Genes and intergenic regions of the genome contribute differentially to a wide array of biological processes, the study of which is called functional genomics. Put simply, functional genomics is a way to understand what roles genes play in an organism.
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Functional genomics deals with how a particular gene dynamically expresses itself in specific contexts. Scientists researching this field use their knowledge of gene function to develop models that are capable of linking genotype to phenotype. Some common functional genomics approaches, based on the level of study, are DNA level (genomics and epigenomics), protein level (proteomics), metabolite level (metabolomics), and RNA level (transcriptomics).
Evolution of functional genomics
The earliest examples of functional genomics involved the study of organisms, such as bacteriophages, bacteria, budding yeast, etc., whose genomes could be mutated at random to identify the different genes associated with various developmental, physiological, or molecular processes. It was, however, difficult to use the same methods in the case of mammals owing to experimental barriers. These barriers arose, mainly, from the nature of the mammalian genome, which is diploid.
Mutating one gene left the copy intact, hindering traditional mutagenesis approaches. Further, mammalian genomes contain less than 2% protein-coding genes, which makes their use inefficient. These challenges have made mammalian biological research difficult for decades. RNAi and CRISPR-Cas9 are two powerful homology-based gene targeting techniques that have helped overcome the above-mentioned barriers.
RNA interference or RNAi
In previous research, scientists have attempted to use antisense RNA to knock down gene expression. They found that when antisense and sense RNA strands were delivered jointly, there were synergistic effects on gene silencing. While the first experiments were conducted on nematodes, the basic machinery applies to all eukaryotic systems.
In higher eukaryotes, the double-stranded RNA (dsRNA)-dependent silencing phenomena require three core components, namely, Drosha, Dicer, and Argonaute (Ago) gene family members. Conversion of various forms of dsRNA to smaller dsRNAs is carried out by Drosha and Dicer proteins. The responsibility of the Ago proteins is to use the sequence of these smaller dsRNAs to identify and target homologous RNAs.
Currently, two types of RNAi triggers are used to inhibit gene function in mammals. There are small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs). siRNAs contain 21-nucleotide of identity to a homologous mRNA target. Additionally, it also contains 19-nucleotide of dsRNA and a 2-nucleotide 3’ overhang. In contrast, Sh RNAs are RNA duplexes that contain a loop-like structure. The duplexes are of 23-29 nucleotides and the loop-like structure joins both strands of the duplex.
CRISPR-Cas9
CRISPR stands for Clustered, Regularly Interspaced, Short Palindromic Repeats, while CRISPR-Cas stands for CRISPR-associated. This pathway acts as an adaptive immune system in bacteria, thereby, providing resistance to genetic parasites.
To target exogenous genomic sequences, it uses a single guide RNA (sgRNA) in a protein effector nuclease. CRISPR-Cas systems are different from RNAi in that they can target and degrade DNA. sgRNA-directed genome editing in prokaryotes and eukaryotes has made use of this property. As an example, the CRISPR-Cas system from Streptococcus pyogenesto elicited robust RNA-guided gene editing in multiple eukaryotes, including mammals.
The CRISPR-Cas system has been shown to consist of two components, namely, a sgRNA and a Cas protein with the sgRNA having two main functions, i.e., target recognition and act as a structural RNA to form the sgRNA-Cas9 complex (tracrRNA). As mentioned earlier, the CRISPR-Cas system has been used in the case of mammalian cells, and in this case, the system requires the expression of a codon-optimized Cas9 gene.
The codon-optimized Cas9 gene, together with a nuclear localization sequence and the expression of sgRNA from an RNA polymerase III promoter, promotes gene editing.
Human functional genomics project
The Human Functional Genomics Project (HFGP) is a large-scale project whose aim is to find out more about the role played by genetics in shaping the human immune response. It seeks to characterize and better understand the heterogeneity in the human immune response using various analyses and functional phenotyping.
The analyses could be wide-ranging, i.e., DNA (genomic), RNA (transcriptomic), metabolites (metabolomics), or microbiome (microbiomics). In an interesting piece of research involving two healthy cohorts of 200 and 500 individuals, scientists investigated which genetic variants influence cytokine production in response to ex vivo stimulation. The subjects were challenged with bacterial, fungal, viral, and non-microbial stimuli.
Scientists observed a strong impact of genetic heritability on cytokine production capacity. Their research provided an extensive idea of the genetic variants that could have an impact on the production of six different cytokines in whole blood, mononuclear cells, and macrophages.
Functional genomics in plants
Functional genomics plays a vital role in plant sciences. It is being used in diverse applications such as producing alternative gluten-free grains, understanding plant processes and their response to climatic changes, etc. Several databases, for example, the Gramene database, are available that help studies related to functional genomics or conduct functional analysis of plants.
The Gramene database provides valuable information, to plant breeders and agricultural scientists, on metabolic pathways of various plant species and genomes, such that they can conduct a comparative study between species.
Functional genomics and drug discovery
The pharmaceutical industries have focused on decreasing the rate of failure associated with the identification of potential disease targets against which effective therapeutic agents would be developed.
In this context, experiments based on functional genomics provide evidence of the relationship between the potential disease target and their related disease, at the DNA/RNA/protein level. Functional genomics has also played an important role in guiding drug discovery for the treatment of cancer.
Sources:
- Functional Genomics. EMBL-EBI. (2021). [Online] Available at: www.ebi.ac.uk/.../
- Functional genomics. Paddison’s Lab. (2021). [Online] Available at: https://research.fredhutch.org/paddison/en/functional-genomics.html
- Li, Y. et al. (2016). A Functional Genomics Approach to Understand Variation in Cytokine Production in Humans. 164(4), pp. P1099-1110.E14,
- Bunnik, E. M., and Le Roch, K. G. (2013). An Introduction to Functional Genomics and Systems Biology. Advances in wound care, 2(9), pp. 490–498. https://doi.org/10.1089/wound.2012.0379
- Wilson, K.E. et al. (2004). Functional genomics and proteomics: application in neurosciences. Journal of Neurology, Neurosurgery & Psychiatry;75, pp.529-538.
Further Reading