CRISPR Interference in Mycobacteria
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What is the purpose of the SapI-based Golden Gate cloning site in the CRISPRi backbones?

  • It enables the transformation of multiple sgRNAs into one plasmid. (correct)
  • It allows for the cloning of a single sgRNA.
  • It provides a restriction site for T4 DNA ligase.
  • It enhances the expression of the plasmid.

    • Which step is NOT part of the Golden Gate cloning procedure?

  • Performing one-pot restriction digestion and ligation.
  • Incubating at 25°C for 30 minutes. (correct)
  • Amplifying the promoter-sgRNA-terminator cassettes.
  • Transforming the Golden Gate reaction into competent cells.

    • What is the main purpose of performing phenotypic assays after transforming the CRISPRi constructs?

  • To validate the complementation of the CRISPRi knockdown phenotypes. (correct)
  • To analyze the plasmid size.
  • To measure the growth rate of E. coli.
  • To confirm the transformation efficiency.

    • What is the recommended temperature for incubating the Golden Gate reaction for 1 hour?

    <p>37°C</p> Signup and view all the answers

    Which of the following components is crucial for the Golden Gate cloning reaction?

    <p>Restriction enzyme SapI</p> Signup and view all the answers

    How many additional sgRNA cassettes can be cloned into a single CRISPRi backbone using the SapI-based Golden Gate approach?

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

    Which of the following primers is used for Sanger sequencing validation of the cloning?

    <p>Both A and C</p> Signup and view all the answers

    What is the purpose of adding kanamycin to the LB broth when plating the transformation?

    <p>To select for kanamycin-resistant clones.</p> Signup and view all the answers

    What temperature should the M.tuberculosis culture be maintained at during the growth phase?

    <p>37 C</p> Signup and view all the answers

    What is the purpose of adding kanamycin to the LB plates?

    <p>To allow selection of kanamycin-resistant clones</p> Signup and view all the answers

    How long should the E.coli cells be recovered after transformation before plating?

    <p>1 hour</p> Signup and view all the answers

    What should be done with the supernatant after centrifugation of the electrocompetent cells?

    <p>It should be discarded.</p> Signup and view all the answers

    At what OD600 should the M.tuberculosis culture be grown to in the initial culture before expansion?

    <p>1.0</p> Signup and view all the answers

    What is the correct voltage setting during the electroporation process?

    <p>2500 V</p> Signup and view all the answers

    How long should the M.tuberculosis culture be grown in the stationary phase to reach saturation?

    <p>3-4 days</p> Signup and view all the answers

    What is the purpose of resuspending the electrocompetent M.tuberculosis cells in 10% glycerol?

    <p>To maintain cell viability during storage</p> Signup and view all the answers

    What is the initial optical density (OD600) to which the overnight culture of M.smegmatis should be diluted?

    <p>0.01 - 0.02</p> Signup and view all the answers

    Which medium is used for recovering transformations in M.smegmatis after electroporation?

    <p>7H9-ADC-Tween80</p> Signup and view all the answers

    What is the purpose of incubating M.smegmatis with kanamycin at a concentration of 20 μg/mL?

    <p>To select for transformants</p> Signup and view all the answers

    What is the optimal temperature for growing M.smegmatis cultures during the preparation of competent cells?

    <p>37 C</p> Signup and view all the answers

    During which phase of growth is the M.smegmatis CRISPRi strain supposed to be diluted for further growth?

    <p>Saturation phase</p> Signup and view all the answers

    What is a suggested advantage of targeting the gene ORF rather than the gene promoter in CRISPRi systems?

    <p>Stronger transcriptional interference</p> Signup and view all the answers

    What should the culture optical density (OD600) reach after growing for 16 hours before preparing electrocompetent cells?

    <p>1.0</p> Signup and view all the answers

    What effect has been noted when using the SpyCas9 system for CRISPRi regarding sgRNA length?

    <p>Optimal length of 20 nt</p> Signup and view all the answers

    What is the role of the protospacer adjacent motif (PAM) in the dCas9 binding process?

    <p>It is recognized first, destabilizing the adjacent DNA duplex.</p> Signup and view all the answers

    How does the dCas9--sgRNA complex interfere with transcription?

    <p>By blocking RNA polymerase from accessing the promoter.</p> Signup and view all the answers

    What advantage does the tunability of the Sth1 dCas9 CRISPRi system provide?

    <p>It allows precise control over gene expression levels.</p> Signup and view all the answers

    Which small molecules can induce the engineered CRISPRi system in M. tuberculosis?

    <p>Anhydrotetracycline and Doxycycline</p> Signup and view all the answers

    What characteristic of dCas9 makes it 'dead' or non-nuclease?

    <p>Mutations that disable its nuclease activity.</p> Signup and view all the answers

    What is significant about the Sth1 dCas9 CRISPRi system in terms of scalability?

    <p>It allows rapid generation of unique sgRNA pools.</p> Signup and view all the answers

    Which of the following best describes the concept of rheostat-like control in gene silencing?

    <p>Variable gene expression across a range of levels.</p> Signup and view all the answers

    In what settings can CRISPRi-mediated control of the M. tuberculosis transcriptome be applied?

    <p>In axenic culture, ex vivo, and in vivo models.</p> Signup and view all the answers

    What is the minimum length of perfect complementarity in sgRNAs that can lead to off-target gene repression?

    <p>9 nucleotides</p> Signup and view all the answers

    Why is it important to check sgRNAs for off-target effects?

    <p>To ensure accurate gene targeting</p> Signup and view all the answers

    What defines a 'bad seed' in the context of sgRNA design?

    <p>A specific 5 nucleotide sequence causing fitness costs</p> Signup and view all the answers

    What is the recommended method to minimize light exposure for ATc?

    <p>Wrap flasks and plates in aluminum foil</p> Signup and view all the answers

    Which component in the GTC buffer plays a role in inhibiting RNases?

    <p>Guanidine thiocyanate</p> Signup and view all the answers

    What is a common frequency range for the occurrence of 'escaper' colonies when using ATc?

    <p>1:500 to 1:1000 transformants</p> Signup and view all the answers

    What is a limitation of monitoring CRISPRi target gene knockdown through protein levels?

    <p>Paucity of antibodies to endogenous proteins</p> Signup and view all the answers

    Which PAM sequence is prevalent in the bacterial chromosome, and is associated with more common off-target gene repression?

    <p>5'-NGG-3'</p> Signup and view all the answers

    What is necessary for the expression vector to be compatible with the CRISPRi backbone?

    <p>It should not use kanamycin resistance for positive selection.</p> Signup and view all the answers

    Which part of the first amplified fragment is essential for proper cloning?

    <p>An upstream overhang that aligns with the second fragment.</p> Signup and view all the answers

    What does lack of complementation by expression of a CRISPRi-sensitive allele indicate?

    <p>It shows that the phenotype is not due to the second allele's expression.</p> Signup and view all the answers

    What can be done if cloning efficiency is low using the Golden Gate approach?

    <p>Perform the processes in sequential steps.</p> Signup and view all the answers

    How many times should the cycling be adjusted for thermocycler conditions when using 6 or more inserts?

    <p>30 times alternating between 37 C and 16 C.</p> Signup and view all the answers

    What may be necessary when cloning multiple sgRNA cassettes into a single Golden Gate site?

    <p>To increase the volume of competent E. coli cells used.</p> Signup and view all the answers

    Which of the following mycobacterial species might the described methods apply to?

    <p>M.abscessus and M.avium complex.</p> Signup and view all the answers

    What role does T4 DNA ligase play in the Golden Gate reaction?

    <p>It digests the sgRNA plasmid and ligates sgRNA cassettes.</p> Signup and view all the answers

    Study Notes

    CRISPR Interference (CRISPRi) for Targeted Gene Silencing in Mycobacteria

    • CRISPRi is a platform for programmable gene silencing in mycobacteria.
    • It offers advantages over traditional genetic approaches, including faster gene regulation and simultaneous modulation of multiple genes.
    • CRISPRi uses a deactivated Cas9 enzyme (dCas9) guided by a single guide RNA (sgRNA) to target and repress gene transcription.
    • The dCas9 used is from Streptococcus thermophilus (Sth1Cas9).
    • The sgRNA targets a specific DNA sequence.
    • The PAM (protospacer adjacent motif) sequence adjacent to the target site is crucial for dCas9 binding, and its sequence influences targeting efficiency.
    • CRISPRi is scalable and cost-effective.
    • The method is applicable to in vitro and in vivo studies of Mycobacterium tuberculosis.

    Introduction

    • Mycobacterium tuberculosis genetic manipulation has seen improvements in the last 20 years.
    • Existing methods include promoter replacement, inducible protein degradation, and gene deletion.
    • These methods are often slow and restricted to non-essential genes.
    • TnSeq allows for genome-wide loss-of function studies but is limited by non-essential gene analysis.
    • CRISPRi enables simultaneous modulation of multiple genes, which facilitates the study of genetic interactions.

    Transcriptional Interference

    • CRISPRi works by blocking RNA polymerase access to promoters or inhibiting elongation.
    • The M. tuberculosis CRISPRi system is inducible using anhydrotetracycline or doxycycline, allowing for flexible manipulation in different experimental settings.

    sgRNA Design

    • Design sgRNAs targeting the non-template strand of the target gene.
    • Use a PAM sequence that increases targeting efficiency (determined experimentally).
    • Selecting nucleotides upstream of PAM (21-24 nucleotides) is important.
    • Initiation nucleotides A or G for sgRNA synthesis are preferable in mycobacteria.

    CRISPRi-Resistant Complementation

    • Complementation with a CRISPRi-resistant allele is important to confirm that the observed phenotype is due to transcriptional knockdown of the targeted gene.
    • Introducing silent mutations in the sgRNA-binding site or PAM can disrupt CRISPRi targeting.

    Electroporation

    • Mycobacteria are made electrocompetent (allows DNA uptake).
    • CRISPRi constructs are electroporated into the bacterial cells.

    Quantification of Transcriptional Knockdown

    • qRT PCR is used to determine the degree of transcriptional knockdown.
    • A housekeeping gene is used for normalization of target gene expression levels.

    Targeting Multiple Genes

    • CRISPRi can target multiple genes in tandem using a single plasmid, using a Sapl-based Golden Gate.

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    CRISPR Interference for Targeted Gene Silencing in Mycobacteria PDF

    Description

    Explore the innovative CRISPR interference (CRISPRi) method for targeted gene silencing specifically in mycobacteria. This quiz delves into the mechanism of CRISPRi utilizing deactivated Cas9 and its advantages over traditional methods. Understand its scalability, cost-effectiveness, and applications in studying Mycobacterium tuberculosis.

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