their promoter regions. We further demonstrated that this observed pattern was not attributed to the gene expression level, expression broadness, or the number of transcription factor binding sites. Interestingly, we found miRNA target sites were significantly enriched in the genes located in differentially methylated regions or partially methylated domains. Finally, we explored the features of DNA methylation and miRNA regulation in cancer genes and found cancer genes tended to have low methylation level and more miRNA target sites. Conclusion This is the first genome-wide investigation of the combined regulation of gene expression. Our results supported a complementary regulation between DNA methylation (transcriptional level) and miRNA function (post-transcriptional level) in the human genome. The results were helpful for our understanding of the evolutionary forces towards organisms' complexity beyond traditional sequence level investigation.</p

Histone modification pattern evolution after yeast gene duplication

<p>Abstract</p> <p>Background</p> <p>Gene duplication and subsequent functional divergence especially expression divergence have been widely considered as main sources for evolutionary innovations. Many studies evidenced that genetic regulatory network evolved rapidly shortly after gene duplication, thus leading to accelerated expression divergence and diversification. However, little is known whether epigenetic factors have mediated the evolution of expression regulation since gene duplication. In this study, we conducted detailed analyses on yeast histone modification (HM), the major epigenetics type in this organism, as well as other available functional genomics data to address this issue.</p> <p>Results</p> <p>Duplicate genes, on average, share more common HM-code patterns than random singleton pairs in their promoters and open reading frames (ORF). Though HM-code divergence between duplicates in both promoter and ORF regions increase with their sequence divergence, the HM-code in ORF region evolves slower than that in promoter region, probably owing to the functional constraints imposed on protein sequences. After excluding the confounding effect of sequence divergence (or evolutionary time), we found the evidence supporting the notion that in yeast, the HM-code may co-evolve with <it>cis</it>- and <it>trans</it>-regulatory factors. Moreover, we observed that deletion of some yeast HM-related enzymes increases the expression divergence between duplicate genes, yet the effect is lower than the case of transcription factor (TF) deletion or environmental stresses.</p> <p>Conclusions</p> <p>Our analyses demonstrate that after gene duplication, yeast histone modification profile between duplicates diverged with evolutionary time, similar to genetic regulatory elements. Moreover, we found the evidence of the co-evolution between genetic and epigenetic elements since gene duplication, together contributing to the expression divergence between duplicate genes.</p

Evolution of alternative splicing after gene duplication

Alternative splicing and gene duplication are two major sources of proteomic function diversity. Here, we study the evolutionary trend of alternative splicing after gene duplication by analyzing the alternative splicing differences between duplicate genes. We observed that duplicate genes have fewer alternative splice (AS) forms than single-copy genes, and that a negative correlation exists between the mean number of AS forms and the gene family size. Interestingly, we found that the loss of alternative splicing in duplicate genes may occur shortly after the gene duplication. These results support the subfunctionization model of alternative splicing in the early stage after gene duplication. Further analysis of the alternative splicing distribution in human duplicate pairs showed the asymmetric evolution of alternative splicing after gene duplications; i.e., the AS forms between duplicates may differ dramatically. We therefore conclude that alternative splicing and gene duplication may not evolve independently. In the early stage after gene duplication, young duplicates may take over a certain amount of protein function diversity that previously was carried out by the alternative splicing mechanism. In the late stage, the gain and loss of alternative splicing seem to be independent between duplicates

Paralog-divergent Features May Help Reduce Off-target Effects of Drugs: Hints from Glucagon Subfamily Analysis

Side effects from targeted drugs remain a serious concern. One reason is the nonselective binding of a drug to unintended proteins such as its paralogs, which are highly homologous in sequences and have similar structures and drug-binding pockets. To identify targetable differences between paralogs, we analyzed two types (type-I and type-II) of functional divergence between two paralogs in the known target protein receptor family G-protein coupled receptors (GPCRs) at the amino acid level. Paralogous protein receptors in glucagon-like subfamily, glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), exhibit divergence in ligands and are clinically validated drug targets for type 2 diabetes. Our data showed that type-II amino acids were significantly enriched in the binding sites of antagonist MK-0893 to GCGR, which had a radical shift in physicochemical properties between GCGR and GLP-1R. We also examined the role of type-I amino acids between GCGR and GLP-1R. The divergent features between GCGR and GLP-1R paralogs may be helpful in their discrimination, thus enabling the identification of binding sites to reduce undesirable side effects and increase the target specificity of drugs