miRNA and siRNA mechanisms

• CRISPR-Cas9 is a bacterial defense system against viral infections, providing immune memory.
• CRISPR (Clustered Regularly Interspaced Palindromic Repeats) is a DNA sequence with palindromic repeats separated by foreign DNA segments.
• Cas9 is an RNA-guided endonuclease that recognizes and cuts DNA, guided by RNA to deactivate viruses.
• Bacteria use CRISPR-Cas9 as a defense by keeping a viral DNA "memory" as a spacer in the CRISPR structure.
• Bacteria use an RNA copy (guide RNA) of the viral DNA memory to recognize and target the virus upon re-infection.
• Scientists use CRISPR-Cas9 in the lab to target and cut any DNA in a cell by modifying the guide RNA for gene editing or deactivation.
• The CRISPR-Cas9 process involves a phage injecting DNA into a bacterium.
• The bacterium incorporates a fragment of the phage DNA into its CRISPR sequence as a new spacer.
• The CRISPR sequence is transcribed into a precursor RNA, which is then cut into guide RNAs (gRNAs).
• gRNAs bind to Cas9 protein to target specific DNA sequences.
• If a phage reinfects the bacterium, the gRNA guides Cas9 to the viral DNA, and Cas9 cuts the viral DNA, preventing replication.
• CRISPR is a precise tool to modify genes by identifying and cutting specific problematic DNA sequences with Cas9 and a guide RNA.

DNA Repair Mechanisms After CRISPR Cut

• Non-homologous end joining (NHEJ) repairs double-strand breaks by directly joining broken ends without a template, often causing mutations.
• Homology-directed repair (HDR) uses a homologous DNA template to correct double-strand breaks, allowing for specific sequence insertion or replacement; it is more precise than NHEJ but less frequent.

CRISPR-Cas9 Delivery Methods

• Combined Cas9/sgRNA plasmid involves a single plasmid coding for both Cas9 and a simplified guide RNA (sgRNA).
• Cas9 transcription is under the control of a Pol-II promoter, while sgRNA is under a Pol-III promoter.
• Cas9/sgRNA plasmid mainly inactivates a gene via random breakage and repair with NHEJ, resulting in mutation.
• A Cas9 plasmid and sgRNA gene fragment system uses two components: a plasmid carrying Cas9 and a fragment coding for the sgRNA, to repair an existing mutation or to introduce a new mutation because a template has been incorporated.
• In vitro transcribed Cas9 and sgRNA involved producing Cas9 and sgRNA directly in vitro.
• Cas9 becomes a functional protein and sgRNA is synthesized into mature RNA.
• Cas9 and sgRNA are injected into the cell.
• The complex Cas9-sgRNA disappears after its action, thus limiting the risks of toxicity, off-target cuts, or unwanted integration.

Specificity and Limitations of CRISPR

• sgRNA contains a 20-nucleotide guide region that pairs with the target DNA sequence and it is most effective at the beginning of the protospacer sequence.
• A short neighboring sequence called PAM (Protospacer Adjacent Motif) is recognized by the Cas9 protein, with TGG in red.
• After sgRNA attaches to the target, Cas9 cuts both DNA strands just before the PAM sequence, creating a double-strand break.

Challenges with CRISPR

• CRISPR is not always precise and can cut unintended regions, which can be hard to identify.
• The Cas9 protein can be mutated to limit its cleavage activity, reducing off-target effects, and concentrating activity on one specific gene.
• Mutating Cas9 to lose all cleavage capacity results in dCas9 (dead Cas9) that can still bind to DNA using sgRNA, but does not cut.

Applications of dCas9

• dCas9 with a transcriptional activator increases the expression of a specific gene by guiding the activator to a specific promoter or regulatory region.
• dCas9-DNMT adds methylation marks to specific DNA regions, epigenetically altering gene expression.
• dCas9-TET removes methylation marks, potentially reactivating silent genes.

Problems & Complications with dCas9

• Activating a target gene using dCas9 with a transcriptional activator domain requires multiple gRNAs for efficient activation.
• Introducing multiple plasmids (for gRNAs and dCas9) into cells is complex and has low delivery efficiency.
• A single plasmid containing both gRNA and dCas9 is a potential solution, but still complex.
• Expressing gRNAs from a Pol-II promoter poses a problem of polyadenylation.
• Using a fluorescent protein linked to an RNA allows addition of a poly-A8 tail, similar to bacterial mechanisms.
• Designing gRNAs as introns inserted into a Pol-II transcript did not work due to intron processing damaging the RNA.

miRNA (MicroRNA)

• RNA interference (RNAi) is a gene silencing mechanism using double-stranded RNAs to inhibit gene expression.
• In eukaryotes, 30–70% of genes are regulated by small inhibitory RNAs at the transcription or translation levels.
• These RNAs can be endogenous (microRNAs) or exogenous (small interfering RNAs).

miRNA Synthesis

• The miRNA gene is transcribed into pri-miRNA, a long RNA molecule with a stem-loop structure.
• In the nucleus, the Drosha-DGCR8 complex cuts pri-miRNA into pre-miRNA.
• Pre-miRNA is transported to the cytoplasm by Exportin-5 protein.
• Dicer enzyme cuts pre-miRNA to generate a miRNA-miRNA* duplex.
• One strand of the duplex is incorporated into the RISC complex (RNA-induced silencing complex) containing the Ago2 protein.
• If miRNA perfectly pairs with the target mRNA, RISC cleaves the mRNA, blocking translation.
• If the pairing is imperfect, the miRNA inhibits translation without degrading the mRNA.
• miRNA serves to turn off certain genes at the right time.
• miRNA pairs with the "Seed Sequence" (positions 2 to 8 of the miRNA) region of the target mRNA for specificity.
• Flank parts on either side of the seed sequence play an accessory role in the stability of the interaction.

Key Features of miRNA

• Primary transcripts (pri-miRNA) are encoded by Pol2/Pol3 promoters and are present in exons and introns.
• Digestion occurs twice during processing; recognition is structural.
• Drosha digests pri-miRNA, separates the stem-loop, and produces a 65-70 base pair pre-miRNA before export to the cytoplasm.
• Dicer digests pre-miRNA to produce a mature miRNA of 21-22 base pairs.
• RISC (RNA-Induced Silencing Complex) exhibits perfect pairing leading to degradation of mRNA, imperfect pairing leads to inhibition of translation.

siRNA (Small Interfering RNA)

• siRNA originates mainly from exogenous sources like viruses or transposons.
• siRNA does not need Drosha processing; Dicer directly cleaves it in the cytoplasm to produce fragments of 21-25 nucleotides.
• siRNA loads into the RISC complex and perfectly pairs with its target sequence on mRNA, resulting in direct degradation of the mRNA.
• miRNA can tolerate mismatches and often inhibits translation, while siRNA is more specific and degrades mRNA directly.

Recognition and Binding Strength of miRNA

• Genomic miRNA identification is based on predicting secondary structures, especially hairpins (stem-loops).
• Homology prediction with targets is based on the seed sequence.
• The human genome has over 1000 genes encoding miRNAs, each potentially regulating hundreds of targets.
• The targeting efficiency of miRNAs depends on the seed sequence and the number of binding sites on the target mRNA.
• A stronger binding occurs if the seed sequence has 8 nucleotides, and G:U wobbles weaken the binding.
• Mismatches at the end of the seed sequence affect the link less than those in the middle.

Evolutionary Origins of Regulatory RNAs

• Regulatory RNAs probably originated as protection against transposons and viruses.
• 45% of the human genome is foreign DNA.
• siRNAs and miRNAs may have evolved to protect the genome by targeting and reducing the expression of foreign elements.
• miRNAs play a role in RNA editing, destabilizing double-stranded RNAs from viruses/retroelements and targeting virus/retroelement expression.
• MicroRNAs have evolved to fine-tune endogenous gene expression and act as key regulators of endogenous gene expression
• miRNAs are crucial for embryonic development, maintenance of cell identity, and fine-tuning of gene expression.
• Dicer suppression leads to embryonic lethality.

miRNA-Associated Diseases

• Decrease in tumor suppressor miRNAs.
• Increase in onco-miRNAs promoting tumor growth.

miRNA Sponges

• miRNA sponges are non-coding RNAs or artificial RNAs that act as "sponges" by binding to miRNAs, preventing them from interacting with their natural targets and blocking regulatory function.

Ribozymes

• Ribozymes are catalytic RNA molecules capable of cleaving RNA targets in cis (same molecule) or trans (different molecule).
• They play an essential role in processes such as RNA splicing, viral replication, and tRNA biosynthesis.
• The ribozyme binds to the target mRNA via base pairing at a specific region.
• Once attached, it catalyzes a cleavage reaction of the phosphodiester backbone at a specific cleavage site.
• This cleavage results in the fragmentation of the mRNA into two pieces, preventing its translation into protein.
• After the reaction, the ribozyme remains intact and can act on other target mRNA molecules, thereby acting catalytically to effectively regulate or inhibit the expression of the targeted genes.

This quiz covers RNA interference (RNAi), focusing on miRNA and siRNA mechanisms. Questions cover gene silencing, RISC complex, miRNA processing, and evolutionary roles. Test your knowledge of gene regulation and RNAi pathways.