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Questions and Answers

What is the purpose of adding additional bases at the 5' ends of both oligos?

  • To enhance the efficiency of the Cas9 protein
  • To increase the on-target score of the gRNA
  • To reduce the likelihood of off-target effects
  • To ensure the DNA oligos can be cloned into a vector (correct)

    • What is the advantage of designing a complementary strand and flipping it to make the reverse strand?

  • It reduces the specificity of the gRNA
  • It increases the likelihood of Cas9 DNA cleavage (correct)
  • It increases the risk of off-target effects
  • It reduces the binding efficiency of the gRNA to the target site

    • What is the format of the cDNA sequence used to design the gRNA?

  • GenBank format
  • SBOL format
  • GFF format
  • FASTA format (correct)

    • What is the purpose of using IDT to get custom gRNA?

    <p>To get a selection of target sequences with on-target scores</p> Signup and view all the answers

    What is the purpose of using a bacterial expression plasmid containing a cDNA for fluorescent protein?

    <p>To allow for fluorescence when transformed into bacteria</p> Signup and view all the answers

    What is the primary function of the Cas9 protein in the described experiment?

    <p>To cut out specific DNA sequences</p> Signup and view all the answers

    What is the purpose of using a complementary strand and flipping it to make the reverse strand?

    <p>To ensure efficient targeting of both strands of DNA</p> Signup and view all the answers

    What type of sequence is used to design the gRNA?

    <p>cDNA sequence</p> Signup and view all the answers

    What is the purpose of adding the 'atag' sequence to the forward oligo?

    <p>To facilitate cloning into a vector</p> Signup and view all the answers

    What is the purpose of using IDT to get custom gRNA?

    <p>To obtain a selection of target sequences with on-target scores</p> Signup and view all the answers

    What is the expected outcome when a bacterial expression plasmid containing a cDNA for fluorescent protein is transformed into bacteria?

    <p>The bacteria will fluoresce</p> Signup and view all the answers

    What is the significance of the on-target score provided by IDT for the target sequences?

    <p>It predicts the efficacy of the gRNA in gene knockout</p> Signup and view all the answers

    What is the purpose of designing a DNA oligo for the Cas9 protein?

    <p>To create a custom gRNA for gene knockout</p> Signup and view all the answers

    What is the advantage of using a cDNA sequence for fluorescent protein in the design of the gRNA?

    <p>It provides a template for the design of the gRNA</p> Signup and view all the answers

    What is the expected outcome of using the Cas9 protein to cut out the fluorescent protein gene?

    <p>The fluorescent protein gene will be knocked out</p> Signup and view all the answers

    What is the primary purpose of using a fluorescent protein in the experiment?

    <p>To identify potential targets for the gRNA</p> Signup and view all the answers

    What is the significance of using both forward and reverse strands in the design of the DNA oligo?

    <p>It ensures efficient targeting of the gene by the Cas9 protein</p> Signup and view all the answers

    What is the role of the on-target score provided by IDT?

    <p>It selects the optimal target sequence for the gRNA</p> Signup and view all the answers

    What is the purpose of transforming the bacterial expression plasmid into bacteria?

    <p>To express the fluorescent protein in the bacteria</p> Signup and view all the answers

    What is the ultimate goal of designing gRNAs to knockout fluorescent protein genes?

    <p>To create a bacterial strain that does not express the fluorescent protein</p> Signup and view all the answers

    Study Notes

    Bacterial Expression Plasmids

    • Contain cDNAs for fluorescent proteins (EGFP or mCherry), leading to fluorescence when transformed into bacteria

    gRNA Design

    • Used IDT to design custom gRNA for chosen protein
    • Imported cDNA sequence in FASTA format
    • Received a selection of target sequences from IDT, each with an on-target score

    Oligo Design for Cas9 Protein

    • Used target sequence as forward strand
    • Designed complementary strand and flipped it to make the reverse strand for efficient targeting
    • Added additional bases at the 5’ ends of both oligos for cloning into a vector
      • Forward oligo: added atag
      • Reverse oligo: added aaac

    Practical Overview

    • Designed gRNAs to knock out fluorescent protein genes using Cas9 protein

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    Description

    This quiz covers the design of bacterial expression plasmids, gRNA design, and oligo design for Cas9 protein. Learn about CRISPR-Cas9 gene editing and its applications in molecular biology.

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