CRISPR/CAS9 Gene Editing Tool

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Questions and Answers

What is the role of the Cas9 protein in the CRISPR/Cas9 system?

To integrate spacers into the genome.

To provide a complementary template for DNA repair.

To inhibit bacterial growth.

To unwind and cut double-stranded DNA. (correct)

Which repair mechanism is involved when no complementary template is available in the CRISPR/Cas9 system?

Trans-activating repair (TAR)

Template-directed repair (TDR)

Non-homologous end joining (NHEJ) (correct)

Homology-directed repair (HDR)

What does the acronym CRISPR stand for?

Clustered Regularly Interspaced Short Palindromic Repeats (correct)

Clustered Random Interspaced Short Palindromic Repeats

Covalently Regulated Interspersed Short Palindromic Repeats

Coded Regularly Interspersed Short Palindromic Repeats

What is the function of the single guide RNA (sgRNA) in the CRISPR/Cas9 mechanism?

To dictate the Cas9 target site. Signup and view all the answers

Which sequence types are found in CRISPR RNA (crRNA)?

Spacer sequences and repeat elements. Signup and view all the answers

What are the two main steps involved in the adaptation phase of the CRISPR/Cas9 system?

Acquisition of new spacers and integration of spacers. Signup and view all the answers

What is tracrRNA and what is its function?

A noncoding RNA that hybridizes to crRNA for the targeting process. Signup and view all the answers

Which statement best describes the evolutionary aspect of CRISPR systems?

Multiple CRISPR-type immune systems have developed over billions of years. Signup and view all the answers

What is the role of the Cas1 and Cas2 proteins in the CRISPR process?

They direct the acquisition of foreign DNA. Signup and view all the answers

Which term describes the foreign DNA that enters the host cell during CRISPR acquisition?

Protospacer Signup and view all the answers

What initiates the transcription of CRISPR RNA (crRNA)?

The leader end of the repeat-spacer array Signup and view all the answers

During interference, what does Cas9 bind to in order to target foreign nucleic acids?

The nontarget strand near the PAM Signup and view all the answers

Which type of DNA repair mechanism is characterized by error-free repair of DNA breaks?

Homology-directed Repair (HDR) Signup and view all the answers

What is a common result of the Non-Homologous End Joining (NHEJ) pathway?

Short random insertions or deletions Signup and view all the answers

What function does the dead variant of Cas9 (dCas9) serve?

It binds to DNA without cleaving it. Signup and view all the answers

What role does the Protospacer Adjacent Motif (PAM) play in the CRISPR process?

It is recognized by Cas9 to initiate targeting. Signup and view all the answers

What is the primary function of dCas9 in transcriptional repression?

To block RNA polymerase access Signup and view all the answers

Knock-ins (KIs) in genetic engineering refer to which of the following?

Gain of gene functions Signup and view all the answers

Which issue is NOT associated with the ethical concerns of CRISPR technology?

Effectiveness of gene therapy Signup and view all the answers

What does nCas9 stand for in the context of CRISPR technology?

Nickase Cas 9 Signup and view all the answers

What is a potential risk when edited genes in CRISPR technology are passed to future generations?

They may cause unforeseen complications. Signup and view all the answers

The use of CRISPR tools in therapeutics is mainly hindered by which factor?

Editing efficiency Signup and view all the answers

In CRISPR applications, what role does a transcriptional activator play when fused with dCas9?

Stimulates transcription of adjacent genes Signup and view all the answers

What complicates moral decision making regarding the use of CRISPR technology?

Uncertainty in risk/benefit analysis Signup and view all the answers

Study Notes

Introduction to CRISPR/Cas9

CRISPR is a natural bacterial defense mechanism against viruses, evolving over billions of years.
Bacteriophages were identified in the late 1800s as antibacterial agents, leading to the discovery of CRISPR.
CRISPR is classified based on associated Cas genes found near its arrays.
Cas9 protein creates double-stranded breaks in DNA at specific target sites identified by custom-designed sgRNA.
DNA repair through non-homologous end joining (NHEJ) can introduce errors, while homology-directed repair (HDR) offers precise editing when a template is present.

CRISPR Locus and Components

"Locus" refers to the location of a gene on a chromosome.
Cas9 unwinds and eliminates large DNA segments.
crRNA is a noncoding RNA that guides the CrRNA:tracrRNA:Cas9 complex to target nucleic acids.
g-RNA is formed by linking crRNA and tracrRNA covalently, crucial for targeting.

Mechanism of CRISPR/Cas9

Adaptation: Acquisition of foreign DNA (protospacer) as new spacers integrated into the host genome, directed by Cas1–Cas2 proteins.
Expression: Transcription of crRNA begins at the leader end of the repeat-spacer array.
Interference: Effector complex recognizes and cleaves foreign nucleic acids. It scans for a PAM site and checks for sequence similarity before executing a double-strand break.

DNA Repair Mechanisms

Homology-directed repair (HDR): Template-dependent, error-free DNA repair, enabling specific gene edits.
Insertion/deletion (indel): Result from NHEJ, causing random changes at the target site.
Mutant Cas9 (dCas9) binds without cleaving, used for transcriptional repression or activation, affecting gene expression.

Applications of CRISPR

Knockouts (KOs): Induce loss of gene function through indels.
Knock-ins (KIs): Introduce new gene functions via targeted genome modifications.
nCas9: A modified Cas9 that cleaves one DNA strand, enhancing precision.

Ethical Concerns Surrounding CRISPR

Concerns exist about the technical limitations of CRISPR, including potential inaccuracies and incomplete edits.
Future implications for modified organisms are uncertain, sparking debates about gene stability and inheritance effects.
The unpredictable outcomes of genetic editing in germline or somatic cells raise ethical questions and complicate benefit-risk assessments.

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Description

This quiz explores the CRISPR/CAS9 gene editing technology, delving into its history, applications, and mechanisms. Learn how this groundbreaking tool evolved from natural bacterial defense systems to revolutionize genetic engineering. Test your knowledge on one of the most significant scientific advancements of our time.