CRISPR and Genetic Engineering

Multiplex Genome Engineering Using CRISPR/Cas Systems

Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

Genome engineering using the CRISPR-Cas9 system

RNA-Guided Human Genome Engineering via Cas9

Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.

The new frontier of genome engineering with CRISPR-Cas9

The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.

Applying evolutionary biology to address global challenges

BACKGROUND Differences among species in their ability to adapt to environmental change threaten biodiversity, human health, food security, and natural resource availability. Pathogens, pests, and cancers often quickly evolve resistance to control measures, whereas crops, livestock, wild species, and human beings often do not adapt fast enough to cope with climate change, habitat loss, toxicants, and lifestyle change. To address these challenges, practices based on evolutionary biology can promote sustainable outcomes via strategic manipulation of genetic, developmental, and environmental factors. Successful strategies effectively slow unwanted evolution and reduce fitness in costly species or improve performance of valued organisms by reducing phenotype-environment mismatch or increasing group productivity. Tactics of applied evolutionary biology range broadly, from common policies that promote public health or preserve habitat for threatened species—but are easily overlooked as having an evolutionary rationale, to the engineering of new genomes. ADVANCES The scope and development of current tactics vary widely. In particular, genetic engineering attracts much attention (and controversy) but now is used mainly for traits under simple genetic control. Human gene therapy, which mainly involves more complex controls, has yet to be applied successfully at large scales. In contrast, other methods to alter complex traits are improving. These include artificial selection for drought- and flood-tolerant crops through bioinformatics and application of “life course” approaches in medicine to reduce human metabolic disorders. Successful control of unwanted evolution depends on governance initiatives that address challenges arising from both natural and social factors. Principal among these challenges are (i) global transfer of genes and selection agents; (ii) interlinked evolution across traditional sectors of society (environment, food, and health); and (iii) conflicts between individual and group incentives that threaten regulation of antibiotic use and crop refuges. Evolutionarily informed practices are a newer prospect in some fields and require more systematic research, as well as ethical consideration—for example, in attempts to protect wild species through assisted migration, in the choice of source populations for restoration, or in genetic engineering. OUTLOOK A more unified platform will better convey the value of evolutionary methods to the public, scientists, and decision-makers. For researchers and practitioners, applications may be expanded to other disciplines, such as in the transfer of refuge strategies that slow resistance evolution in agriculture to slow unwanted evolution elsewhere (for example, cancer resistance or harvest-induced evolution). For policy-makers, adoption of practices that minimize unwanted evolution and reduce phenotype-environment mismatch in valued species is likely essential to achieve the forthcoming Sustainable Development Goals and the 2020 Aichi Biodiversity Targets.

The integrative biology of genetic dominance

Dominance is a basic property of inheritance systems describing the link between a diploid genotype at a single locus and the resulting phenotype. Models for the evolution of dominance have long been framed as an opposition between the irreconcilable views of Fisher in 1928 supporting the role of largely elusive dominance modifiers and Wright in 1929, who viewed dominance as an emerging property of the structure of enzymatic pathways. Recent theoretical and empirical advances however suggest that these opposing views can be reconciled, notably using models investigating the regulation of gene expression and developmental processes. In this more comprehensive framework, phenotypic dominance emerges from departures from linearity between any levels of integration in the genotype-to-phenotype map. Here, we review how these different models illuminate the emergence and evolution of dominance. We then detail recent empirical studies shedding new light on the diversity of molecular and physiological mechanisms underlying dominance and its evolution. By reconciling population genetics and functional biology, we hope our review will facilitate cross-talk among research fields in the integrative study of dominance evolution.

Comparative Molecular Biology Approaches for the Production of Poliovirus Virus-Like Particles Using <i>Pichia pastoris</i>

Although the current poliovirus immunization program has been extremely successful in reducing the number of cases of paralytic polio worldwide, now more cases are caused by vaccine-derived polioviruses than by wild poliovirus. Switching to inactivated poliovirus vaccines will reduce this over time; however, their production requires the growth of large amounts of virus. This biosafety concern can be addressed by producing just the virus capsid. The capsid serves to protect the genetic material, which causes disease when introduced into a cell. Therefore, empty capsids (virus-like particles [VLPs]), which lack the viral RNA genome, are safe both to make and to use. We exploit yeast as a versatile model expression system to produce VLPs, and here we specifically highlight the potential of this system to supply next-generation poliovirus vaccines to secure a polio-free world for the future.

Gene-Environment Interactions and Epigenetic Regulation in Autism Etiology through Multi-Omics Integration and Computational Biology Approaches

Autism Spectrum Disorder (ASD) is a multifactorial neurodevelopmental condition characterized by substantial genetic heterogeneity and complex environmental influences. Emerging evidence suggests that gene-environment interactions, mediated through dynamic epigenetic mechanisms, play a critical role in modulating neurodevelopmental trajectories implicated in ASD. This review synthesizes current advances in understanding the etiological interplay between genetic variants, environmental exposures, and epigenetic regulation, with a focus on DNA methylation, histone modifications, and non-coding RNAs. We explore how these layers of molecular control intersect to dysregulate neurodevelopmental gene networks and contribute to ASD pathophysiology. Central to this investigation is the integration of multi-omics platforms— encompassing genomics, transcriptomics, epigenomics, proteomics, and metabolomics—supported by computational biology, machine learning, and systems-level modeling frameworks. These technologies facilitate the identification of molecular subtypes, predictive biomarkers, and regulatory circuits associated with ASD. Furthermore, we examine the translational implications of these findings in the context of precision medicine, including early diagnosis, patient stratification, and individualized therapeutic development. Despite the challenges of data heterogeneity, scalability, and interpretability, the integration of high-dimensional biological data holds transformative potential for elucidating ASD etiology and advancing targeted interventions.

University students’ knowledge and readiness to practice genomic nursing in Nigeria

Genetic nursing education provides knowledge of traits and inherited diseases. This has not been well integrated into nursing practice in Nigeria. This study evaluated university nursing students’ knowledge of genomic concepts and readiness to practice genomic nursing in Nigeria. A cross-sectional study was conducted. Three universities were purposively selected in Nigeria. A total of 136 participants were recruited using convenient sampling technique. A modified Genetic Nursing Concept Inventory questionnaire was distributed to participants in their classrooms. Data were analyzed with SPSS (23); descriptive data were presented in tables and figures with their mean and standard deviations. Chi-square test and multivariate analysis were used to ascertain association between variables at p < 0.05 level of significance. Findings indicated that participants have poor knowledge (89%) and lack readiness (66%) to practice genomic nursing in Nigeria. Their knowledge influenced their readiness (χ2 = 21.033, df = 1, p = 0.001). Institution type was the most consistent predictor of knowledge (χ2 = 48.586, df = 2, p = 0.001) and readiness (OR = 14.817, p = 0.326, C.I. = 3.190, 319.57) as those in federal institution were more knowledgeable and prepared to practice genetic nursing. Participants perceived that poor funding, lack of trained personnel, and social/environmental factors could affect their readiness to practice genetic nursing. The study has brought to the fore that nursing students have low knowledge and were not ready to practice genetic nursing, efforts should be made to look into the adequacy of nursing training on genetic nursing and strategies needed for its integration in education and practice.