What is genome-wide siRNA screening?
Here we describe a genome-wide siRNA screen to identify genes regulating the human macrophage TNF-α response to LPS. We include a secondary validation screen conducted with six independent siRNAs per gene to facilitate removal of off-target screen hits.
What is siRNA screening?
Like genetic screening, RNAi screening allows for identification of genes relevant to a given pathway, structure or function via association of a mutant phenotype with gene knockdown. Like chemical screening, RNAi screening is amenable to miniaturization and automation, facilitating high-throughput studies.
What is genome-wide RNAi screen?
Abstract. Genome-wide RNA interference (RNAi) screening is an emerging and powerful technique for genetic screens, which can be divided into arrayed RNAi screen and pooled RNAi screen/selection based on different screening strategies.
What are the characteristics of siRNA?
Structure. Naturally occurring siRNAs have a well-defined structure that is a short (usually 20 to 24-bp) double-stranded RNA (dsRNA) with phosphorylated 5′ ends and hydroxylated 3′ ends with two overhanging nucleotides. The Dicer enzyme catalyzes production of siRNAs from long dsRNAs and small hairpin RNAs.
What is shRNA knockdown?
shRNA molecules are processed within the cell to form siRNA which in turn knock down gene expression. The benefit of shRNA is that they can be incorporated into plasmid vectors and integrated into genomic DNA for longer-term or stable expression, and thus longer knockdown of the target mRNA.
How does RNA interference regulate gene expression?
RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression.
What is the role of siRNA?
siRNAs. siRNAs are highly specific and usually synthesized to reduce the translation of specific messenger RNAs (mRNAs). This is done to reduce the synthesis of particular proteins. They form from double-stranded RNA transcribed and then cut to size in the nucleus before releasing into the cytoplasm.
What is the difference between siRNA and miRNA?
Discovered a little over two decades ago, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are noncoding RNAs with important roles in gene regulation. The major difference between siRNAs and miRNAs is that the former are highly specific with only one mRNA target, whereas the latter have multiple targets.
How are genome wide CRISPR Cas9 knockout screens used?
Genome-wide CRISPR-Cas9 knockout screens aim to elucidate the relationship between genotype and phenotype by ablating gene expression on a genome-wide scale and studying the resulting phenotypic alterations.
How is the specificity of the sgRNA determined?
The specificity of the sgRNA is determined by a 20-nt sequence, homologous to the genomic locus of interest, and the binding to Cas9 is mediated by a constant scaffold region of the sgRNA. The desired target site must be immediately followed (5’ to 3’) by a conserved 3 nucleotide protospacer adjacent motif (PAM).
How is CRISPR used for genome wide loss of function?
Targeted gene knockout using CRISPR/Cas9 requires the use of a delivery system to introduce the sgRNA and Cas9 into the cell. Although a number of different delivery systems are potentially available for CRISPR, genome-wide loss-of-function screens are predominantly carried out using third generation lentiviral vectors.
How are sgRNAs created to prevent false positives?
Multiple sgRNAs (at least 4-6) should be created against every single gene to limit false-positive detection, and negative control sgRNAs with no known targets should be included. The sgRNAs are then created by in situ synthesis, amplified by PCR, and cloned into a vector delivery system.