Functional Genomics Screening
Functional genomics enables the identification and understanding of gene functions and their involvement in various biochemical, cellular, and physiological pathways. With the availability of complete genome sequences and advanced genome-editing tools, functional genomics allows large-scale analysis of gene function. By performing genomic screens, researchers can systematically alter gene activity to observe resulting phenotypic changes, helping to unravel complex biological pathways and disease mechanisms. This approach facilitates the discovery of novel drug targets and therapeutic interventions. Two key methods in functional genomics are forward genetic screening, where genes are modified to select for desired phenotypes, and reverse genetic screening, which analyzes the effects of disrupting specific genes or gene combinations on cellular or organismal traits.
Overview
Advances in Functional Genomic Screens
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.