CRISPR-Cas9 System

CRISPR-Cas9 System

• CRISPR-Cas9 is a bacterial defense system providing adaptive immunity against viruses (bacteriophages).
• CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a DNA sequence with palindromic repeats separated by viral DNA segments.
• Cas9 is an RNA-guided endonuclease that recognizes and cuts DNA corresponding to a guide sequence.
• Cas9 protein acts like molecular scissors, guided by RNA to attach to and deactivate viral DNA.
• It is a defense mechanism where bacteria store viral DNA fragments as "memory" spacers within the CRISPR structure.
• When the same virus attacks, bacteria use an RNA copy (guide RNA) of the stored viral DNA to target and disable the virus's DNA.
• Scientists have adapted this mechanism to target and cut any DNA in a cell by modifying the guide RNA.
• This allows for gene repair, modification, or deactivation in various organisms.

CRISPR-Cas9 Process

• A phage injects its DNA into a bacterium.
• The bacterium identifies the phage DNA, extracts a fragment, and inserts it into its CRISPR sequence as a new spacer between repeats.
• The CRISPR sequence, now containing spacers, transcribes into a long precursor RNA.
• This precursor RNA is cut into small fragments, called guide RNAs (gRNAs).
• Each gRNA corresponds to a specific spacer and retains the information to target the phage DNA.
• gRNAs bind to the Cas9 protein, forming an active complex that targets specific DNA sequences.
• If the phage reinfects, the gRNA guides Cas9 to the viral DNA corresponding to its spacer.
• The Cas9 protein cuts the phage DNA, preventing replication and protecting the bacterium.

CRISPR-Cas9 for Gene Editing

• CRISPR is a tool in medicine for precise and targeted gene modification.
• With Cas9 protein and a guide RNA, a specific DNA sequence responsible for a genetic disease can be identified and cut.
• After DNA is cut, the cell repairs the break via non-homologous end joining (NHEJ) or homology-directed repair (HDR).
• NHEJ is a natural, imprecise DNA repair mechanism leading to insertions or deletions (mutations), potentially inactivating genes causing nonspecific modifications.
• HDR is a precise DNA repair mechanism which uses a homologous DNA template to correct double-strand breaks, allowing specific sequence insertion or replacement but requires specific conditions and occurs less frequently than NHEJ.

CRISPR-Cas9 Delivery Methods

• Combined Cas9/sgRNA plasmid: A single plasmid codes for both Cas9 protein and a simplified guide RNA (sgRNA), resulting in mutation and is mainly used to inactivate a gene via random breakage and repair with NHEJ.
• Cas9 plasmid + sgRNA gene fragment: Uses a plasmid carrying Cas9 and a fragment coding for the sgRNA which repairs an existing mutation or introduces a new mutation after template incorporation.
• In vitro transcribed Cas9 and sgRNA: Cas9 and sgRNA are produced directly in vitro, then injected into the cell, bypassing transcription. The complex disappears after its action limiting toxicity, off-target cuts, or unwanted integration.

sgRNA and Cas9 Mechanics

• The sgRNA contains a 20-nucleotide guide region that pairs with the target DNA sequence.
• The Cas9 protein recognizes a short neighboring sequence called PAM (Protospacer Adjacent Motif).
• Once the sgRNA attaches to the target, Cas9 cuts both DNA strands just before the PAM sequence, creating a double-strand break.
• Off-target cutting is a problem with CRISPR, because Cas-9 can cut other unintended regions and is difficult to identify.
• Mutations to the Cas9 protein suppress its cleavage activity at all potential sites and limit accidental or off-target cuts.
• Modifying Cas9 to target only one specific gene concentrates activity and allows the chromosome to remain intact reducing adverse effects.
• Complete mutation of Cas9 yields an inactive version called dCas9 (dead Cas9), which still binds to DNA via sgRNA but cannot cut.

Applications of dCas9

• dCas9 allows for targeted applications when specific effectors are attached
• dCas9 with a transcriptional activator attached increases the expression of a specific gene by guiding the activator.
• dCas9-DNMT, when fused with a methyltransferase enzyme, adds methylation marks to specific DNA regions, epigenetically altering gene expression.
• dCas9-TET, when fused to a demethylase enzyme, removes methylation marks from specific regions, potentially reactivating silent genes.

Problems and Complications with CRISPR

• If dead Cas9 (dCas9) is used coupled with a transcriptional activator domain to target a promoter region, this can activate a target genes expression.
• Efficient activation requires targeting several gRNAs simultaneously (multiplexed RNA), because a single gRNA is generally not enough to produce significant activation.
• The necessity of introducing multiple plasmids (4 for gRNAs, 1 for dCas9) into cells is the main issue because the difficulty lies in the efficiency of delivery of the plasmids, thus limiting the systems overall efficiency.
• An alternative solution is designing a single plasmid that contains both gRNA and dCas9, but this also remains complex.
• Expressing gRNAs from a Pol-II promoter poses a problem of polyadenylation, which interferes with gRNA function, although advantageous because Pol-Il promoters express in most cells
• A proposed solution involves using a fluorescent protein linked to an RNA, allowing for poly-A8 tail addition (similar to the bacterial mechanism).
• Another attempt designed gRNAs as introns inserted into a Pol-II transcript for generation after splicing; which did not work because intron processing damaged the RNA and made the gRNAs nonfunctional.

miRNA (MicroRNA) Overview

• RNA interference (RNAi), discovered by Andrew Fire and Craig Mello in 2006, is a gene silencing mechanism using double-stranded RNAs.
• 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 transcribes into pri-miRNA, a long RNA molecule with a stem-loop structure.
• In the nucleus, the Drosha-DGCR8 complex cuts pri-miRNA into a shorter precursor called pre-miRNA.
• Exportin-5 transports pre-miRNA out of the nucleus to the cytoplasm.
• In the cytoplasm, Dicer cuts pre-miRNA to generate a miRNA-miRNA* duplex (two complementary RNA strands).
• One strand of the duplex incorporates into the RISC complex (RNA-induced silencing complex) containing the Ago2 protein.
• Perfect miRNA pairing with the target mRNA leads to RISC cleaving the mRNA blocking translation. Imperfect pairing inhibits translation without degrading the mRNA.
• This step silences certain genes at the right time and allows the cell to function properly and adapt to its environment.
• miRNA pairs with a specific region of the target mRNA, called the "Seed Sequence" (positions 2 to 8 of the miRNA).
• The seed sequence is important for targeting. The Flank parts on either side of the seed sequence play an accessory role in the stability of the interaction.

Characteristics of miRNA

• G:U mismatches (wobble) are tolerated and influence inhibition efficiency.
• Target sequences are short and can tolerate mismatches therefore each miRNA can regulate many mRNAs, and each mRNA can be targeted by several different miRNAs which creates a large regulatory network in cells.

miRNA Summary

• Primary transcripts created are encoded by Pol2/Pol3 promoters and are present in exons and introns.
• Digestion occurs twice and recognition is structural.
• Drosha digests the pri-miRNA, separates the stem-loop structure, and produces a 65-70 base pair pre-miRNA which is exported into the cytoplasm.
• Dicer digests the pre-miRNA and produces a mature miRNA of 21-22 base pairs.
• RISC (RNA-Induced Silencing Complex) results in degradation of mRNA with perfect pairing or inhibition of translation with imperfect pairing.

siRNA (Small Interfering RNA)

• siRNA (small interfering RNA) is a small double-stranded RNA that, originates from exogenous sources (viruses, transposons) with a possibility of endogenous origin.
• Unlike miRNA, siRNA doesn't require the Drosha processing step and is cleaved by Dicer in the cytoplasm into fragments of 21-25 nucleotides.
• Once loaded into the RISC complex (like miRNA), siRNA pairs perfectly with its target mRNA, usually directly degrading the mRNA.
• miRNA can tolerate mismatches and often inhibits translation thus siRNA is more specific in its action, while miRNA often regulates multiple targets because of partial complementarity.

Recognition and Binding Strength

• Genomic miRNA identification is based on predicting the secondary structure, identifying hairpins (stem-loops).
• Homology prediction with targets relies on the seed sequence, a key region for recognition.
• The human genome contains over 1000 genes encoding miRNAs and each can potentially regulate hundreds of targets.
• The targeting efficiency (binding strength) of miRNAs depends on with the seed sequence and the number of binding sites.
• Strength depends on the length of the seed sequence, the binding is stronger if there are 8 nucleotides compared to 6, with the G:U wobble weakening the binding. The affect depends on location; If the mismatch is at the end of the seed, it affects the link less than if it is in the middle.
• The more binding sites present on the target mRNA the better, with 8 nucleotides being preferable to 7, which is better than 6.

Evolutionary Origins of Regulatory RNAs

• Regulatory RNAs likely protect against transposons and viruses
• 45% of the human genome is foreign DNA!
• siRNAs and miRNAs protect the genome by targeting and reducing the expression of foreign elements, preventing their proliferation or deleterious effects.
• Regulatory RNAs influence RNA editing
• miRNAs and siRNAs recognize and cleave double-stranded RNAs (dsRNAs) from viruses/retroelements, preventing their translation and limiting infection or integration into the genome.
• miRNAs specifically regulate genes associated with retroelements reducing their impact on genome stability
• MicroRNAs fine-tune endogenous gene expression
• Initially a defense against genetic invaders, miRNAs have evolved to regulate endogenous genes.
• MicroRNAs influence tissue-specific expression, embryonic development, maintaining cell identity, and fine-tuning of gene expression.

miRNA-Dependent Regulation

• Tissue-Specific miRNAs: Crucial for embryonic development, maintenance of cell identity, and fine-tuning of gene expression. Dicer suppression leads to embryonic lethality.
• Cancer: Decrease in tumor suppressor miRNAs or an Increase in onco-miRNAs promoting tumor growth.
• miRNA sponges are molecules (non-coding RNAs or artificial RNAs) that bind to miRNAs via complementary sequences and prevent them from interacting with their natural targets (mRNAs).
• This modulation of expression in genes regulated by miRNAs example is an application where A reporter gene (lacZ) is fused to a target sequence complementary to miR-196a and When the miRNA is present and active, it cleaves or inhibits the expression of lacZ, reducing the blue coloration allowing visualization of the areas where miR-196a is expressed, revealing its spatial and temporal role in embryonic development.

Additional Information

• miRNA does not completely silence genes; it acts as a fine-tuning regulator, reducing expression by about 95% even with a perfect match and decreases further if mismatches are present.
• Ribozymes are catalytic RNA molecules able to cleave RNA targets in cis (same molecule) or trans (different molecule).
• They have an essential role in processes such as RNA splicing, viral replication, and tRNA biosynthesis.

Ribozyme Action

• Binds to the target mRNA via base pairing.
• Catalyzes cleavage of the phosphodiester backbone at a specific site.
• Cleavage fragments the mRNA into two pieces, preventing its translation.
• After the reaction, the ribozyme remains intact and can act on other target mRNA molecules, regulating/inhibiting gene expression.

Explore the CRISPR-Cas9 system's function in bacteria, guide RNA roles, and viral DNA incorporation. Learn about modifications for gene editing, event sequences during viral infections, and the role of spacer sequences in bacterial immunity.