CRISPR-Cas9 System

CRISPR-Cas9 System

• CRISPR-Cas9 is a bacterial defense system providing adaptive immunity against phages (viruses).
• 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 target DNA based on a guide sequence.
• It functions like molecular scissors, guided by RNA to precisely target and deactivate viral DNA.
• Bacteria use CRISPR-Cas9 as a defense mechanism, retaining a portion of viral DNA as a "memory" in the form of a spacer within the CRISPR structure.
• If the same virus attacks again, the bacteria uses an RNA copy (guide RNA) of this memory to target the virus's DNA.

CRISPR-Cas9 in the lab

• Scientists have adapted the CRISPR-Cas9 mechanism for use in the laboratory.
• By modifying the guide RNA, any DNA in a cell can be targeted and cut.
• This enables scientists to "repair", "modify", or "deactivate" specific genes within an organism's genome.

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.
• The CRISPR sequence containing the spacers transcribes into a long precursor RNA.
• Processing steps then cut the precursor RNA into smaller guide RNAs (gRNAs).
• Each gRNA corresponds to a specific spacer and can target the phage DNA.
• The gRNAs bind to the Cas9 protein, forming an active complex.
• When a phage reinfects the bacterium, gRNA guides Cas9 to the viral DNA.
• Cas9 then cuts the phage DNA, preventing replication.

CRISPR-Cas9 for Gene Editing

• CRISPR is a tool in medicine, facilitating precise and targeted gene modification.
• Guided by the Cas9 protein and a guide RNA, specific DNA sequences responsible for genetic diseases can be identified and cut.
• Once DNA is cut, the cell repairs the break via:
  • Non-homologous end joining (NHEJ): repairs double-strand breaks by directly joining broken ends without a template, leading to mutations via insertions or deletions that can inactivate genes or cause non-specific modifications.
  • Homology-directed repair (HDR): utilizes a homologous DNA template to correct double-strand breaks, allowing specific sequences to be inserted or replaced. HDR is more precise than NHEJ but requires specific conditions and occurs less frequently.

CRISPR-Cas9 Methods

• Combined Cas9/sgRNA plasmid involves a single plasmid containing genes for both the Cas9 protein and a simplified guide RNA (sgRNA), mainly used to inactivate a gene via random breakage and repair with NHEJ.
• The transcription of Cas9 is regulated by a Pol-II promoter, and sgRNA is regulated by a Pol-III promoter for transcribing small structural RNAs.
• Cas9 plasmid + sgRNA gene fragment uses two components and can repair an existing mutation or introduce a new mutation if a template has been incorporated
• In vitro transcribed Cas9 and sgRNA: Cas9 and sgRNA are directly produced and injected into cells.
• The complex Cas9-sgRNA disappears after its action, thus limiting toxicity and off-target issues.

sgRNA components in gene editing

• The sgRNA contains a 20-nucleotide guide region.
• This pairs with the target sequence in the DNA.
• The Cas9 protein recognizes a short neighboring sequence called PAM (Protospacer Adjacent Motif).
• Once the sgRNA attaches, Cas9 cuts both DNA strands just before the PAM sequence, creating a double-strand break.
• The lack of precision remains problematic since Cas-9 can cut regions we do not want to target
• Cas9 can be mutated to suppress its cleavage activity at all potential sites, limiting off-target cuts.
• Cas9 can be modified to target one specific gene.
• Mutation of Cas9 leads to dead Cas9 (dCas9), which can still bind to DNA but does not cut.
• Specific effectors can attach to dCas9 for targeted applications.
  • dCas9 with a transcriptional activator: Increases the expression of a specific gene by guiding the activator.
  • dCas9-DNMT: Alters gene expression by adding methylation marks to DNA.
  • dCas9-TET: Reactivates silent genes by removing methylation marks from DNA.

Problems and Complications with CRISPR-Cas9

• When using dCas9 with a transcriptional activator, multiple gRNAs are needed for efficient activation.
• The introduction of multiple plasmids in the cell (for gRNAs and dCas9) is problematic.
• A single plasmid containing both gRNA and dCas9 can be designed, but efficiency of plasmid delivery into cells can cause problems.
• Solutions include expressing gRNAs from a Pol-II promoter (problematic due to polyadenylation) or using a fluorescent protein linked to an RNA for poly-A8 tail addition.
• Designing gRNAs as introns inserted into a Pol-II transcript was attempted but failed due to RNA damage during intron processing.

miRNA

• RNA interference (RNAi) by Andrew Fire and Craig Mello in 2006 is a gene silencing mechanism using double-stranded RNAs.
• In eukaryotes, small inhibitory RNAs regulate 30–70% of genes through transcription or translation.
• These RNAs are either endogenous (microRNAs) or exogenous (small interfering RNAs).

miRNA Synthesis

• The miRNA gene transcribes into pri-miRNA, forming a stem-loop structure.
• In the Drosha-DGCR8 complex, the pri-miRNA is cut to produce pre-miRNA.
• Exportin-5 transports the pre-miRNA to the cytoplasm.
• Dicer cuts the pre-miRNA into a miRNA-miRNA* duplex.
• One of the two duplex strands incorporates into the RISC complex (containing the Ago2 protein).
• With perfect mRNA pairing, the RISC cleaves the mRNA, blocking translation.
• With imperfect pairing, the miRNA inhibits translation without degrading the mRNA.
• It controls the production of proteins by targeting mRNAs.
• Instead of pairing with the entire mRNA, it pairs with the "Seed Sequence" (positions 2 to 8 of the miRNA).
• Flank regions on either side of the seed sequence play a role in stability.

miRNA Summarized

• Primary transcripts (pri-miRNA) are encoded by Pol2/Pol3 promoters and present in exons and introns.
• Drosha digests the pri-miRNA, separating the stem-loop structure and producing a 65-70 base pair pre-miRNA; this is then exported into the cytoplasm.
• Dicer digests the pre-miRNA to produce a mature miRNA of 21-22 base pairs.
• RISC (RNA-Induced Silencing Complex): Perfect pairing causes degradation of mRNA, while imperfect pairing inhibits translation.

siRNA

• siRNA (small interfering RNA) is a small double-stranded RNA from exogenous sources like viruses or transposons.
• Unlike miRNA, siRNA is cleaved by Dicer in the cytoplasm into 21-25 nucleotide fragments.
• Once in the RISC complex, siRNA perfectly pairs with mRNA, leading to mRNA degradation.
• MiRNA tolerates mismatches, inhibiting translation rather than degradation.
• siRNA is more specific, while miRNA regulates multiple targets.

Recognition and binding strength

• Genomic miRNAs are identified by identifying hairpins (stem-loops) in their secondary structures.
• Homology to targets is predicted primarily based on the seed sequence.
• The human genome contains over 1000 genes encoding miRNAs.
• The targeting efficiency of miRNAs relies on the seed sequence and the number of binding.
  • Seed Sequence: stronger binding with 8 nucleotides than 6.
  • G:U wobble weakens binding
  • Mismatch at the end of the seed is less impactful

Evolutionary Origins of RNA

• Protection against transposons and viruses
  • SiRNAs and miRNAs protect against foreign DNA and regulate gene expression.
• RNA editing and the role of miRNAs
  • Destabilization of double-stranded RNAs (dsRNAs) from viruses/retroelements
  • Targeting virus/retroelement expression limits infectious activity or integration into the genome.
• Fine-tuning of gene expression

miRNA-Dependent Regulation

• Tissue-Specific miRNAs
  • Vital for embryonic development, cell identity maintenance, and gene expression.
  • Dicer suppression leads to embryonic lethality.
• Cancer
  • Decreased tumor suppressor miRNAs.
  • Increased onco-miRNAs promote tumor growth.
• miRNA sponges are non-coding RNAs or artificial RNAs that bind to miRNAs, preventing their interactions with natural targets.
• Example uses a reporter gene fused to a target sequence; miRNA presence indicates cleavage or inhibited expression.

Ribozymes

• Ribozymes are catalytic RNA molecules that cleave RNA targets
• They bind to the target mRNA, catalyze a cleavage reaction, and fragment the mRNA, preventing translation.
• The ribozyme remains intact and can act on other target mRNA molecules.

Explore CRISPR-Cas9 system's precision using mutated Cas9 and targeted dCas9 applications. Identify challenges in multiplexed gRNA delivery and Pol-II promoter expression. Understand the function of Cas9 protein and guide RNA (gRNA) in bacterial immunity.