YB - Gene Editing Flashcards
(15 cards)
Q1: What are historical examples of human-driven genetic or phenotypic modification? (2)
- Artificial cranial deformation – practiced in ancient cultures to reshape infants’ skulls for status or tradition.
- Selective breeding – early humans modified animals and plants (e.g., domestic dogs, corn) for desired traits long before modern gene editing existed.
Q2: What are the major modern gene-editing tools and how do they work? (4)
- Meganucleases – enzymes that cut DNA at specific, long recognition sequences; require protein engineering for each target site.
- Zinc Finger Nucleases (ZFNs) – fusion of a zinc finger DNA-binding domain to a FokI nuclease; allow precise editing but are difficult to design.
- TALENs – DNA-binding domains fused to nucleases; easier to design than ZFNs, useful in multiple organisms.
- CRISPR/Cas9 – RNA-guided nuclease system that creates targeted double-strand DNA breaks using customizable guide RNAs.
Q3: How does the CRISPR/Cas9 system naturally function in bacteria and archaea? (2 phases)
A. Adaptation (1st infection):
- Foreign DNA (e.g., from viruses) is recognized and fragmented by Cas1 and Cas2 proteins.
- These fragments (spacers) are integrated into the bacterial CRISPR locus, forming a genetic “memory” of infection.
B. Interference (2nd infection):
- CRISPR locus is transcribed into pre-crRNA, which is processed into crRNAs.
- In Type II systems, tracrRNA pairs with crRNA to guide Cas9 to matching DNA.
- Cas9 cleaves the DNA at the target site near a PAM sequence, inactivating the invader.
Q4: What are the different types of CRISPR systems used in gene editing? (4)
- Type II – Cas9: most widely used, cleaves dsDNA.
- Type V-A – Cpf1: cleaves dsDNA with staggered ends.
- Type V-B – C2c1: also cleaves DNA, less common.
- Type VI – C2c2: targets single-stranded RNA using HEPN domains, rather than DNA.
Q5: What are the components and differences between wild-type and lab-adapted CRISPR/Cas9 systems? (4)
Wild-Type:
- Uses Cas9, crRNA (guides Cas9), and tracrRNA (helps process crRNA and form complex).
- Cas9 cuts DNA at a site adjacent to a PAM sequence.
Lab-Adapted System:
- Uses Cas9 + single guide RNA (sgRNA), a fusion of crRNA and tracrRNA.
- sgRNA simplifies gene editing while maintaining specificity for target DNA near a PAM.
- The PAM sequence is not part of the sgRNA, but remains required in the DNA target.
Q6: What are the two major cellular pathways for repairing CRISPR-induced DNA breaks? (8)
Non-Homologous End Joining (NHEJ):
- Error-prone repair that ligates DNA ends directly.
- Involves Ku70/80, DNA-PKCS, and leads to mutations or deletions.
- Often used for gene knockout.
Homology Directed Repair (HDR):
- Uses a donor DNA template with flanking homology arms.
- Leads to precise gene correction or insertion (e.g., knock-in reporter genes).
- Requires dividing cells.
Q7: How is CRISPR/Cas9 used to generate reporter mice? (4)
- A double-strand break is introduced at a specific locus (e.g., Nanog).
- A donor plasmid with a fluorescent marker (e.g., Cherry) and homology arms is provided.
- HDR enables insertion of the reporter gene into the locus.
- Mice can be used to study gene expression or lineage tracing.
Q8: What are some applications of CRISPR/Cas9 gene editing? (5)
Medicine:
- Drug development
- Gene surgery for inherited diseases
Biology:
- Creation of animal models
- Study of genetic variation
Biotech:
- Modification of materials, food, and biofuels
Q9: How is CRISPR used to identify genes involved in cancer metastasis? (6 steps)
- NSCLC cell line is modified to stably express Cas9-EGFP.
- Cells are infected with a library of 67,405 sgRNAs targeting different genes.
- The mutant pool of cells is generated.
- Cells are injected into mice (subcutaneously).
- Tumors are collected at early (2-week) and late (6-week) stages.
- sgRNAs are sequenced to identify which gene knockouts promote or prevent metastasis.
Q10: How is CRISPR/Cas9 used to repress gene transcription? (2 methods)
Initiation Block:
- Dead Cas9 (dCas9) is guided to the promoter or TF binding sites.
- Physically blocks RNA polymerase or TFs from initiating transcription.
Elongation Block:
- dCas9 is targeted within the gene body (near ATG start codon).
- Blocks RNA polymerase progression → stops transcription mid-way.
Q11: What are methods to activate transcription using CRISPR/dCas9? (2)
Constitutive Activation:
- dCas9 is fused to VP64 (transcription activator).
- Guided to the promoter → recruits transcription machinery for constant expression.
Light-Induced Activation:
- dCas9 fused to CIB1, and CRY2 fused to activator.
- Blue light causes CIB1-CRY2 interaction, bringing the activator to the promoter.
- Enables precise temporal control over gene activation.
Q12: How can CRISPR be used to modify the epigenome? (2 systems)
dCas9-LSD1:
- dCas9 targets LSD1 to a gene.
- Demethylates histones → leads to gene repression.
dCas9-p300:
- p300 adds acetyl groups to histones.
- Promotes gene activation by opening chromatin.
Q13: What are some examples of CRISPR/Cas9-based clinical trials from 2016–2018? (3)
- China (2016): PD-1 gene edited in T cells for lung cancer.
- USA (2017): In vivo editing to inactivate HPV.
- UK (2017–18): UCART19 trials using CAR-T cells + PD-1 knockout.
Q14: What are some genetic targets for upcoming CRISPR clinical trials? (6)
- Crygc – cataracts
- DMD – Duchenne muscular dystrophy
- HBB – β-thalassemia
- CFTR – cystic fibrosis
- HIV-1 LTR – HIV latency
- EBV – Epstein-Barr virus
Q15: What is Casgevy and what condition does it treat? (3)
- First CRISPR-based gene therapy approved by the FDA (Dec 8, 2023).
- Treats sickle cell disease (SCD) caused by mutations in the HBB gene.
- Aims to correct red blood cell shape and prevent pain, anemia, and organ damage.
