CRISPR-Cas9 and Genome Editing — MCQs
Which of the following techniques can be used to detect single base pair substitutions?
Which of the following doesn't occur in 5' to 3' direction?
What is the repair mechanism associated with CRISPR-cas9?
Prader-Willi syndrome and Angelman syndrome are examples of what genetic phenomenon?
Mutations are due to changes in:
Which type of RNA is primarily involved in gene silencing?
Which of the following statements about sickle cell disease is true?
Which malformation is associated with mutations in the HOX gene?
In CRISPR-Cas9 system, which repair mechanism is predominantly used for genome editing?
When a gene is expressed exclusively from the allele inherited from one parent while the allele from the other parent is silenced, what is this phenomenon known as?
CRISPR-Cas9 and Genome Editing Indian Medical PG Practice Questions and MCQs
Question 1: Which of the following techniques can be used to detect single base pair substitutions?
- A. FISH
- B. Southern blot
- C. PCR (Correct Answer)
- D. Restriction Fragment Length Polymorphism (RFLP)
Explanation: ***PCR (with sequencing or allele-specific methods)*** - **PCR-based techniques** are the most versatile methods for detecting single base pair substitutions (point mutations) - **Allele-specific PCR** can directly detect known point mutations by using primers specific to mutant or wild-type alleles - **PCR followed by Sanger sequencing** is the gold standard for identifying any single base pair substitution - **High-resolution melting (HRM) analysis** after PCR can detect mutations based on melting curve differences - PCR amplification is the foundation that enables these detection methods *FISH (Fluorescence in situ hybridization)* - FISH detects **large chromosomal abnormalities** such as aneuploidy, translocations, large deletions, and duplications - It visualizes chromosomal-level changes using fluorescent probes - **Not sensitive enough** to detect single base pair changes, as these are too small to visualize cytogenetically *Southern blot* - Southern blot detects **large DNA rearrangements**, insertions, deletions, or copy number variations - Analyzes restriction enzyme fragments separated by gel electrophoresis - **Generally cannot detect** single base pair substitutions unless they create or abolish a restriction enzyme recognition site - Even when applicable, PCR-based methods are more efficient and sensitive *Restriction Fragment Length Polymorphism (RFLP)* - RFLP can detect single base pair substitutions **only if** they create or abolish a **restriction enzyme recognition site** - Classic example: **Sickle cell mutation** (GAG→GTG in β-globin gene) abolishes an MstII restriction site - **Limited applicability** - can only detect the subset of point mutations that affect restriction sites - PCR-based methods are preferred as they can detect **any** single base pair substitution, not just those affecting restriction sites
Question 2: Which of the following doesn't occur in 5' to 3' direction?
- A. DNA repair
- B. Transcription
- C. DNA replication
- D. RNA editing (Correct Answer)
Explanation: ***RNA editing*** - **RNA editing** involves modifications to **RNA molecules** after transcription, such as base insertions, deletions, or substitutions. - This process does not follow a 5' to 3' synthesis direction, unlike DNA or RNA synthesis. *DNA repair* - **DNA repair mechanisms**, such as **excision repair**, involve synthesizing new DNA to replace damaged sections. - This synthesis occurs in the **5' to 3' direction** by **DNA polymerases**. *Transcription* - **Transcription** is the process where **RNA polymerase** synthesizes an **RNA molecule** from a **DNA template**. - This synthesis always occurs in the **5' to 3' direction**, adding nucleotides to the 3' end of the growing RNA strand. *DNA replication* - **DNA replication** involves the synthesis of new **DNA strands** from a **template strand**. - **DNA polymerase** adds nucleotides exclusively in the **5' to 3' direction**, requiring a primer for initiation.
Question 3: What is the repair mechanism associated with CRISPR-cas9?
- A. Mismatch repair
- B. Non-homologous end joining (Correct Answer)
- C. Nucleotide excision repair
- D. Base excision repair
Explanation: ***Non-homologous end joining*** - **CRISPR-Cas9** creates a **double-strand break (DSB)** in DNA, which is primarily repaired by **non-homologous end joining (NHEJ)**. - **NHEJ** is the **predominant repair pathway** in most cells, accounting for 60-90% of DSB repairs. - **NHEJ** is an error-prone repair mechanism that ligates the broken ends directly, often leading to small **insertions or deletions (indels)** causing gene knockout. - Note: **Homology-directed repair (HDR)** is another CRISPR-associated mechanism used for precise editing when a donor template is provided, but **NHEJ is the primary endogenous repair pathway**. *Mismatch repair* - This mechanism corrects errors that arise during **DNA replication**, such as incorrect base pairing. - It does not repair **double-strand breaks** induced by CRISPR-Cas9. *Nucleotide excision repair* - This pathway removes **bulky DNA adducts** and lesions, such as those caused by UV radiation. - It is not involved in repairing **CRISPR-Cas9 induced double-strand breaks**. *Base excision repair* - This mechanism corrects **damaged or modified bases**, typically single base changes, without affecting the sugar-phosphate backbone significantly. - It handles different types of DNA damage than the **double-strand breaks** generated by CRISPR-Cas9.
Question 4: Prader-Willi syndrome and Angelman syndrome are examples of what genetic phenomenon?
- A. Gene Knockout
- B. Impaired DNA repair
- C. Genomic Imprinting (Correct Answer)
- D. RNA interference
Explanation: ***Genomic Imprinting*** - **Genomic imprinting** is an epigenetic phenomenon where certain genes are expressed in a **parent-of-origin-specific manner**. - In Prader-Willi syndrome, the disease results from the loss of function of specific genes on chromosome 15 (15q11-q13) inherited from the father, while Angelman syndrome results from the loss of function of a different gene (UBE3A) in the same region, but inherited from the mother. *RNA interference* - **RNA interference** is a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules. - This process is not directly responsible for the parent-of-origin-specific expression patterns observed in these syndromes. *Gene Knockout* - A **gene knockout** is a genetic technique in which an organism's genes are made inoperative. - While it involves modifying gene function, it does not explain the differential expression based on parental origin. *Impaired DNA repair* - **Impaired DNA repair** refers to defects in the mechanisms that correct DNA damage. - This can lead to increased mutations and conditions like cancer, but it is not the underlying mechanism for Prader-Willi or Angelman syndromes.
Question 5: Mutations are due to changes in:
- A. DNA nucleotide sequence (Correct Answer)
- B. RNA nucleotide sequence
- C. Amino acid sequence of ribonuclease
- D. Cell membrane
Explanation: ***DNA nucleotide sequence*** - **Mutations** are defined as changes in the **genetic material**, which is primarily composed of **DNA**. - These changes in the **nucleotide sequence** of DNA can alter the genetic code, leading to changes in **protein structure and function**. *RNA nucleotide sequence* - While RNA can have its nucleotide sequence altered, these changes are generally not considered true **mutations** in the heritable sense for most organisms. - RNA is typically a temporary molecule, and changes to its sequence are usually not passed down to subsequent generations. *Amino acid sequence of ribonuclease* - An altered **amino acid sequence** in a protein like ribonuclease is a consequence of a **mutation in the DNA**, not the mutation itself. - **Ribonucleases** are enzymes that catalyze the degradation of RNA, and their structure is determined by the **DNA sequence**. *Cell membrane* - The cell membrane is a **lipid bilayer** with embedded proteins that regulates cellular transport and communication. - While its components can be affected by genetic mutations, alterations in the cell membrane itself do not constitute the primary definition of a **mutation**.
Question 6: Which type of RNA is primarily involved in gene silencing?
- A. rRNA
- B. tRNA
- C. miRNA (Correct Answer)
- D. mRNA
Explanation: ***miRNA*** - **miRNA** (microRNA) is a small non-coding RNA molecule that plays a crucial role in **post-transcriptional regulation of gene expression**. - It functions by binding to complementary messenger RNA (mRNA) molecules, leading to **mRNA degradation** or **inhibition of translation**, thereby silencing genes. - miRNA is the primary RNA type involved in **gene silencing** through the RNA interference (RNAi) pathway. *rRNA* - **rRNA** (ribosomal RNA) is a primary component of **ribosomes**, the cellular machinery responsible for protein synthesis. - Its main function is to **catalyze peptide bond formation** and provide structural integrity to the ribosome, not gene silencing. *tRNA* - **tRNA** (transfer RNA) is responsible for carrying specific **amino acids** to the ribosome during protein synthesis. - It acts as an adapter molecule, translating the **genetic code** in mRNA into an amino acid sequence. *mRNA* - **mRNA** (messenger RNA) carries genetic information from **DNA to ribosomes** for protein synthesis. - While mRNA can be targeted by gene silencing mechanisms (like miRNA), it is not the RNA type that performs the silencing function itself.
Question 7: Which of the following statements about sickle cell disease is true?
- A. Sickling is completely reversible with oxygenation, making it clinically insignificant.
- B. Sickling leads to a significant increase in overall MCHC levels in the blood.
- C. Fetal hemoglobin inhibits sickling. (Correct Answer)
- D. Sickling occurs exclusively in the homozygous state and never in the heterozygous state.
Explanation: ***Sickling is reversible with oxygenation*** - When oxygen tension is restored, hemoglobin S can re-hydrate and revert to its normal shape, reducing sickling. - This reversible process is essential for managing episodes of vaso-occlusive crisis in sickle cell disease. *Fetal hemoglobin facilitates Sickling* - Fetal hemoglobin (HbF) actually inhibits sickling by stabilizing the erythrocyte shape and reducing the proportion of hemoglobin S [1]. - Individuals with higher levels of HbF experience fewer sickling-related complications [1]. *Sickling occurs both in heterozygous and homozygous state* - Sickling primarily occurs in the homozygous state (HbSS); heterozygotes (HbAS) usually do not experience significant sickling effects [1]. - Heterozygous individuals may have a selective advantage against malaria, but they are not prone to sickle cell crises. *Sickling Leads to decreased MCHC* - Sickling does not directly lead to decreased mean corpuscular hemoglobin concentration (MCHC); MCHC is typically normal in sickle cell patients. - In fact, sickle cell disease often results in hemolysis and can lead to increased MCHC in some cases.
Question 8: Which malformation is associated with mutations in the HOX gene?
- A. Polysyndactyly (Correct Answer)
- B. Holoprosencephaly
- C. Mayer Rokitansky syndrome
- D. Gorlin syndrome
Explanation: ***Polysyndactyly*** - The **HOX gene** plays a critical role in limb development and is associated with the malformation of **polysyndactyly**, which is characterized by extra fingers or toes [1]. - This condition is due to the disruption of the normal **patterning** during limb formation, directly involving the action of HOX genes [1]. *Gorlin syndrome* - Gorlin syndrome is primarily caused by mutations in the **PTCH1 gene**, linked to **basal cell carcinoma** and other abnormalities. - It does not involve HOX gene mutations, hence is **not** related to limb malformations. *Holoprosencephaly* - Holoprosencephaly is a developmental condition often linked to **chromosomal anomalies** and abnormal embryonic development, **not specifically** HOX gene mutations. - It refers to the incomplete separation of the forebrain, distinct from the **limb malformations** associated with HOX genes. *Mayer Rokitansky syndrome* - Mayer-Rokitansky syndrome involves **agenesis** or **hypoplasia** of the uterus and upper two-thirds of the vagina, which is due to other genetic factors. - This condition is not related to the functions of the **HOX genes** in limb or skeletal development. **References:** [1] Kumar V, Abbas AK, et al.. Robbins and Cotran Pathologic Basis of Disease. 9th ed. Bones, Joints, and Soft Tissue Tumors, p. 1186.
Question 9: In CRISPR-Cas9 system, which repair mechanism is predominantly used for genome editing?
- A. Nucleotide excision repair
- B. Mismatch repair
- C. Homology-Directed Repair (HDR)
- D. Non-Homologous End Joining (NHEJ) (Correct Answer)
Explanation: ***Non-Homologous End Joining (NHEJ)*** - **NHEJ** is the most common and error-prone repair pathway in mammalian cells, directly ligating the broken DNA ends created by **Cas9**. - This pathway often results in **insertions** or **deletions (indels)** at the cut site, leading to gene knockout by causing frameshifts. *Nucleotide excision repair* - **Nucleotide excision repair (NER)** is primarily involved in removing bulky DNA adducts and pyrimidine dimers caused by UV radiation. - It involves excising a segment of DNA around the damage, not repairing double-strand breaks induced by CRISPR-Cas9. *Homology-Directed Repair (HDR)* - **HDR** is a precise repair mechanism that uses a homologous DNA template to repair double-strand breaks, allowing for precise gene editing (e.g., specific base changes, gene insertion). - While it can be leveraged in **CRISPR-Cas9**, it is less efficient and less common than **NHEJ** in most mammalian cells, especially when no exogenous template is provided. *Mismatch repair* - **Mismatch repair (MMR)** systems correct base-pair mismatches and small insertion/deletion loops that arise during DNA replication. - This mechanism is not involved in repairing the double-strand breaks generated by the **CRISPR-Cas9** system.
Question 10: When a gene is expressed exclusively from the allele inherited from one parent while the allele from the other parent is silenced, what is this phenomenon known as?
- A. Genomic imprinting (Correct Answer)
- B. Mosaicism
- C. Alleles
- D. Chimerism
Explanation: ***Genomic imprinting*** - **Genomic imprinting** is an epigenetic phenomenon where certain genes are expressed in a **parent-of-origin-specific manner**. - This means that depending on whether the gene was inherited from the **mother or the father**, only one copy (maternal or paternal) is expressed, while the other is silenced. *Mosaicism* - **Mosaicism** describes the presence of **two or more cell lines** with different genotypes within a single individual, originating from a single zygote. - This typically arises from a **post-zygotic mutation** or chromosomal abnormality during early embryonic development. *Alleles* - **Alleles** are different forms of a **single gene** located at the same locus on homologous chromosomes. - An individual inherits **two alleles** for each gene, one from each parent, but both are usually expressed unless one is recessive. *Chimerism* - **Chimerism** refers to an individual composed of cells from **two or more different zygotes**, meaning the cells originate from different genetic lineages. - This can occur through processes like **fusion of two embryos** or organ transplantation.
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