CRISPR/Cas Systems and Genome Editing
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Questions and Answers

What role does the CRISPR/Cas system primarily serve in bacteria?

  • Facilitating cell division
  • Providing immunity against phages (correct)
  • Storing energy for metabolic processes
  • Enhancing protein synthesis

    • What happens to elements of phage DNA in the CRISPR/Cas system?

  • They are discarded by the bacterium
  • They become part of the CRISPR array (correct)
  • They are converted into proteins
  • They trigger oxidative stress in the bacterium

    • Which component is synthesized from the CRISPR array to target foreign DNA?

  • tRNA
  • rRNA
  • crRNA (correct)
  • mRNA

    • Which of the following describes a characteristic of the CRISPR/Cas9 mechanism in genome editing?

    <p>It targets specific DNA sequences using crRNA</p> Signup and view all the answers

    What is one significant application of CRISPR/Cas9 technology in biotechnology?

    <p>Editing genes to eliminate genetic diseases</p> Signup and view all the answers

    What is a common application of the CRISPR/Cas9 system in the context of animal genetics?

    <p>Generation of transgenic animals by gene editing</p> Signup and view all the answers

    Which of the following reflects a key advantage of CRISPR components in genome editing?

    <p>They are now available in single plasmids.</p> Signup and view all the answers

    How has the standard practice for gene editing changed in plants since the introduction of CRISPR/Cas9?

    <p>It is now considered a standard lab practice.</p> Signup and view all the answers

    What aspect of genome editing does the CRISPR/Cas9 system simplify the most?

    <p>The accessibility of editing tools in eukaryotic organisms</p> Signup and view all the answers

    What is a significant benefit of using the CRISPR/Cas9 system in scientific research?

    <p>It allows for precise and efficient genetic modifications.</p> Signup and view all the answers

    What are the two domains in Cas9 responsible for the nuclease activity?

    <p>HNH and RuvC domains</p> Signup and view all the answers

    Where does the RuvC domain cut the DNA strand in Cas9?

    <p>At a position adjacent to the PAM</p> Signup and view all the answers

    What is a unique feature of Cpf1's catalytic activity compared to Cas9?

    <p>Cpf1 has a single catalytic cleavage site for both DNA strands</p> Signup and view all the answers

    What role does the Nuc domain in Cpf1 serve?

    <p>It facilitates DNA binding</p> Signup and view all the answers

    Which domain in Cpf1 is responsible for the cleavage mechanism?

    <p>RuvC domain</p> Signup and view all the answers

    Which of the following statements about the HNH domain is correct?

    <p>It cuts the strand to which gRNA is hybridized.</p> Signup and view all the answers

    How does Cpf1 differ from Cas9 in terms of DNA strand cleavage?

    <p>Cpf1 cleaves to both strands at a single site</p> Signup and view all the answers

    Which statement correctly describes the cleavage activity of Cas9?

    <p>Each domain cuts a different strand.</p> Signup and view all the answers

    What is the primary role of Cas9 in gene editing?

    <p>Nuclease activity</p> Signup and view all the answers

    Why is it important to inhibit Cas9 activity after a specified period?

    <p>To minimize off-target effects</p> Signup and view all the answers

    Which method is suggested for reducing the activity of Cas9?

    <p>Administer specific proteins</p> Signup and view all the answers

    What happens when the active site (HNH) of Cas9 is altered or occluded?

    <p>Dramatically reduces catalytic activity</p> Signup and view all the answers

    Which of the following proteins is noted for its effective interaction with Cas9?

    <p>AcrIIC1</p> Signup and view all the answers

    What is a potential consequence of Cas9's unchecked activity?

    <p>Serious consequences to the organism</p> Signup and view all the answers

    Stable interactions between Cas9 and inhibitory proteins are beneficial because they:

    <p>Maintain a tight link reducing unwanted cleavage</p> Signup and view all the answers

    What role do proteins administered as viral DNA play in relation to Cas9?

    <p>They inhibit Cas9 activity</p> Signup and view all the answers

    What is the primary advantage of using double nickase activity in the Cas9 system?

    <p>It allows for specific mutation with reduced off-target effects.</p> Signup and view all the answers

    Base editors provide scientists the ability to make what kind of changes to DNA?

    <p>Single base changes without inducing indels.</p> Signup and view all the answers

    What role does the cytosine deaminase play in the cytosine base editor (CBE) mechanism?

    <p>It converts cytosine to uracil.</p> Signup and view all the answers

    What is a significant limitation of homology-directed repair (HDR) in genetic engineering?

    <p>It results in low efficiency and high indels.</p> Signup and view all the answers

    How does the protein UGI contribute to the function of base editors?

    <p>It overrides the cell's base excision repair (BER) system.</p> Signup and view all the answers

    Which of the following statements is true regarding the efficiency of base editors compared to traditional methods?

    <p>Base editors offer higher efficiency and fewer off-target effects.</p> Signup and view all the answers

    In the context of Cas9 and Cpf1, what distinguishes them as editing tools?

    <p>Both can be utilized in base editing strategies with unique functions.</p> Signup and view all the answers

    Why is reducing off-target effects critical in genetic engineering?

    <p>It minimizes unintended mutations that could disrupt other genes.</p> Signup and view all the answers

    What specific feature allows base editors to avoid inducing indels during DNA editing?

    <p>The design of the base editor proteins.</p> Signup and view all the answers

    What is the consequence of using single-stranded breaks instead of double-stranded breaks in genome editing?

    <p>It decreases the chances of off-target mutations.</p> Signup and view all the answers

    Study Notes

    Bacterial Immunity

    • CRISPR/Cas system provides bacterial immunity against phages
    • CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats
    • CRISPR/Cas systems create an array of phage DNA fragments
    • These DNA fragments are replicated to synthesize crRNA
    • crRNA targets effector proteins to foreign DNA

    Mechanism of DNA cleavage

    • Cas9 enzyme contains two domains that possess nuclease activity
    • These are the RuvC and HNH domains
    • Each of these domains cuts one strand of DNA
    • The RuvC domain cuts the strand adjacent to the PAM
    • The HNH domain cuts the strand to which the gRNA hybridizes

    CRISPR/Cas9 in Eukaryote Genome Editing

    • CRISPR/Cas9 has many potential applications
    • CRISPR/Cas9 can be used to generate transgenic animals and plants
    • CRISPR/Cas9 is easily accessible on single plasmids

    Inhibition of Cas9 Activity

    • Cas9 activity is pivotal to gene editing
    • Uncontrolled Cas9 activity can have serious consequences for the organism
    • Methods to alter, reduce, or eliminate Cas9 activity are important to reduce off-target effects
    • Viral DNA can be administered to inactivate Cas9
    • Proteins can target the DNA binding and cleavage sites of Cas9
    • The active site (HNH) of Cas9 can be occluded by proteins

    Double Nickase Activity

    • Nicking at two sites creates a staggered break instead of double-strand break cleavage
    • This is achieved by mutating Cas9 catalytic sites and using two sgRNAs
    • Single nicking reduces the chance of off-target effects

    CRISPR Base Editors

    • Base editors combine several proteins
    • Base editors allow scientists to alter a single base without inducing indels
    • Base editors are more precise because they do not induce indels

    Cytosine Base Editor (CBE) Protein Detail

    • Both gRNA and Cas9 or Cpf1 target the machinery to a specific DNA sequence
    • Cytosine deaminase converts C to U
    • UGI (Uracil glycosylase inhibitor) overrides the cell's BER (Base Excision Repair) system

    Base Editors are Less Error-Prone

    • HDR (homology-directed repair) is not efficient and results in indels
    • CBE has higher efficiency and induces fewer indels
    • Both Cas9 and Cpf1 can be used with base editors

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    Related Documents

    Genome Editing II CRISPR/Cas9 PDF

    Description

    This quiz covers the fundamentals of the CRISPR/Cas system, including its role in bacterial immunity and its application in eukaryotic genome editing. Learn about the mechanism of DNA cleavage by the Cas9 enzyme and the importance of regulating its activity for safe genetic modifications.

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