A primer on the application of biotechnology for conservation: technology, tools, and targets
We have devised this report to highlight the potential for biotechnology and synthetic biology to revolutionize conservation practice in the world’s oceans. We define these two important fields below:
Biotechnology broadly refers to the methods and processes by which living organisms are modified by humans for human purposes. This can include more historic processes such as animal and plant domestication and subsequent artificial selection to enhance particular traits. Currently, it now includes more advanced methods such as genetic engineering and cell and tissue culture techniques. Biotechnology is largely informed by a variety of fields ranging from molecular biology, to chemical engineering and computer science.
Synthetic biology refers to the application of biotechnology toward the development of artificial biological systems for research, engineering, consumer, medical, and increasingly, conservation applications. Synthetic biology in conservation may include targeted engineering of DNA sequence to enhance species fitness (i.e. facilitated adaptation), creation of novel microbial organisms capable of degrading environmental pollutants (bioremediation), and the development of viable alternatives to animal products (i.e. clean-meat, rFC, polycarbonate or cellulose sponges). Advancements in synthetic biology are largely contingent on understanding how cells are naturally programmed to do what they inherently do, so that we may reprogram them to function in specific ways that are tailored to a particular outcome.
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Bioinformatics is an interdisciplinary field that incorporates biology, computer science, engineering, mathematics and statistics in order to understand biological data. Bioinformaticians produce software and software pipelines that enable efficient processing of genomic data, such that sequence reads can be assembled into genomes, genomes can be annotated, and genetic variants can be identified, which can then be used in specific analyses such as population assignment or identification of adaptive variants, to name a few.
A genome-wide association study (GWAS), also known as whole genome association study (WGAS), is an observational study of a genome-wide set of genetic variants in different individuals to see if any variant is associated with a trait. GWASs typically focus on associations between single-nucleotide polymorphisms (SNPs) and traits like major human diseases, but can equally be applied to any other genetic variants of any organism. GWA studies investigate the entire genome, in contrast to methods that specifically test a small number of pre-specified genetic regions. Any two genomes differ in millions of different ways including small variations in the individual nucleotides of the genomes (SNPs) as well as many larger variations, such as deletions, insertions, and copy number variations. Any of these may cause alterations in an individual’s traits. Most GWA studies to date have been targeted at biomedical research, but are increasingly used to study wild species. For example, a genome-wide survey of SNPs in bottlenose dolphins allowed researchers to identify five candidate genes involved in host resistance to cetacean morbillivirus (Batley et al., 2014), providing potential targets for vaccine or therapy design.
A genoscape is a map of genetically distinct populations across geographical space. Genoscapes correlate genetic variants to dynamic population movements from breeding grounds to dispersal patterns. Genoscapes can be informative for delineating boundaries for protected areas, sustainable harvests, and restocking efforts. A genoscape also directly informs the field of landscape genomics, whereby the geographic distribution of genetic variation can be mapped and used to understand how gene flow between populations is related to specific features of the landscape, including anthropogenic barriers and habitat fragmentation. It may also inform the distribution of critical adaptive traits such as disease resistance or climate change tolerance.
Population genetics is the study of genetic differences within and between populations. In theory, a population’s genetic composition can be predicted over time if there is no gene flow, no selection, no genetic drift, and very large (infinite) population size. No natural population meets all these conditions, so allele frequencies change over time causing populations to develop differences. Genetic diversity can be lost within a population due to random sampling of alleles across generations, a process known as drift, and this occurs more rapidly in small populations. Many species exist as a network of several connected populations that are linked by dispersal, thereby replacing diversity that may be lost locally over time. Population genomics offers a higher resolution for estimating neutral genetic diversity, as well as the opportunity to understand how selection shapes genetic differences between populations.
Several tools and technologies have proven critical in synthetic biology as they allow us to investigate and compare the entire complement of DNA and RNA sequences between organisms across the tree of life, and allow us to cultivate and preserve cells in very particular ways. These technologies are described below:
Ancient DNA is a term used to describe genetic material preserved in degraded post-mortem biological remains such as museum specimens, including that of extinct species. Ancient DNA is an incredibly useful means of describing a variety of historic ecological attributes on the scales of both individual species and communities. Samples can be used to reconstruct former communities and species interactions (Bellemain et al., 2013), clarify the demographic history of a population (Barnes, 2002), gain insight into genomic sequences of extinct species (Miller et al., 2008), and reveal historic haplotypes and alleles that may have been lost in a population over time (Campos et al., 2010).
DNA barcoding uses short genetic markers to identify specific organisms. It is possible to take an unknown tissue sample and query small fragments of its DNA sequence against reference databases of known genetic barcodes to identify the exact species from which the tissue was derived. The Barcode of Life Data Systems (BOLD) is the largest reference library available for annotating sequences of unknown origin. DNA barcoding can be broadly divided into two main approaches: a single-species approach (targeted barcoding) or a multispecies approach (metabarcoding).
Targeted barcoding is aimed at detecting a single species, through the use of a primer that selectively targets that species’ DNA. It is useful for detecting the presence of species of interest, such as rare and endangered or invasive species by sampling environments (i.e. soil, water; see eDNA below) where they potentially occur.
Metabarcoding, by contrast, simultaneously identifies multiple taxa from a sample without the need for a priori knowledge of the species that are likely to be present. The difference between the two can be informally described as “see if a certain species is here” (single-species barcoding) versus “see what species are here” (metabarcoding). Metabarcoding thus allows for characterization of full communities, dietary diversity (fecal metagenomics), or to passively monitor for new/unexpected invasive species or pathogens.
Cryopreservation is the process of preserving cells, tissues or whole organisms by cooling them to temperatures between -80 °C and -196°C. At these temperatures, chemical and enzymatic activity is largely halted. Cryoprotectants are injected into or used to coat the sample to prevent the formation of ice crystals that would otherwise damage cell tissues. Cryopreservation has already acted as an effective insurance policy to maintain the genetic diversity of many wildlife and agriculture species (Rall 1993; Zhang 2011; Woelders and Hiemstra 2011). The hope is that in the future these cells such as egg, sperm, larvae, etc. can be returned to an active state to carry out their biological functions. Cryopreservation facilities currently harbor a very small number of marine taxonomic groups. Of these, commercial species, along with a few model species and species of special concern dominate banked collections (Martínez-Páramo et al., 2017). However, these focused efforts have led to major gaps across the marine tree of life, and there are currently large groups of taxa whose specimen are rarely or never preserved.
Environmental DNA (eDNA) is a non-invasive method for detecting and identifying species that were recently present in a specific location as indicated from cells detected in air, soil, and water samples taken from the environment. Small volumes of ocean water contain cells from all species that were recently in the vicinity of the collection area. Paired with DNA barcoding, eDNA samples can identify all cells within a sample often to the species level (Stat et al., 2017). Thus, eDNA can establish reliable distribution information on all present species, including rare, cryptic, nocturnal, microscopic, and even visibly indistinguishable species (Boussarie et al., 2018; Fukumoto et al., 2015; Kumar et al., 2009; Laramie et al., 2015) without sampling them directly. This technique has several conservation applications including determining the habitat preferences of species of special concern (Laramie et al., 2015), and for invasive species management (Deiner et al., 2015; Smart et al., 2015). Another benefit is that eDNA sequencing is much less costly to use than traditional biological surveys, which can require extensive resources to implement. Finally, eDNA can often be collected in tandem with other surveys, rather than requiring a dedicated sampling trip.
Genomic DNA sequences are fixed, but rates of transcription into RNA and translation into proteins fluctuate widely and rapidly depending on environmental and physiological needs. Recent advances in molecular techniques have enabled researchers to quantify the precise amount of RNA molecules and proteins that are present in a sample at a particular time, which are major contributors to the appearance and behavior of an organism. Comparing samples from individuals under different circumstances such as environmental stressors, life history stages, and exhibiting different physical characteristics thus facilitates a new understanding of how genes and proteins are involved in the adaptive response to stimuli (Storey and Wu, 2013). Such knowledge has important conservation implications as it can help predict how species will respond to environmental changes and can be used to inform selective breeding and genetic engineering.
DNA sequencing is simply determining the order of nucleotides in a particular string of DNA. Sequencing all of the DNA of an organism is called whole genome sequencing, which provides perhaps the most critical information for documenting and understanding processes of biodiversity. Sequences allow researchers to calculate relatedness both within and between species, identify physical traits and geographic associations with genetic sequences, and quantify genetic variation on multiple scales, from individuals, to populations, to entire communities. Knowing a genomic sequence can increase speed and efficiency of species monitoring by allowing researchers to look for signals of selection (Therkildsen et al., 2013), estimate the nature and timing of demographic events (Excoffier et al., 2013), and identify the geographic origin for any given sample within a species (Helyar et al., 2011). Decreased costs of sequencing and technological innovations that take advantage of the 3-D structure of DNA enable entire genomes to be sequenced at low cost and assembled to chromosome-level resolution. It is now common and affordable to investigate many thousands of markers across hundreds of individuals using whole genome resequencing (WGR); targeted capture, or reduced representation library sequencing.
An important component of synthetic biology involves the direct manipulations of genomes so that cells and organisms can be programmed to perform in a specific manner, as well as recapitulation of biologic environments for the production cells and cellular products (i.e. cell culture). There are several important tools and mechanisms relevant to conservation. These are defined below:
Genome editing refers to the process by which DNA is inserted, deleted, modified or replaced at a specific location in the genome of a living organism. Technologies that enable this process include TALENS, Zinc fingers, and most recently CRISPR. Genome editing makes it possible to study gene functions in plants and animals and advance the field of synthetic biology by modifying genes and genomes in very specific ways and toward very specific goals.
Cell culture involves the growth of cells under controlled conditions, generally outside of their natural environment. The cells are provided a media containing nutrients and potentially specific chemical and mechanical stimuli which mimic true biologic and physiochemical conditions that direct the development of the cells toward a desired end point or phenotype. Generally, primary cells harvested directly from a living organism have a finite lifespan. Immortalized cell lines have been established for a variety of species and tissue types. Cell culture directly supports recombinant DNA/gene editing technology, vaccine development, and tissue engineering for biomedical and commercial purposes.
Cellular agriculture refers to the use of molecular and cell-culture methods toward the production of agricultural products that are otherwise obtained through the harvest of whole organisms, and generally falls into two main categories of production. Tissue-engineering involves the harvesting and culturing of primordial cells (i.e. stem cells) which can be grown on a scaffold with the right chemical signals to stimulate differentiation and growth into consumable tissue (i.e. muscle). Acellular production, on the other hand, involves the use of recombinant DNA technology to engineer microbes to produce desirable organic molecules such as proteins and fats (i.e. Yamashita et al. 1987). The latter approach relies on commonly used industrial biotechnologies and so may be more immediately scalable, whereas tissue engineering requires a greater research investment to develop cell culturing protocols that properly mimic the chemical and biological environments of the target species or tissue (Stephens, et al. (2018)
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is the technology that is revolutionizing genomic engineering. CRISPRs are short sequences that originate from viral genomes and have been incorporated into the bacterial genome to protect against viruses. Specialized proteins (CRISPR associated proteins- Cas) process these sequences and cut matching viral DNA sequences. Plasmids containing Cas genes and specifically constructed CRISPRs can be introduced into eukaryotic cells so that the eukaryotic genome can be cut at any desired position. The resulting DNA cut can then be repaired by natural cellular processes using the sequence of a different allele, or even a gene from another species, to make a permanent change to the genome.
A gene drive is a genetic engineering technology that manipulates sexually reproducing species, excluding viruses and bacteria) to prophet a particular suite of genes through a population. The technique can be used to add, delete, disrupt, or modify genes. Gene drives have been proposed as a means to genetically modify specific populations and entire species, specifically problematic species such as disease-vectoring insects, invasive species, and pesticide-resistant pest species.
Given the opposition to genetically modified organisms, gene drive technology has yet to be tested in the wild. However, it has proven incredibly powerful in laboratory settings. For example, female fertility genes were identified and knocked out in malaria-carrying mosquitoes using a gene drive carrying a CRISPR/Cas9 system. This sterility genotype was then passed on to between 91% and 99% of progeny, leading to significantly fewer offspring in the population and promising results for disease control (Hammond et al 2016). Similarly, a rodent pest control study estimated that a single introduction of just 100 mice carrying a gene drive sequence could eradicate a population of 50,000 mice within four to five years (Prowse et al 2017).
Researchers and environmentalists worry that gene drives could become invasive, spreading unintentionally far in nature with undesired effects on non-target species through hybridization. Despite their clear potential to improve ecosystem function, these cutting-edge techniques need to be fine-tuned and thoughtfully evaluated before gene-edited organisms are released in to the wild (Mout et al 2017; Spicer and Molnar 2018).
Guidelines for research on gene drives were spelled out in a June 2016 report from the National Academy of Sciences, titled Gene Drives on the Horizon: Advancing Science, Navigating Uncertainty, and Aligning Research with Public Values. The report concludes that “the potential benefits of gene drives for basic and applied research are significant and justify proceeding with laboratory research and highly controlled field trials.”
RNA interference, or RNAi, silences or suppresses the expression of specific genes of interest (often referred to as gene-silencing). Silencing of a single salivary gene, for instance, was shown to be lethal in pea aphids (Mutti et al., 2006). Knockdown of a sex determination gene led to the production of an entirely male progeny of red flour beetles (Shukla and Palli, 2012) and crustaceans exposed to heat stress exhibited much lower survival rates when their heat shock protein transcripts had been silenced compared to unaltered transcriptomes (Iryani et al., 2017).
Technologies that enable the cloning and production of individuals to augment diversity and population size:
In Vitro Fertilization (IVF) is currently the most universal pathway used to aid in the conservation of mammals, particularly for species that do not readily reproduce in captivity.
Interspecies Somatic Nuclear Transfer (iSCNT) involves the transfer of a nucleus from any cell type into a donor oocyte of an appropriate surrogate. The power of this technology is that non-germ line tissues, which are often the only type preserved in biobanks, can be used to recapitulate genomes from individuals that cannot directly contribute to the gene pool.
Stem Cell Embryogenesis (SCE) has been proven to be feasible in laboratory mice and could overtake SCNT cloning as the leading mammalian reproductive technology in the coming decades. The revolutionary potential of the technology is that it can reduce the reproductive resources needed to yield offspring. In SCE, a skin cell is first transformed into a stem cell (called an induced pluripotent stem cell, or iPSC). Once in stem cell form, it can be reprogrammed to develop into sperm or egg cells. Once sperm and egg cells are made scientists can use IVF to generate a genetically diverse embryo, implant the embryo into a surrogate mother and give birth to a new unique individual. This process is currently being pioneered to attempt to save the Northern White Rhinoceros from extinction.
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