lecture 4 - RNAi and CRISPR Flashcards
statements about CRISPR-Cas9
It leads to cleavage of DNA
The site of cleavage is directed by the guide RNA sequence
Mutations in DNA sequence do not always lead to changes in protein sequence
what are Practical issues with CRISPR-Cas applications?
Off target cleavage
Delivery method; ex vivo versus in vivo approaches
NHEJ mutations variable
HDR inefficient in dividing cells, absent (?) in terminally differentiated, non-dividing cells
Several CRISPR clinical trials in the pipeline including: improving the efficiency of cancer immunotherapy; preventing HPV from causing cervical cancer.
what is the Example of therapeutic application of CRISPR-Cas:Duchenne Muscular Dystrophy?
Affects mostly males at a rate of 1 in 3,500 births
DMD is the most severe and common type of muscular dystrophy
Characterised by the wasting away of muscles – voluntary muscles, heart and breathing muscles.
Death usually occurs by age 30 due to congestive heart failure.
Caused by a range of mutations in the X-linked dystrophin gene.
how is CRISPR-Cas9 used for genome editing?
Very simple system – Cas9 and a 98 base sgRNA
The protospacer RNA guides the Cas9 to the corresponding sequence in the genome
Cas9 then cuts both strands of the genomic DNA
what is CRISPR/Cas systems?
CRISPR: clustered regularly interspaced short palindromic repeat
Cas: CRISPR associated genes
In 2005, 3 separate groups discovered a novel system of bacterial adaptive immunity, to defend against bacteriophages.
2012: Doudna and Charpentier showed that Cas9 from S. pyrogenes is a programmable RNA-guided DNA endonuclease
2013: Several synthetic biology groups used single guide RNAs to edit genomes
How does the miRNA (miHtt) produced by the AAV vector result in less HTT mRNA and protein being produced?
The miHTT binds to mRNA, affecting mRNA stability and translation efficiency
The AAV study used a negative control vector that didn’t express the miHTT. What is this control for?
To see if the vector backbone had an effect
These two papers are important because they showed clear improvements in the mice, and use the AAV vector, which is the most promising one for human therapy.
what is RNA interference (RNAi)?
Regulation of translation by small RNAs in Eukaryotes
Micro RNAs (miRNA) : Inhibit mRNA translation
Small interfering RNA (siRNA): Induce mRNA degradation
what are the Practical issues with RNA inteference?
Specificity – is siRNA only targeting the gene of interest?
Interferon response – a non-specific cellular response to dsRNA
Need to use negative control siRNA, and repeat with different siRNA triggers.
Incomplete knockdown; Reversible
Delivery in humans – how to stably get the siRNA to the correct location?
what are the Normal biological roles of miRNA?
Each microRNA (miRNA) can bind to many different mRNAs as mismatches are permitted.
miRNAs are important for development and differentiation
what is an Example of therapeutic application of RNAi - Huntington’s disease
Rare and fatal inherited disease of the central nervous system
Caused by a single dominant allele, which means heterozygous individuals will develop the disease
The disease is caused by aggregation of a mutant form of the protein Huntingtin (Htt), resulting in damage to brain cells.
This leads to gradual loss of co-ordination, decline in mental ability and changes in personality.
Onset is typically between 30 and 50 years of age, with death 15 to 20 years later.
Compare NHEJ and HR…
Homology directed repair
Can insert new transgene or correct version of mutant gene if provided
No errors introduced
Can only occur in the G2 stage of the cell cycle.
Non-homologous end joining
Introduces errors as bases removed during processing of the break.
Results in mutations or frameshifts
Not great for normal cells, BUT very useful for knocking out proteins
Can occur at any stage in the cell cycle
what is dystrophin?
-important for muscle strength, and acts like a molecular shock absorber during movement
-Loss of dystrophin makes cells fragile, and muscle cells and fibres degenerate
where do mutations occur?
anywhere along the dystrophin gene
Severe phenotypes result from frameshifts, leading to premature termination of translation, and thus loss of function protein.
what is exon skipping?
Exon skipping is a viable therapeutic approach in DMD, as loss of some of the exons in the rod domain does not affect the function of the protein
what is Adeno-associated virus ?
Infects dividing and non-dividing cells.
Doesn’t replicate in human cells
Version used for gene therapy doesn’t integrate into the genome
No apparent pathogenic activity
Only small DNA constructs possible
what is miHtt?
improvements in behavioural deficits
describe the differences in non-homologous directed repair and homologous
Homology-Directed Repair (HDR) and Non-Homologous End Joining (NHEJ) are two mechanisms for repairing double-strand breaks (DSBs) in DNA:
Homology-Directed Repair (HDR)
Uses a homologous DNA template (e.g., sister chromatid) to accurately repair DSBs.
Error-free or has minimal mutations.
Active in the S and G2 phases of the cell cycle.
Requires more time and cellular resources.
Non-Homologous End Joining (NHEJ)
Directly ligates broken DNA ends without a template.
Error-prone, often leading to insertions or deletions (indels).
Functions throughout the cell cycle, especially in G1.
Faster but less accurate.
HDR is precise but limited to specific cell cycle phases, while NHEJ is faster but prone to errors.
what do cellular DNA repair processes do?
repair the double strand break
what is the use of siRNA/shRNA for Protein Knockdown in Research
siRNA/shRNA for Protein Knockdown in Research
Small interfering RNA (siRNA) and short hairpin RNA (shRNA) are widely used tools for gene silencing in research by targeting messenger RNA (mRNA) for degradation, leading to reduced protein expression.
siRNA (Small Interfering RNA)
Synthetic, short double-stranded RNA (~21-25 nucleotides).
Introduced transiently into cells to degrade specific mRNA.
Provides short-term knockdown, useful for quick experiments.
shRNA (Short Hairpin RNA)
DNA-encoded RNA that forms a hairpin structure.
Expressed continuously via plasmids or viral vectors.
Enables stable, long-term gene silencing.
These methods are essential for studying gene function, validating drug targets, and modeling diseases by reducing specific protein levels.
