Speciation and adaptation research meets genome editing.

Advances in forest tree genomics

Does evolution proceed in small steps or large leaps? How repeatable is evolution? How constrained is the evolutionary process? Answering these long-standing questions in evolutionary biology is indispensable for both understanding how extant biodiversity has evolved and predicting how organisms and ecosystems will respond to changing environments in the future. Understanding the genetic basis of phenotypic diversification and speciation in natural populations is key to properly answering these questions. The leap forward in genome sequencing technologies has made it increasingly easier to not only investigate the genetic architecture but also identify the variant sites underlying adaptation and speciation in natural populations. Furthermore, recent advances in genome editing technologies are making it possible to investigate the functions of each candidate gene in organisms from natural populations. In this article, we discuss how these recent technological advances enable the analysis of causative genes and mutations and how such analysis can help answer long-standing evolutionary biology questions.This article is part of the theme issue ‘Genetic basis of adaptation and speciation: from loci to causative mutations’.

The adaptation to a new habitat often results in a confounding between genomewide genotype and beneficial alleles. When the confounding is strong, or the allelic effects is weak, it is a major statistical challenge to detect the adaptive polymorphisms. We describe a novel approach to dissect polygenic traits in natural populations. First, candidate adaptive loci are identified by screening for loci directly associated with the adaptive trait or the expression of genes known to affect it. Then, a multilocus genetic architecture is inferred using a backward elimination association analysis across all candidate loci with an adaptive false discovery rate-based threshold. Effects of population stratification are controlled by accounting for genomic kinship in both steps of the analysis and also by simultaneously testing all candidate loci in the multilocus model. We illustrate the method by exploring the polygenic basis of an important adaptive trait, flowering time in Arabidopsis thaliana, using public data from the 1,001 genomes project. We revealed associations between 33 (29) loci and flowering time at 10 (16)°C in this collection of natural accessions, where standard genomewide association analysis methods detected five (3) loci. The 33 (29) loci explained approximately 55.1 (48.7)% of the total phenotypic variance of the respective traits. Our work illustrates how the genetic basis of highly polygenic adaptive traits in natural populations can be explored in much greater detail using new multilocus mapping approaches taking advantage of prior biological information, genome and transcriptome data.

COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites.

Manipulating genomes by traditional targeted genome editing technique( gene targeting) is inefficient,making it impractical or difficult to use the technique as a gene-therapy approach to cure diseases and decipher gene functions. To overcome this problem,next-generation targeted gene-editing techniques were developed to achieve higher efficiency for gene correction,specific locus integration or knock-in and high throughput gene knock-out. The progress of new techniques for targeted genome-editing tools were reviewed, including zinc finger nucleases( ZFN), transcription activator-like effector nucleases( TALENs), and a clustered regularly interspaced short palindromic repeats( CRISPR) / Cas system. A brief summary of the history, recent structure,progress,and future prospects was presented. After comparing these tools,it was found that CRISPR systems offer an advantage over ZFN and TALEN.

Unintended on-target chromosomal instability following CRISPR/Cas9 single gene targeting

It is clear that while there is significant potential for cardiovascular genome-editing therapies to eventually be translated to patients, there are substantial technical obstacles that remain to be addressed, and discussions about the acceptability

Skeletal muscle (SKM) is an energetic organ with a high degree of plasticity. Different environmental stimuli as exercise or cold, but also physical inactivity, lead to complex molecular regulations that result in metabolic adaptations of the SKM and the whole body. Key factors in SKM plasticity and whole body energy homeostasis are the peroxisome proliferator-activated receptor (PPAR) γ coactivator-1 (PGC-1) family including three members, PGC-1α, PGC-1β and PGC-related coactivator (PRC). The PGC-1s are coactivators and hence use transcription factor binding partners (TFBP) in order to regulate their target genes. The complexity of transcriptional control might even be increased by epigenetic alterations, mainly DNA methylation. The aim of my thesis was to study the regulation of global molecular mechanisms by SKM PGC-1α and PGC-1β leading to muscle plasticity in various environmental contexts. We combined diverse experimental, computational and multi-omics approaches such as chromatin immunoprecipitation sequencing (ChIPseq), RNA sequencing (RNAseq), reduced representation bisulfite sequencing (RRBS) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated proteins) genome editing technology in skeletal muscle systems in vitro and in vivo and investigated the effect of external stimuli as cold or exercise in different PGC-1α/β genotypes. Our data show that various interventions like acute and chronic exercise have different methylation profiles or combined with cold-induced muscle shivering, individual transcript profiles in wild type (WT) mice. A time-dependent correlation of DNA methylation with gene expression was observed, however dissimilar in acute and chronic exercise. Furthermore, we dissected potential memory marks on the DNA by methylation following chronic training in mice. In addition, we could show for the first time a role of PGC-1α, not only in exercise performance but as well in altered transcriptome and methylome profiles subsequent to exercise and changed transcription profile to cold stimulation, by using muscle-specific PGC-1α knockout (MKO) mice. Thus, PGC-1α is a major contributor in global metabolic control by the regulation of a transcriptional network through multiple TF interactions and its involvement in epigenetic alterations. To further investigate the PGC-1α network, the Ppargc1a locus multiplex epitope tag knock-in mouse, which we generated by the CRISPR/Cas technology, will serve as a platform for future studies. This genetic mouse model allows now detailed evaluation of PGC-1α isoforms as well as the identification of new TFBPs under diverse contexts and in different tissues, due to non-tissue-specific epitope tags at the proximal PGC-1α promoter. However, in C2C12 myotubes we could show that PGC-1α regulates its target genes either by direct TF binding or indirectly. Even more, the genomic context of guanine-cytosine (GC) and cytosine-phosphate-guanine (CpG) amount affects PGC-1α recruitment and allows the estrogen-related receptor α (ERRα), a known TFBP of PGC-1α in the regulation of mitochondrial biogenesis, to regulate PGC-1α target genes without coactivation by PGC-1α but by interaction with the TF specificity protein 1 (SP1). Contrarily, we observed that PGC-1β acts mostly indirect on its target genes and only to a very small extent direct on the DNA by TF binding. Taken together, our data provide new knowledge of the functional role of PGC-1α and PGC-1β in SKM metabolism. The involvement of transcriptional regulation and epigenetic control under basal, acute and chronic exercise conditions as well as in cold-induced muscle shivering, adds a next piece of puzzle to the complex network regulated by these coactivators. Our findings help to understand the mechanism of SKM plasticity and open new signaling pathways and targets, which will, complemented with further studies, support the development of novel therapeutic strategies to cure myopathies and fight against metabolic disorders and other pathophysiological conditions.

Cas9, the RNA-guided DNA endonuclease from the CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) system, has been adapted for genome editing and gene regulation in multiple model organisms. Here we characterize a Cas9 ortholog from Streptococcus thermophilus LMG18311 (LMG18311 Cas9). In vitro reconstitution of this system confirms that LMG18311 Cas9 together with a trans-activating RNA (tracrRNA) and a CRISPR RNA (crRNA) cleaves double-stranded DNA with a specificity dictated by the sequence of the crRNA. Cleavage requires not only complementarity between crRNA and target but also the presence of a short motif called the PAM. Here we determine the sequence requirements of the PAM for LMG18311 Cas9. We also show that both the efficiency of DNA target cleavage and the location of the cleavage sites vary based on the position of the PAM sequence.

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

In It Together

Genome editing can be used to create new wheat varieties with enhanced performance. Clustered regularly interspaced short palindromic repeat (CRISPR) is a powerful tool for knockout generation, precise modification, multiplex engineering, and the activation and repression of target genes. Targeted mutagenesis via RNA-guided genome editing using type II CRISPR-Cas9 is highly efficient in some plant species, but not in others. One possible solution is to use newly discovered variants of genome editing enzymes such as the class 2 system component Cpf1 (CRISPR from Prevotella and Francisella 1) in place of the more commonly used Cas9. We compared the editing efficiency of Cas9 and two Cpf1 orthologs, AsCpf1 (Acidaminococcus spp. BV3L6) and LbCpf1 (Lachnospiraceae bacterium ND2006) in wheat (Triticum aestivum). LbCpf1 had a higher editing efficiency for the target gene TaPDS than AsCpf1 and Cas9, and Cas9 induced more off-target mutations than AsCpf1 and LbCpf1, suggesting that CRISPR-LbCpf1 is a powerful genome editing tool for polyploid plants such as wheat.

Genome editing is a cutting-edge technology that generates DNA double strand breaks at the specific genomic DNA sequence through nuclease recognition and cleavage, and then achieves insertion, replacement, or deletion of the target gene via endogenous DNA repair mechanisms, such as non-homologous end joining, homology directed repair, and homologous recombination. So far, more than 600 human hereditary eye diseases and systemic hereditary diseases with ocular phenotypes have been found. However, most of these diseases are of incompletely elucidated pathogenesis and without effective therapies. Genome editing technology can precisely target and alter the genomes of animals, establish animal models of the hereditary diseases, and elucidate the relationship between the target gene and the disease phenotype, thereby providing a powerful approach to studying the pathogenic mechanisms underlying the hereditary eye diseases. In addition, correction of gene mutations by the genome editing brings a new hope to gene therapy for the hereditary eye diseases. This review introduces the molecular characteristics of 4 major enzymes used in the genome editing, including homing endonucleases, zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9), and summarizes the current applications of this technology in investigating the pathogenic mechanisms underlying the hereditary eye diseases. (Chin J Ophthalmol, 2017, 53: 386-371).

The genetic basis of reproductive isolation is a key problem in evolutionary biology, and house mice in the genus Mus provide a good model for addressing this problem. M. musculus musculus and M. m. domesticus diverged recently, hybridize in nature, are easily crossed in the lab, and are isolated largely by hybrid male sterility. Many genetic and genomic tools exist for mice, making them particularly tractable from a genetic standpoint. We have been studying the genetic basis of reproductive isolation in house mice through a combination of genome-wide analyses of introgression in natural populations and crosses in the laboratory using wild-derived inbred strains. In these crosses, the reduced fertility of hybrid males appears to be due mainly to post-meiotic disruptions of spermatogenesis. These males show no gross abnormalities in the structure or frequency of primary spermatocytes, but they have smaller testes, fewer sperm, more abnormal sperm head morphologies, and reduced fertility and fecundity. Both field and laboratory studies reveal a major role for the X chromosome in reproductive isolation. Construction of near-isogenic lines of mice through repeated backcrossing has helped identify regions of the X chromosome that are likely to underlie hybrid male sterility. Laboratory crosses also show that hybrid male sterility has a surprisingly complex genetic basis, despite the fact that these species diverged recently. Finally, sterile hybrid males show widespread over-expression of the X chromosome, raising the possibility that mis-regulation of gene expression on the X chromosome during spermatogenesis may be a major factor in the evolution of reproductive isolation. This work was supported by grants from NSF (DEB 0749004 and DEB 0642778) and NIH (GM074245-01) to MWN. (platform)

SummarySexual selection has been studied intensively, and is often strong in natural populations. Theoretical models, comparative studies and laboratory selection experiments all suggest that evolution driven by sexual selection should be rapid and common.However, there are relatively few documented cases of the contemporary evolution of secondary sexual traits in natural populations. Moreover, the causes are not always due to sexual selection, but often due to altered natural selection regimes, or an altered balance between natural and sexual selection.Here we discuss recent empirical studies that have demonstrated the contemporary evolution of secondary sexual traits in natural populations. Our examples include both continuous traits and discrete polymorphisms. Taxa in which the contemporary (rapid) evolution of secondary sexual traits have been demonstrated include fish, insects, mammals, reptiles and birds. The evolutionary rates of these changes range from 0·005 to 0·570 haldanes (arithmetic mean = 0·14; geometric mean = 0·095; median = 0·12).The relative rarity of examples could be explained by different genetic architectures of sexually selected traits compared with naturally selected traits, or by sexual selection regimes not being sustained in the long term. These factors could potentially slow down evolutionary rates of secondary sexual traits and make the detection of the contemporary evolution empirically more difficult.Promising and underutilized approaches to sexual selection research include reciprocal transplant experiments and estimation of sexual isolation between conspecific populations.