CRISPR - Genetics

1. What does CRISPR stand for? Who discovered/developed it?

• CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats.

• Discovered in 1987 by Yoshizumi Ishino and later developed as a genome-editing tool by Emmanuelle Charpentier and Jennifer Doudna.

2. In what organism was it discovered?

CRISPR was discovered in the bacteria Escherichia coli.

3. Why did the system originally evolve in prokaryotes?

CRISPR evolved as a bacterial immune system to protect against viral infections by recognizing and cutting the DNA of invading phages.

4. Name at least two ways this system can be used.

1. Gene editing: Modifying or correcting genetic sequences in organisms.

2. Gene regulation: Turning genes on or off by modifying promoters or other regulatory regions.

5. What are guide RNAs?

Guide RNAs (gRNAs) are synthetic or natural RNA molecules that direct the Cas9 enzyme to a specific DNA sequence for cutting.

6. What does the CAS protein enzyme do?

Cas (CRISPR-associated) proteins, such as Cas9, act as molecular scissors, cutting DNA at precise locations specified by the guide RNA.

7. Why is this such a powerful technology?

CRISPR allows for precise, efficient, and cost-effective editing of specific DNA sequences, enabling breakthroughs in genetic research, medicine, and agriculture.

8. What ethical questions does this technology raise?

1. Use in human germline editing, potentially altering future generations.

2. Concerns about designer babies.

3. Risks of unintended genetic consequences or ecological impacts.

4. Unequal access to CRISPR technology.

9. Name the two ways that CRISPR technology can be used in cells.

1. Knockout mutations: Disabling specific genes to study their function.

2. Gene correction: Repairing faulty genes to treat genetic disorders.

10. For what human disorders is CRISPR now being used as a technology?

1. Sickle cell anemia.

2. Beta-thalassemia.

3. Leber’s congenital amaurosis (a genetic eye disorder).

4. Certain cancers.

5. Huntington's disease.

11. Nobel Prize for CRISPR development:

Winners: Emmanuelle Charpentier and Jennifer Doudna won the Nobel Prize in Chemistry in 2020 for developing CRISPR-Cas9 as a genome-editing tool.

12. What is a plasmid? What types of genes are commonly found there?

• Plasmid: A circular, double-stranded DNA molecule independent of chromosomal DNA in bacteria.

• Common genes: Antibiotic resistance, metabolic enzymes, or virulence factors.

13. How are plasmids involved in antibiotic resistance?

Plasmids often carry antibiotic resistance genes and can transfer them between bacteria via horizontal gene transfer.

23. How can plasmids be acquired by bacteria?

1. Conjugation: Transfer through direct cell-to-cell contact.

2. Transformation: Uptake from the environment.

3. Transduction: Transfer by bacteriophages.

24. Mechanisms of antibiotic resistance in bacteria:

1. Efflux pumps: Remove antibiotics from the cell.

2. Enzymatic degradation: Break down antibiotics (e.g., β-lactamases).

3. Target modification: Alter the antibiotic’s binding site.

4. Reduced permeability: Limit antibiotic entry into the cell.

25. Definitions:

• Lawn of cells: Uniform bacterial growth on a petri dish.

• Phages: Viruses that infect bacteria.

• Plaque: Clear zones on a bacterial lawn where phages have lysed cells.

26. Why are phages potentially better than antibiotics?

1. Specificity: Target specific bacteria without harming others.

2. Resistance: Can evolve to counteract bacterial resistance.

3. Low side effects: Less likely to disrupt the microbiome.

27. CRISPR vs. Telomerase and Ribosome:

CRISPR’s guide RNA is similar to:

• Telomerase RNA: Guides telomerase to the ends of chromosomes.

• Ribosomal RNA: Positions the ribosome on mRNA for protein synthesis.

28. What are FISH probes?

Fluorescent In Situ Hybridization (FISH) probes are labeled DNA or RNA sequences that bind to specific genomic regions. Detection is via fluorescence microscopy.

29. Why do interphase nuclei stain differently than prophase-metaphase nuclei in FISH?

• Interphase nuclei: Chromatin is less condensed, showing dispersed signals.

• Prophase-metaphase nuclei: Chromosomes are highly condensed, showing discrete signals.

30. Why do human vs. mouse genomes look different in FISH? Define 'synteny.'

• Human and mouse chromosomes differ due to evolutionary rearrangements.

• Synteny: Conserved blocks of genetic loci across different species.

31. Muntjacs from China and India producing viable offspring—will they be fertile?

• Viable offspring are possible due to compatible genetic material, but fertility depends on chromosome pairing during meiosis, which may be impaired due to structural differences.

32. Genome evolution in:

a. Humans vs. great apes: Fewer chromosomal rearrangements; notable example is the fusion of two ancestral ape chromosomes to form human chromosome 2.
b. Humans vs. mice: Significant rearrangements, including translocations, inversions, and duplications.

33. What is a Robertsonian translocation?

A chromosomal rearrangement where two acrocentric chromosomes fuse at their centromeres, forming a single chromosome.

34. Why are Robertsonian Translocations tolerated mitotically but not meiotically?

• Mitotically: Cells can function with altered chromosome numbers.

• Meiotically: Chromosomal misalignment during tetrad formation can lead to nonviable gametes or aneuploidy.