RNA Interference (RNAi) Quiz

RNA Interference: Mechanisms and Applications

• Gene silencing via antisense involves inserting a copy of the target gene in reverse orientation, producing sense and antisense mRNA that base pair and impede translation.
• In plants, antisense RNA was less efficient in suppressing gene expression than sense RNA, leading to the discovery of cosuppression mediated by short interfering RNA (siRNA) molecules.
• RNA interference (RNAi) is a gene silencing method that prevents mRNA translation using siRNA or microRNA (miRNA), with miRNA being endogenous and siRNA being introduced into the cell.
• miRNA and siRNA are small RNA molecules involved in natural gene silencing by inhibiting translation or promoting mRNA degradation.
• In nature, RNAi controls developmental timing, protects against RNA viruses, and regulates transposon activity.
• miRNA synthesis involves transcription by RNA Polymerase II/III, processing to produce mature miRNA, and subsequent binding to promote target mRNA degradation.
• Drosha and Dicer are ribonucleases that produce siRNA from pri- and pre-miRNA, with Drosha functioning in the nucleus and Dicer in the cytosol as part of the RNA-induced silencing complex (RISC).
• Eukaryotic genomes contain miRNAs that target mRNAs for gene silencing.
• The structure of RNAi gene and transcript includes portions identical and complementary to the mRNA target, allowing for identification and degradation of the target mRNA.
• RNAi is a powerful tool in molecular biology, used in biotechnology for gene silencing and regulation of gene expression.
• Dicer can recognize and cleave externally supplied dsRNAs to produce siRNA, which regulates gene expression through degradation or translation impeding of targeted mRNA.
• In nematodes, RNA-dependent RNA Polymerases (rdRP) can amplify siRNA to strengthen gene silencing, while in human cells, RNAi can be induced by introducing shRNA that is identical/homologous to the target gene.

Gene Editing Tools: TALENs, ZFNs, and CRISPR/Cas9

• Transcription Activator-Like Effector (TALE) proteins are secreted by Xanthomonas bacteria during plant infections and are known for their precision in recognizing single base pairs through repeat variable residues.
• TALEs are combined with Fok1 endonuclease to create TALENs, which enable targeted DNA cleavage at specific locations.
• TALENs induce double-strand breaks with sticky ends, providing advantages in ease of design for new genomic targets and higher specificity compared to other technologies.
• Zinc-Finger Nucleases (ZFNs) play a crucial role in genome editing by inducing double-strand breaks at precise genomic locations, allowing targeted mutations or insertion of desired sequences during repair mechanisms.
• ZFNs consist of zinc-finger modules or motifs and a FokI-derived cleavage domain, allowing for independent manipulation of binding and cleavage domains.
• ZFNs are considered broad editors, but some zinc-finger modules display unreliable binding or off-target effects, posing challenges in their application with standardized plasmids.
• The CRISPR system, particularly the type II CRISPR system, employs the Cas9 protein and a guiding RNA molecule to induce precise DNA cleavage.
• The CRISPR system utilizes a bacterial defense mechanism against viral infections, where specific fragments of the invader's genetic material are captured as "spacers" between identical "repeats" to form a memory bank.
• The CRISPR/Cas9 complex patrols the bacterial cell, searching for target DNA sequences that match the guide RNA, recognizing a specific region called PAM (protospacer adjacent motif) in the DNA to ensure specificity.
• When the complex locates its target, the Cas protein introduces a double-strand break in the DNA, activating the cell's natural repair mechanisms, which include non-homologous end joining (NHEJ) or homology-directed repair (HDR).
• The key advantage of CRISPR lies in its adaptability and efficiency in guiding cleavage, allowing for the production of a single RNA molecule that guides cleavage in conjunction with Cas9, offering a straightforward and effective way to edit genes.
• CRISPR's simplicity allows for the production of a single RNA molecule that guides cleavage in conjunction with Cas9, offering a straightforward and effective way to edit genes.

Gene Editing Tools: TALENs, ZFNs, and CRISPR/Cas9

• Transcription Activator-Like Effector (TALE) proteins are secreted by Xanthomonas bacteria during plant infections and are known for their precision in recognizing single base pairs through repeat variable residues.
• TALEs are combined with Fok1 endonuclease to create TALENs, which enable targeted DNA cleavage at specific locations.
• TALENs induce double-strand breaks with sticky ends, providing advantages in ease of design for new genomic targets and higher specificity compared to other technologies.
• Zinc-Finger Nucleases (ZFNs) play a crucial role in genome editing by inducing double-strand breaks at precise genomic locations, allowing targeted mutations or insertion of desired sequences during repair mechanisms.
• ZFNs consist of zinc-finger modules or motifs and a FokI-derived cleavage domain, allowing for independent manipulation of binding and cleavage domains.
• ZFNs are considered broad editors, but some zinc-finger modules display unreliable binding or off-target effects, posing challenges in their application with standardized plasmids.
• The CRISPR system, particularly the type II CRISPR system, employs the Cas9 protein and a guiding RNA molecule to induce precise DNA cleavage.
• The CRISPR system utilizes a bacterial defense mechanism against viral infections, where specific fragments of the invader's genetic material are captured as "spacers" between identical "repeats" to form a memory bank.
• The CRISPR/Cas9 complex patrols the bacterial cell, searching for target DNA sequences that match the guide RNA, recognizing a specific region called PAM (protospacer adjacent motif) in the DNA to ensure specificity.
• When the complex locates its target, the Cas protein introduces a double-strand break in the DNA, activating the cell's natural repair mechanisms, which include non-homologous end joining (NHEJ) or homology-directed repair (HDR).
• The key advantage of CRISPR lies in its adaptability and efficiency in guiding cleavage, allowing for the production of a single RNA molecule that guides cleavage in conjunction with Cas9, offering a straightforward and effective way to edit genes.
• CRISPR's simplicity allows for the production of a single RNA molecule that guides cleavage in conjunction with Cas9, offering a straightforward and effective way to edit genes.