Currently for multicellular systems and organisms, our technology is closely aligned with natural reproduction: we edit gametes or embryos, and rely on natural processes to differentiate genetically-identical cells into tissues. Gene editing methodology allows substantial improvements and inclusion of novel biochemical and molecular changes. Today our engineering abilities in plants are limited to stable integration of small genetic circuits (fewer than 200 kilobases). Examples of engineered modification of animals include genome editing of chicken embryos to produce virus resistance and the inactivation of porcine endogenous retroviruses in pigs for human organs transplants, but significant work is necessary before we are able to selectively edit and modify multicellular eukaryotes with confidence and consistency.
Generally, we need a better understanding of cell-to-cell interactions and to establish stable modifications within multicellular systems. Current state-of-the-art in multicellular engineering includes tools and technologies for some plants and fungi (such as Aspergillus), but most advances toward this goal have occurred in the engineering of a single cell type within a multicellular organism (for example, the introduction of the Polled trait into dairy cattle breeds) and germline engineering. An emerging technology in multicellular system engineering is cell-scaffolding (loading specialized cells onto engineered matrices) and enabled control over the three-dimensional shape and structure of a system. Advancements in engineering for tissue- and organ-on-a-chip technologies are also helping to bring about advancements in this area.
For related reading, please see Biomolecule, Pathway, and Circuit Engineering: Holistic, integrated design of multi-part genetic systems (i.e., circuits and pathways).
Breakthrough Capabilities & Milestones
Ability to control differentiation and de-differentiation of cells within a population.
On-demand, reproducible functionalization of simple micro-tissues or micro-consortia made up of two or more engineered cell types.
Variations in cell culture quality between trials and institutions.
Potential Solution: Investigate incubation conditions to identify and mitigate environmental sources of variability in cell behavior and growth.
Potential Solution: Identify informative biomarkers.
Programmable and regulatable pathways that can be induced to differentiate or de-differentiate somatic cells.
Bottleneck/Challenge: Gaps in understanding which genes and networks can be altered to control cell behavior.
Potential Solution: Experimental tools to co-regulate any desired set of multiple gene targets, such as via engineered transcription factors.
Ability to characterize and control the three-dimensional (3D) architecture of multicellular systems.
Characterize existing tissue components and standardize measurements to evaluate function.
Bottleneck/Challenge: Inconsistent reporting of matrix and cell performance resulting from differences in composition and tissue geometry.
Potential Solution: Generate a library of known cell types, matrices, and exogenous signalling molecules and characterize all combinations under identical conditions and geometries; measurements should include characteristics such as stress-strain response, degradation rates in serum, and immunogenicity.
Bottleneck/Challenge: Discovery of novel exogenous signalling molecules to regulate cell and tissue behavior.
Potential Solution: Evaluate specific factors (via high-throughput chemical screening) that can be supplemented to existing 3D matrices/scaffolds to induce drastic changes in cellular behavior (such as morphology, differentiation, tissue composition, matrix alignment).
Identification of novel 3D scaffold designs that can lead to desirable cellular properties.
Bottleneck/Challenge: Limited capacity for nutrient delivery (~100µm by diffusion alone).
Potential Solution: Develop synthetic microvascular networks, either by self-assembly of endothelial cells and pericytes or 3D patterning of tissues; these networks must be able to support cell growth within the scaffold and be robust to changes in tissue composition (including the introduction of additional cell types, mechanical forces, or chemical factors).
Bottleneck/Challenge: Heterogeneous cell seeding within a large scaffold.
Potential Solution: Enable and advance formation and production of extracellular matrix and methods to improve seeding (for example, forced flow of cells into tissue).
Create modular, synthetic communication circuits that can be implemented in tissues to allow for control of new or existing cellular communication systems.
Bottleneck/Challenge: Cell heterogeneity within tissue and ability to target only the cell type(s) of interest.
Potential Solution: Link integration or expression of a circuit to expression of a synthetic molecule in the target cell type.
Bottleneck/Challenge: Robustness against perturbations, including the various signalling molecules expressed in tissue.
Potential Solution: Build in redundant control to genetic circuits, and leverage advances in biomolecular design to use components with large induction ratios and minimal cross-talk with other circuit components.
Bottom-up design and construction of whole organs at the centimeter-length scale.
Bottleneck/Challenge: Nutrient delivery in organs.
Potential Solution: Assemble synthetic arterioles and venuoles to interface with capillaries, then couple these synthetic vascular networks with cell culture media perfusion strategies.
Bottleneck/Challenge: Large-scale assembly of substructures into complete tissues.
Potential Solution: Development of hierarchical assembly methods (either spatial isolation or stepwise assembly) to generate specific sub-tissues with complex geometries and defined cell compositions.
Bottleneck/Challenge: Understanding the principles of organ design; most efforts to date focus on recapitulation of existing organs or a reduced set of functions performed by a given organ.
Potential Solution: Prototyping synthetic organs to substitute for, complement, or enhance native organ function in a manner beyond recapitulation of evolved biology.
Ability to achieve stable non-heritable changes in somatic cells.
Routine delivery of biomolecule “effectors” (i.e., DNA, RNA, proteins) into slowly-dividing or non-dividing cells.
Bottleneck/Challenge: Lack of technologies for homogenous delivery of macromolecules into tissues.
Potential Solution: Further development of cell-penetrating nanoparticles and exosomes.
Generation of effective artificial epigenetic chromosomal states and maturation of the emerging field of chromatin engineering.
Bottleneck/Challenge: Incomplete functional characterization of natural chromatin.
Potential Solution: Engineered platforms to rapidly interrogate hundreds of structural, enzymatic, and synthetic chromatin proteins.
Bottleneck/Challenge: Uncertain causal relationship between genome/epigenome and cell behavior.
Potential Solution: Coordination of statistical genome/ epigenome association studies (GWAS/ EWAS) with experimental reconstruction of states to test and validate associations.
Ability to generate cell states that are stable and effective after the inducer/effector is removed in certain model tissues.
Bottleneck/Challenge: Gaps in understanding cell “homeostasis” and how biochemical processes inside cells are interconnected and reinforce each other.
Potential Solution: Development of experimentally-supported predictive (systems biology) models to predict the long-term impact of an artificial perturbation.
Bottleneck/Challenge: Gene-editing dependency; too much focus on transcriptional regulation in the nucleus.
Potential Solution: New tools to control self-perpetuating “post-translational” states (such as RNA and protein modification).
Potential Solution: Advances in organelle engineering (especially for mitochondria).
Nimble adaptation of somatic cell engineering technologies to any natural tissue at any developmental stage.
Bottleneck/Challenge: Gaps in understanding cell-type and tissue-type-specific barriers that enable cells to resist conversions.
Potential Solution: Further advancement of systems biology methods to quickly identify appropriate target genes, proteins, and molecular networks.
Ability to make predictable and precise, targeted, heritable changes through germline editing.
Complete sequence of select host genomes to allow design of targets for gene editing.
Bottleneck/Challenge: Genetic variation.
Potential Solution: Sequencing of specific transformation target lines.
Define and validate tissue-specific DNA parts in plants.
Bottleneck/Challenge: There has been a dearth of plant DNA parts (e.g., promoters) that have been systematically characterized. Although many genes have been described from transcriptome datasets as tissue-specific, the validation and characterization of their tissue specificity will be required for future plant synthetic biology efforts.
Potential Solution: Systematic characterization of various tissue-specific promoters in various plant species.
Efficient germline transformation systems developed in targeted hosts.
Bottleneck/Challenge: Transformation systems are limited and optimization is slow; efficiency is such that the molecular analysis burden is high.
Potential Solution: Increase transformation efficiency through new vector design components that will stimulate cell division during the time the DNA is introduced into the cell or enable improvement in high-throughput molecular analysis platforms to screen for those with the correct edits.
Ability to deliver transgene constructs to most (>90%) somatic cells in a higher eukaryote organism to rapidly prototype transgenic phenotypes.
Bottleneck/Challenge: Higher eukaryotes have relatively long timescales of organism development, making phenotype development too slow for effective research.
Potential Solution: Improve somatic cell nuclear transfer to embryos to speed multi-locus genome engineering for non-model organisms with long generation times.
Bottleneck/Challenge: Existing gene delivery technologies reach only a subset of cells in an intact organism.
Potential Solution: Enhance gene delivery technologies to approach organism-scale delivery.
Temporally controlled transgene expression that works on the scale of generations.
For example, kill switches that are activated only after a defined number of generations.
Bottleneck/Challenge: Robust molecular time-keeping methods.
Potential Solution: Design and implementation of robust synthetic cell cycle oscillators and other molecular timers.
Bottleneck/Challenge: Gene expression platforms that confer stable expression across multiple cell divisions (such as in primary cells).
Potential Solution: Development of stable, controllable, heritable extra-genomic expression platforms, including artificial chromosomes.
Bottleneck/Challenge: Spontaneous silencing of transgene constructs being expressed over long periods of time.
Potential Solution: Synthetic epigenetic mechanisms that interfere with or block natural silencing mechanisms.
Efficient gene editing in differentiated cells.
Bottleneck/Challenge: DNA folding is a physical blockade against gene-editing enzymes.
Potential Solution: Engineering the editing enzymes and/or helper proteins to unfold DNA.
Bottleneck/Challenge: Bacterial CRISPR particles induce an immunogenic response.
Potential Solution: Discovery of non-immunogenic variants.
Potential Solution: Development of “coating” particles or chemical tags.
Bottleneck/Challenge: Some cell types carry heterogeneous, naturally-modified genomes (such as immune cells).
Potential Solution: Delivery of gene expression cassettes that are not integrated into the chromosome.
Ability to domesticate engineered biological parts to confer immune tolerance in immunocompetent organisms.
Bottleneck/Challenge: Introduction of foreign proteins can induce immune rejection in immunocompetent organisms; the rules governing how such rejection is elicited by synthetic biology parts and how it may be circumvented are not yet clear.
Potential Solution: Generation toolboxes of “stealthed” parts that are unlikely to elicit immune rejection.
Potential Solution: Development of strategies for inducting active immune tolerance of synthetic biology parts.
Ability to coordinate engineered multicellular functions in intact organisms via orthogonal communication systems.
Bottleneck/Challenge: Generating synthetic analogs of coordinated processes, such as wound healing or immune protection, will likely require communication between engineered cells; co-opting native cell-cell signaling mechanisms is likely to exhibit cross-talk and cross-regulation with native systems.
Potential Solution: Generation of libraries of mutually orthogonal synthetic signaling molecules and receptors that can confer coordination across various length scales within an organism.
On-demand gene editing of organisms with desired traits.
Bottleneck/Challenge: Gene editing efficiency is low for multiple edits; limitations in what sequences can be edited due to CRISPR target-recognition constraints.
Potential Solution: Develop new CRISPR or other engineered enzymes that have expanded recognition sequences and efficiencies.
Routine, on-demand, efficient germline editing for any targeted hosts of interest at high-throughput scale.
Bottleneck/Challenge: Different and diverse transformation systems needed across species.
Potential Solution: Develop a process that is automated from preparation of embryos to transformation to selection/identification of successfully-edited embryos.
- Looi, F. Y., Baker, M. L., Townson, T., Richard, M., Novak, B., Doran, T. J., & Short, K. R. (2018). Creating disease resistant chickens: A viable solution to avian influenza? Viruses, 10(10). View publication.; Sid, H., & Schusser, B. (2018). Applications of gene editing in chickens: A new era is on the horizon. Frontiers in Genetics, 9, 456. View publication.
- Niu, D., Wei, H.-J., Lin, L., George, H., Wang, T., Lee, I.-H., … Yang, L. (2017). Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science, 357(6357), 1303–1307. View publication.; Ross, M. J., & Coates, P. T. (2018). Using CRISPR to inactivate endogenous retroviruses in pigs: an important step toward safe xenotransplantation? Kidney International, 93(1), 4–6. View publication.
- Farré, G., Blancquaert, D., Capell, T., Van Der Straeten, D., Christou, P., & Zhu, C. (2014). Engineering complex metabolic pathways in plants. Annual Review of Plant Biology, 65, 187–223. View publication.
- Lubertozzi, D., & Keasling, J. D. (2009). Developing Aspergillus as a host for heterologous expression. Biotechnology Advances, 27(1), 53–75. View publication.
- Van Enennaam, A. (2018, June 12). Use of Gene Editing to Introduce the Polled Trait into Elite Germplasm. Retrieved from https://www.dairyherd.com/article/use-gene-editing-introduce-polled-trait-elite-germplasm.
Last updated: June 19, 2019