Functional Genomics Screening
Functional genomics enables the discovery of gene function and involvement in biochemical, cellular, and physiological pathways. The availability of complete genome sequences, combined with readily programmable tools to modify the genome, allows these analyses to be performed on a genome-wide scale. The basic aim of a genomic screen is to understand the emergence of a specific phenotype by modifying gene function in a targeted, purposeful manner. When individual genes are deleted or modulated in a cell or organism, changes in phenotype or behavior can be directly or indirectly observed through carefully planned experiments. Functional genomics screening allows this analysis to be performed in a systematic and parallelized manner, elucidating intricate pathways and disease states and facilitating novel drug target identification.
There are two fundamental ways that functional genomics can link genetics to phenotype. Forward genetic screening involves modifying many genes, selecting for the cells or organisms with the phenotype of interest, and then identifying the genes whose modulation triggered the phenotypic change. Reverse genetic screening analyzes the phenotype of cells or organisms following the disruption of a specific gene or combination of genes.
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Advances in gene editing, gene silencing, gene modulation, next generation sequencing (NGS), and phenotypic screening technologies enable efficient execution of functional genomic screens in a wide variety of model systems.
- RNA interference (RNAi): Several types of RNAi reagents can be employed for gene silencing, including long double-stranded RNA (dsRNA), synthetic small interfering RNA (siRNA), and short hairpin RNA (shRNA). These RNAi reagents are introduced to cells by direct transfection of the modulating factor (siRNAs and dsRNAs), by transfection of DNA encoding a promoter-driven shRNA, or by viral transduction methods using lentiviral constructs with cloned shRNA cassettes. dsRNA and siRNA can be used in arrayed screens for high-throughput screening, while shRNAs can be introduced to cell populations in arrayed or pooled screens for high-throughput analysis, with pooled screens using next generation sequencing (NGS) for deconvolution.
- CRISPR-Cas systems: CRISPR (clustered regularly interspaced short palindromic repeat) systems can be used to manipulate the genomes, transcriptomes, and epigenomes of mammalian cells. In CRISPR-Cas9 gene editing, a Cas9 nuclease is targeted to a specific locus using a guide RNA. Depending on the Cas9 variant employed, CRISPR can be used to genetically silence transcript production by introducing frameshift mutations, repressing transcription machinery, recruiting transcription factors to activate expression, inducing targeted point mutations, or modifying epigenetic markers. Similar to RNAi, CRISPR can be introduced directly as an RNP complex in arrayed screens or as plasmid DNA or lentivirus for both pooled and arrayed screening applications. CRISPR pools, libraries, and arrays facilitate exceptionally versatile, high-throughput screening of genes for functional analysis. Genome modulation screening is also possible with a nuclease-free CRISPR system that utilizes enzymatically inactive dCas9 combined with transcriptional effectors that either activate (CRISPRa) or inhibit (CRISPRi) gene transcription, leading to an increase or decrease in gene expression.
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