CRISPR Interference in Mycobacteria

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Questions and Answers

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 (B)</p> Signup and view all the answers

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

<p>Restriction enzyme SapI (C)</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 (D)</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 (A)</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. (B)</p> Signup and view all the answers

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

<p>37 C (B)</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 (A)</p> Signup and view all the answers

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

<p>1 hour (B)</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. (B)</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 (B)</p> Signup and view all the answers

What is the correct voltage setting during the electroporation process?

<p>2500 V (B)</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 (D)</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 (A)</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 (C)</p> Signup and view all the answers

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

<p>7H9-ADC-Tween80 (D)</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 (D)</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 (D)</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 (C)</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 (C)</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 (D)</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 (C)</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. (D)</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. (B)</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. (A)</p> Signup and view all the answers

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

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

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

<p>Mutations that disable its nuclease activity. (C)</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. (D)</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. (A)</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. (D)</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 (A)</p> Signup and view all the answers

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

<p>To ensure accurate gene targeting (C)</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 (B)</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 (A)</p> Signup and view all the answers

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

<p>Guanidine thiocyanate (A)</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 (B)</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 (B)</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' (D)</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. (A)</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. (C)</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. (A)</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. (C)</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. (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. (B)</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. (D)</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. (C)</p> Signup and view all the answers

Flashcards

dCas9

A deactivated version of Cas9 protein, used in CRISPR-based gene regulation.

sgRNA

A type of RNA molecule that guides the dCas9 protein to a specific target DNA sequence.

PAM

A short DNA sequence near the target site, recognized by dCas9 for binding.

CRISPRi

The process of using CRISPR-based tools to repress or silence gene expression without altering the DNA sequence.

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CRISPRi Tunability

The ability to adjust the level of gene silencing by modifying the strength of the PAM or the length of the sgRNA.

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Allelic Series

Using CRISPRi to create different levels of gene expression, allowing researchers to study subtle effects on cell function.

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M.tuberculosis CRISPRi

A method of manipulating gene expression in Mycobacterium tuberculosis using a CRISPR-based system.

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CRISPRi for Essential Gene Study

Using CRISPRi to investigate essential genes in vitro by partially silencing their expression.

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Transformation

A technique used to insert foreign DNA into bacteria, creating genetically modified bacteria.

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Chemically competent E. coli cells

A solution containing bacteria that have been treated to make them more receptive to foreign DNA.

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Electrocompetent bacteria

A solution containing bacteria that are capable of taking up foreign DNA through electroporation.

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Electroporation

A process used to introduce DNA into cells using an electrical pulse.

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Miniprep

The process of isolating and purifying DNA from cells.

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Oligo

A short sequence of DNA that serves as a specific target for a restriction enzyme.

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Sanger Sequencing

A method used to determine the sequence of DNA.

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Cloning

A technique used to create a genetically modified organism.

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Multi-sgRNA CRISPRi

A method for constructing multiple sgRNAs on a single plasmid, utilizing a SapI-based Golden Gate cloning approach.

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Golden Gate Cloning

A specialized cloning technique allowing for the simultaneous restriction digestion and ligation of multiple DNA fragments in a single reaction.

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SapI Restriction Site

A DNA sequence recognized by restriction enzymes, used for cutting DNA fragments.

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Golden Gate Handles

A set of oligonucleotides containing SapI restriction sites and compatible DNA overhangs for use in Golden Gate cloning.

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PCR Amplification of sgRNA Cassettes

A type of PCR amplification specifically designed for generating sgRNA cassettes containing promoters, sgRNA sequences, and terminators.

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Electrocompetent Cells

The process of introducing genetic material into cells by using specialized chemical treatments to make the cell membrane more permeable.

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Phenotype Confirmation

A technique using microscopy, growth, and survival assays to assess the effectiveness of CRISPRi in altering gene expression.

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Selective Growth of Bacteria

Growing bacteria in a liquid medium containing nutrients and antibiotics, like kanamycin, which allows only the modified bacteria to grow.

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CRISPRi-compatible expression vector

A type of plasmid that is compatible with the CRISPRi system and can be used to express a specific gene of interest.

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sgRNA Seed Sequence

The 9 nucleotide sequence at the 5' end of the sgRNA that is critical for target recognition and off-target effects.

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Bad Seed

A specific 5 nucleotide sequence within the sgRNA seed sequence that can cause fitness costs even when not targeting any specific gene.

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GTC Buffer

A special buffer used for cell lysis during DNA extraction, containing guanidine thiocyanate to lyse cells and inhibit RNases, N-lauryl sarcosine to prevent new transcription, and sodium citrate to protect RNA from degradation.

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CRISPRi Transformation Assay

A method of testing CRISPRi effectiveness by observing the growth inhibition of bacteria transformed with plasmids encoding sgRNAs that target essential genes.

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Escaper Colonies

Colonies that arise in the presence of ATc, indicating that CRISPRi is not completely effective in inhibiting the target gene.

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Off-target Identification

The process of identifying potential off-target sites for sgRNAs, where the sgRNA seed sequence perfectly matches other locations in the genome.

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sgRNA Design Principle

The design principle of avoiding sgRNAs with seed sequences that have perfect matches to the bacterial chromosome, particularly in ORFs or promoters, to minimize off-target effects.

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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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