CRISPR Cas9 MCQs - Technology

Which specific risk is most associated with CRISPR-based gene drives in the wild?

Increased atmospheric carbon dioxide levels
Unintended ecological collapse and permanent genomic changes
The development of human immunity to mosquitoes
Rapid extinction of non-target species

Explanation: Because gene drives spread traits through entire wild populations, they pose the risk of permanent, irreversible ecological impacts if the modification behaves differently than predicted.

Which protein is responsible for processing long pre-crRNA transcripts into mature, individual crRNAs in Type II systems?

Cas9
RNase III
Helicase
Cas6

Explanation: In Type II systems (like Cas9), the host enzyme RNase III cleaves the double-stranded RNA formed by the tracrRNA and pre-crRNA to create mature guides.

Which clinical trial marked the first in-human systemic (in vivo) delivery of CRISPR-Cas9 to treat Transthyretin Amyloidosis?

EDIT-101
CTX001
NTLA-2001
SAR445136

Explanation: NTLA-2001 used lipid nanoparticles to deliver CRISPR components to the liver to knock down the TTR gene, marking a milestone for in vivo gene editing.

Which CRISPR protein is classified as a Type V system and recognizes a T-rich PAM (TTTN)?

CasX
Cas13b
Cas9
Cas12a

Explanation: Cas12a (Cpf1) belongs to the Type V class, utilizing a single RuvC domain and recognizing T-rich PAM sequences, unlike the G-rich PAM used by Cas9.

In the context of 'Gene Drives', what is the function of the 'Cargo' gene?

The guide RNA
The desired trait
The resistance marker
The Cas9 enzyme

Explanation: In a gene drive, the 'cargo' is the specific genetic modification (e.g., malaria resistance) that is linked to the CRISPR machinery to be spread through a population.

Which mechanism does Prime Editing utilize to integrate the desired genetic edit into the host genome?

Reverse transcription
Base excision repair
Non-homologous end joining
Homology Directed Repair

Explanation: Prime editing uses a reverse transcriptase fused to a Cas9 nickase to write new genetic information directly from a pegRNA template into the DNA.

Which CRISPR-associated protein is the smallest known nuclease, facilitating easier delivery via viral vectors?

Cas9
Cas14
Cas12a
Cas3

Explanation: Cas14 is exceptionally small (about 400-700 amino acids), roughly half the size of Cas9, making it highly attractive for compact gene-delivery systems.

In 'Prime Editing', the pegRNA (prime editing guide RNA) contains which unique structural feature at its 3' end?

G-quadruplex
RuvC binding loop
Poly-A tail
Primer Binding Site

Explanation: The pegRNA includes a Primer Binding Site (PBS) and a reverse transcriptase template at its 3' end, which are essential for synthesizing the new DNA sequence.

The 'CRISPR-interference' (CRISPRi) technique utilizes dCas9 to achieve gene silencing primarily through:

mRNA degradation
Epigenetic methylation
Histone acetylation
Transcriptional blocking

Explanation: dCas9 binds to the promoter or coding region and acts as a physical roadblock, preventing RNA polymerase from initiating or continuing transcription.

The 'Prime Editing' method is often described as a 'search-and-replace' tool. Which additional enzyme does it use alongside Cas9?

Reverse Transcriptase
DNA Ligase
Restriction Endonuclease
RNA Polymerase

Explanation: Prime editing uses a 'nickase' Cas9 fused to a reverse transcriptase. It uses a prime editing guide RNA (pegRNA) to both find the target and write new DNA directly into the genome.

Which specific safety mechanism is often proposed to stop or reverse the spread of a CRISPR gene drive in the wild?

Antibiotic resistance
Thermal activation
UV-degradation
Daisy-chain drive

Explanation: A daisy-chain drive is a self-limiting gene drive that eventually 'runs out of steam' because its components are linked in a chain that gets diluted over generations, preventing it from spreading globally.

Which revolutionary CRISPR-based therapy was the first to receive FDA approval (in 2023) for treating Sickle Cell Disease and Beta-thalassemia?

Zolgensma
Luxturna
Casgevy
Glybera

Explanation: Casgevy (exa-cel) is the first approved therapy using CRISPR-Cas9. It edits a patient’s own bone marrow stem cells to produce fetal hemoglobin, compensating for defective adult hemoglobin.

A 'Nickase' version of Cas9 (nCas9) is typically generated by inactivating which specific catalytic domain?

HNH or RuvC
BH or PI
REC2 or REC3
PAM or WED

Explanation: Inactivating one of the two nuclease domains (HNH or RuvC) converts Cas9 into a nickase, which cuts only one strand of the DNA double helix instead of two.

In engineered CRISPR systems, the 'sgRNA' (single guide RNA) is a synthetic fusion of which two natural molecules?

tRNA and snRNA
crRNA and tracrRNA
mRNA and tRNA
crRNA and mRNA

Explanation: For laboratory use, scientists typically combine the target-specific crRNA and the scaffold tracrRNA into a single molecule called the single-guide RNA (sgRNA).

What is the fundamental difference between the DNA cuts made by Cas9 and those made by Cas12a (Cpf1)?

Self vs. Non-self recognition
Blunt vs. Staggered ends
RNA vs. DNA cleavage
Exonuclease vs. Endonuclease

Explanation: Cas9 produces blunt ends by cutting both strands at the same position, whereas Cas12a produces staggered (sticky) ends with a 4-5 nucleotide overhang.

Why is the HDR (Homology Directed Repair) pathway considered difficult to utilize in non-dividing cells like neurons?

Cas9 cannot cross the nuclear membrane of a neuron
HDR is largely restricted to the S and G2 phases of the cell cycle
The gRNA is unstable in the neural environment
Neurons do not contain DNA repair enzymes

Explanation: HDR requires a sister chromatid as a template, which is only present when a cell is preparing to divide. This makes precise gene correction in non-dividing (post-mitotic) cells very challenging.

Which specific domain of the Cas9 protein is responsible for recognizing the PAM sequence on the non-target DNA strand?

BH domain
REC1 domain
PI domain
WED domain

Explanation: The PAM-Interacting (PI) domain at the C-terminus of Cas9 is responsible for identifying and binding the Protospacer Adjacent Motif, initiating DNA unwinding.

What is the biological role of the 'Cas1-Cas2' complex across almost all CRISPR-Cas systems?

Spacer acquisition
Signal transduction
Target interference
crRNA biogenesis

Explanation: Cas1 and Cas2 are the most conserved CRISPR proteins; they function as an integrase complex to capture and insert new viral DNA spacers into the genome.

In the CRISPR-Cas9 system, which component acts as the 'molecular scissors' that performs the physical cut of the DNA?

PAM motif
Spacer DNA
Guide RNA (gRNA)
Cas9 protein

Explanation: The Cas9 protein is a nuclease, an enzyme capable of creating double-strand breaks in the DNA backbone at a location specified by the guide RNA.

In terms of molecular diagnostics, the CRISPR system acts as a highly sensitive sensor primarily because of its ability to:

Glow in the dark naturally
Synthesize new DNA
Recognize specific genetic sequences with high fidelity
Kill all bacteria in a sample

Explanation: The precision of RNA-guided targeting allows CRISPR-based tools to detect even single-nucleotide differences in viral or bacterial DNA/RNA, making them powerful for rapid diagnostics.

Which of the following describes the 'Acquisition' phase of the natural CRISPR-Cas immunity?

Spacer integration
Target cleavage
Viral adsorption
RNA maturation

Explanation: Acquisition is the first stage where the Cas1-Cas2 complex identifies foreign DNA and integrates a segment of it into the CRISPR array as a new spacer.

In the CRISPR locus, what is stored in the 'spacer' regions between the repeats?

Genetic 'memory' from previously encountered viruses
Sequences that trigger cell death
A library of common human mutations
The code for the Cas9 protein

Explanation: Spacers are short segments of DNA captured from invading phages. These 'mugshots' allow the bacteria to recognize and target the same virus if it attacks again.

Which specific RNA structure is formed when crRNA and tracrRNA base-pair in the native bacterial system?

Dual-RNA complex
Stem-loop precursor
Triple-helix motif
Single-guide RNA

Explanation: In nature, the crRNA and tracrRNA exist as two separate molecules that form a dual-RNA complex. The synthetic 'sgRNA' used in labs is a man-made fusion of these two.

Which type of gene editing involves making changes to embryos or reproductive cells, resulting in heritable genetic modifications?

In vivo gene editing
Somatic gene editing
Ex vivo gene editing
Germline gene editing

Explanation: Germline editing affects the DNA in sperm, eggs, or embryos, meaning the changes are passed down to all future generations, raising significant ethical and regulatory concerns.

Which CRISPR variant is notable for its 'collateral' cleavage activity, which is widely utilized in diagnostic tools like SHERLOCK?

Cas9
Cas3
Cas13
Cas12a

Explanation: Cas13 targets RNA. Once it binds to its target, it becomes 'activated' and starts cutting any nearby RNA molecules (collateral cleavage), which can be used to release fluorescent signals for diagnostics.

Which CRISPR application uses a dCas9 fused to a transcriptional activator (like VP64) to turn on a silent gene?

CRISPRi
CRISPR-Tag
CRISPRa
CRISPR-GO

Explanation: CRISPR activation (CRISPRa) utilizes dCas9 to recruit transcriptional machinery to a promoter, inducing the expression of the target gene.

The Cas12a (Cpf1) variant differs from Cas9 in that it creates which type of cut in the DNA?

Blunt ends
Staggered 'sticky' ends
Circularized fragments
Single-strand nicks only

Explanation: While Cas9 creates blunt-end cuts, Cas12a creates staggered cuts with short overhanging 'sticky' ends, which can be more useful for certain types of DNA cloning and assembly.

Which Anti-CRISPR protein (Acr) inhibits SpCas9 by acting as a DNA mimic that blocks the PAM-recognition site?

AcrVA1
AcrIIA1
AcrF1
AcrIIA4

Explanation: AcrIIA4 mimics the negative charge of DNA to occupy the PAM-interacting domain of Cas9, preventing the enzyme from binding to its genomic target.

The Cas9 nuclease has two catalytic domains. One is the HNH domain; which is the other?

Ligase
Polymerase
RuvC
Helicase

Explanation: Cas9 uses two domains to cut DNA: the HNH domain cuts the strand complementary to the guide RNA, and the RuvC domain cuts the non-complementary strand.

In the context of CRISPR diagnostics, 'collateral cleavage' (trans-cleavage) is a defining catalytic property of which nuclease?

dCas9
Cas3
Cas13
Cas9

Explanation: Once Cas13 binds its specific RNA target, it undergoes a conformational change that activates non-specific RNase activity, cleaving nearby RNA molecules.

Which term refers to the CRISPR system's ability to target multiple genes simultaneously by using several different guide RNAs?

Cascading
Duplexing
Poly-editing
Multiplexing

Explanation: Multiplexing allows for complex genetic engineering by targeting multiple sites in the genome at once, which is useful for studying polygenic diseases or complex metabolic pathways.

What is the natural evolutionary origin of the CRISPR-Cas9 system in prokaryotes?

An adaptive immune system against bacteriophages
A regulatory system for metabolic pathways
A mechanism for horizontal gene transfer
A machinery for DNA replication and repair

Explanation: CRISPR-Cas9 evolved as an adaptive immune system in bacteria and archaea, allowing them to recognize and cleave foreign genetic material from invading viruses (bacteriophages).

What is the primary role of the 'Bridge Helix' (BH) in the Cas9 structural architecture?

Recognizing the PAM
Connecting REC and NUC
Anchoring the gRNA
Unwinding the DNA

Explanation: The Bridge Helix is an arginine-rich alpha-helix that links the Recognition (REC) lobe and the Nuclease (NUC) lobe, coordinating conformational changes.

Which type of CRISPR-Cas system is characterized by the presence of a 'Signature Protein' called Cas3?

Type II
Type III
Type I
Type VI

Explanation: Type I systems utilize the multi-subunit Cascade complex for targeting and the Cas3 protein, which has both helicase and nuclease activity, for DNA degradation.

Who were the two scientists awarded the 2020 Nobel Prize in Chemistry for their work on CRISPR-Cas9?

Yoshizumi Ishino and Francisco Mojica
Feng Zhang and George Church
James Watson and Francis Crick
Emmanuelle Charpentier and Jennifer Doudna

Explanation: Charpentier and Doudna were recognized for their 2012 study demonstrating that the CRISPR-Cas9 system could be reprogrammed as a versatile tool for genome editing.

Which DNA repair mechanism is preferentially used by 'microhomology-mediated end joining' (MMEJ) following a CRISPR cut?

Classical NHEJ
Alternative NHEJ
Mismatch repair
Conservative HDR

Explanation: MMEJ (also known as Alt-NHEJ) uses short flanking microhomologies to repair breaks, often resulting in predictable deletions rather than random indels.

In 'Base Editing', the fusion of a cytidine deaminase to a dCas9 allows for which specific chemical transition?

G-C to T-A
C-G to A-T
C-G to T-A
A-T to G-C

Explanation: Cytosine base editors (CBEs) deaminate cytosine to uracil, which is then read as thymine by DNA polymerase, effectively converting C-G to T-A base pairs.

In 'Epigenetic Editing', a dCas9 fused to a methyltransferase is used to do what?

Creating a copy of the gene on a different chromosome
Silencing a gene by adding chemical markers without changing the sequence
Change the base sequence from G to A
Cut the DNA into smaller fragments

Explanation: Epigenetic editing modifies the chemical 'tags' (like methyl groups) on DNA. This can turn genes on or off without altering the underlying genetic code, making the changes potentially reversible.

What is the primary function of the 'tracrRNA' in the native Type II CRISPR-Cas system?

Acquiring new spacers
Degrading viral proteins
Cleaving the target DNA
Scaffolding crRNA to Cas9

Explanation: The trans-activating CRISPR RNA (tracrRNA) hybridized with the crRNA to form a dual-RNA structure that loads the Cas9 protein and activates its nuclease activity.

Which term describes the unintentional cleavage of DNA at genomic sites that have a sequence similar to the intended target?

On-target mutation
Off-target effect
Gene drive
Epigenetic silencing

Explanation: Off-target effects occur when the guide RNA binds to a DNA sequence that is a near-match to the intended target, leading to unintended mutations elsewhere in the genome.

Which term describes a CRISPR system that utilizes an effector complex composed of multiple different Cas protein subunits?

Type II system
Type V system
Class 1 system
Class 2 system

Explanation: CRISPR systems are divided into two classes: Class 1 uses multi-subunit effector complexes (Types I, III, IV), while Class 2 uses a single-protein effector (Types II, V, VI).

The 'Seed Sequence' of a guide RNA, critical for Cas9 specificity, is typically located at which position?

PAM-interacting loop
5' end of spacer
3' end of spacer
Middle of scaffold

Explanation: The 8-12 nucleotides at the 3' end of the spacer (closest to the PAM) are called the 'seed sequence'. Mismatches here usually prevent Cas9 from cutting.

The SpCas9 protein (from Streptococcus pyogenes) requires a specific Protospacer Adjacent Motif (PAM) to recognize the target. What is this sequence?

AAAA
NGG
TATA
GATTACA

Explanation: SpCas9 specifically seeks out the 5'-NGG-3' PAM sequence. If this motif is not present immediately downstream of the target site, Cas9 will not bind or cut the DNA.

To achieve a precise gene 'knock-in' or specific sequence correction, which cellular pathway must be successfully utilized?

NHEJ
Base Excision Repair
Photoreactivation
HDR

Explanation: Homology Directed Repair (HDR) uses a homologous DNA template (provided by the researcher) to precisely repair a double-strand break, allowing for specific sequence changes.

Which off-target detection method utilizes the integration of a double-stranded oligodeoxynucleotide (dsODN) into Cas9-induced breaks?

DIME-seq
GUIDE-seq
SITE-seq
CIRCLE-seq

Explanation: GUIDE-seq (Genome-wide Unbiased Identification of DSBs Enabled by sequencing) tags double-strand breaks with dsODNs to map where Cas9 cuts across the genome.

In the structural study of Cas9, the 'REC' lobe is primarily involved in which function?

Generating ATP
Recognizing the PAM
Binding the gRNA
Cleaving the DNA

Explanation: The Recognition (REC) lobe is a large, alpha-helical domain that serves as a scaffold to hold the guide RNA and the target DNA heteroduplex in place.

A 'Gene Drive' powered by CRISPR-Cas9 is designed to achieve which of the following?

Protect crops from all known fungal infections
Increase the lifespan of laboratory animals
Accelerate the speed of DNA sequencing
Ensure a specific trait is inherited by nearly all offspring

Explanation: Gene drives use CRISPR to copy themselves onto the homologous chromosome, bypassing standard Mendelian inheritance and ensuring the trait spreads rapidly through a population.

Which CRISPR-based technology allows for the chemical conversion of one DNA base to another (e.g., C to T) without creating a double-strand break?

RNA Interference
Gene Drive
Prime Editing
Base Editing

Explanation: Base editing uses a modified Cas9 fused to a deaminase enzyme to chemically transform one nitrogenous base into another, offering high precision for point mutations.

What is the primary function of the PAM sequence in the CRISPR-Cas9 mechanism?

To signal the end of the transcription process
To differentiate between self and non-self DNA
To provide energy for the DNA cut
To act as a template for DNA repair

Explanation: The PAM is essential for distinguishing the bacteria's own CRISPR locus (which lacks the PAM) from the invading viral DNA, preventing the system from destroying its own genome.

Which organization recently established global standards for the governance and oversight of human genome editing following the 2018 'CRISPR babies' scandal?

World Health Organization (WHO)
World Trade Organization (WTO)
UNESCO
Interpol

Explanation: The WHO formed an expert committee to create a framework for governing human genome editing to ensure it is used safely, ethically, and transparently across the globe.

What is the primary advantage of using Lipid Nanoparticles (LNPs) over AAV vectors for CRISPR delivery?

Higher cell specificity
Lower manufacturing cost
Transient expression
Permanent integration

Explanation: LNPs deliver mRNA or RNP complexes that degrade quickly, ensuring the Cas9 is only present briefly, which significantly reduces the risk of off-target mutations.

Which DNA repair pathway is most likely to be triggered after a Cas9 cut if no donor DNA template is provided?

Non-Homologous End Joining (NHEJ)
Nucleotide Excision Repair
Mismatch Repair
Homology Directed Repair (HDR)

Explanation: NHEJ is the default, error-prone repair mechanism that joins broken DNA ends. It often results in small insertions or deletions (indels) that disrupt the gene's function.

What does 'Ex vivo' gene editing refer to in a clinical context?

Editing the genetic code of deceased tissue for research
Editing genes directly inside the patient's body
Editing the genes of pathogens before they infect humans
Editing cells outside the body and then re-infusing them

Explanation: Ex vivo (out of the living) involves removing a patient's cells, editing them in a lab, and then returning the modified cells to the patient, as seen in many blood disorder treatments.

The 'Schrems-like' scrutiny of the Broad Institute vs. UC Berkeley patent war primarily centered on which legal distinction?

Sequence length specificity
Non-obviousness in eukaryotes
First-to-file vs. First-to-invent
Prior art in prokaryotes

Explanation: The Patent Trial and Appeal Board (PTAB) ruled that using CRISPR in eukaryotic cells was a distinct, non-obvious invention from the original work in test tubes.

What is 'dCas9' (dead Cas9) and what is its primary application in research?

A Cas9 variant that only cuts RNA
A catalytically inactive Cas9 used for gene regulation
A version of Cas9 that cuts more efficiently
A Cas9 protein used in forensic DNA analysis

Explanation: dCas9 can bind to a target DNA sequence but cannot cut it. It is often fused to other proteins to turn genes on or off (CRISPRa/CRISPRi) without changing the DNA sequence.

In the context of the natural bacterial system, what is the role of the tracrRNA?

It is the sequence that gets integrated into the host genome
It acts as a scaffold linking crRNA to the Cas9 enzyme
It provides the template for DNA repair
It codes for the Cas9 protein

Explanation: The trans-activating CRISPR RNA (tracrRNA) base-pairs with the crRNA to form a structure that is recognized and bound by the Cas9 protein.

The 'anti-CRISPR' proteins (Acr) are naturally produced by which of the following?

Human immune cells
Bacteriophages
Mitochondria
Synthetic biology labs

Explanation: Anti-CRISPR proteins are an evolutionary counter-defense developed by viruses to block the bacterial CRISPR-Cas system and allow the virus to replicate successfully.

Which of the following is a major limitation of 'Homology Directed Repair' (HDR) in a clinical therapeutic context?

Low efficiency
Viral toxicity
High indel rate
Small cargo limit

Explanation: HDR is naturally much less efficient than NHEJ in most human cells, especially in non-dividing tissues like the brain or muscles, making gene correction difficult.

Which specific nucleobase modification is targeted by 'Adenine Base Editors' (ABEs)?

C to T
T to A
G to C
A to G

Explanation: ABEs use an engineered deoxyadenosine deaminase to convert adenine to inosine, which the cell's machinery treats as guanine, resulting in an A-to-G transition.

What is the primary technical limitation of using Adeno-associated viruses (AAV) for delivering CRISPR-Cas9 components in vivo?

AAVs have a very limited cargo capacity (approx. 4.7 kb)
AAVs replicate too fast for the Cas9 to work
AAVs are highly toxic to humans
AAVs can only infect skin cells

Explanation: The standard SpCas9 and its guide RNA barely fit into an AAV vector. This has led researchers to seek smaller Cas variants, like SaCas9 (from S. aureus), for better delivery.

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