CRISPR Interference in Mycobacteria

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Questions and Answers

What is one primary outcome of developing CRISPR interference (CRISPRi) for Mycobacterium tuberculosis?

  • It eliminates the requirement for genetic manipulation.
  • It improves the understanding of the pathogen's genetic basis. (correct)
  • It simplifies the protein expression process.
  • It allows for the rapid gene replacement.

What is a significant limitation of current genetic methods like promoter replacement in M. tuberculosis?

  • They are too expensive for common use.
  • They do not allow for any type of regulation.
  • They can only target non-essential genes.
  • They take a long time to implement. (correct)

Which genetic approach has been developed to enhance the efficiency of genetic engineering in M. tuberculosis?

  • Promoter replacement
  • ORBIT (correct)
  • Transposon-sequencing
  • CRISPR/Cas9

What does TnSeq allow for in the context of M. tuberculosis?

<p>Simultaneous assessment of numerous loss-of-function mutants. (C)</p> Signup and view all the answers

Which of these does CRISPRi specifically allow researchers to do?

<p>Silence multiple genes simultaneously. (D)</p> Signup and view all the answers

Why does CRISPRi represent a complementary tool to existing genetic methodologies?

<p>It provides a quicker alternative to gene deletion. (B)</p> Signup and view all the answers

What type of Cas9 enzyme does the novel CRISPRi system utilize for mycobacteria?

<p>Cas9 from S. pyogenes. (C)</p> Signup and view all the answers

Which of the following is not a feature of the CRISPR interference (CRISPRi) system?

<p>It replaces the need for all genetic tools. (B)</p> Signup and view all the answers

What is the main function of the dCas9 protein in the CRISPRi system?

<p>To interfere with transcription by blocking access (C)</p> Signup and view all the answers

What role does the PAM play in the targeting specificity of the dCas9-sgRNA complex?

<p>To serve as the binding site for dCas9 (C)</p> Signup and view all the answers

How does tunability in the Sth1 dCas9 CRISPRi system benefit gene silencing?

<p>It allows for precise control over gene expression levels (D)</p> Signup and view all the answers

Which small molecules have been engineered to induce CRISPRi in M.tuberculosis?

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

What advantage does the Sth1 dCas9 CRISPRi system have regarding gene silencing magnitude?

<p>It can adjust PAM strength and sgRNA length for fine control (D)</p> Signup and view all the answers

What does the binding of the dCas9-sgRNA complex result in?

<p>Inhibition of RNA polymerase access to the promoter (B)</p> Signup and view all the answers

What type of genetic method does CRISPRi represent for M.tuberculosis?

<p>A powerful tool for gene silencing and manipulation (B)</p> Signup and view all the answers

What feature allows CRISPRi to generate large pools of unique sgRNA sequences efficiently?

<p>Array-based synthesis advancements (C)</p> Signup and view all the answers

Flashcards

CRISPRi

A technique for targeted gene silencing in mycobacteria, utilizing a CRISPR-Cas system to inhibit gene expression.

Mycobacteria

A type of bacteria that includes the pathogen responsible for tuberculosis (Mycobacterium tuberculosis).

Cas9 enzymes

A family of DNA-targeting enzymes used in CRISPR-Cas systems, known for their ability to cut DNA sequences.

Transposon-sequencing (TnSeq)

A method for studying the effect of gene inactivation by inserting a transposon (a mobile DNA element) into a gene, disrupting its function.

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Promoter replacement

A technique that uses a DNA-binding protein to promote the expression of a specific gene.

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Inducible protein degradation systems

A system that allows controlled degradation of a target protein, often using a 'degradation tag' attached to the protein.

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ORBIT

A genetic engineering approach that uses a transposon to deliver genes into a cell.

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TnSeq in M.tuberculosis

A method for identifying essential genes by measuring the growth of a population of cells after introducing random mutations.

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CRISPRi in M. tuberculosis

A CRISPR-based system for gene silencing in Mycobacterium tuberculosis. It involves using a deactivated Cas9 protein (dCas9) to block RNA polymerase access to target genes.

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Single guide RNA (sgRNA)

A chimeric RNA molecule that guides dCas9 to the target gene in CRISPRi. It contains a guide sequence that matches the target DNA and a scaffold sequence that binds to dCas9.

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Protospacer adjacent motif (PAM)

A short DNA sequence located next to the sgRNA target sequence. It is required for dCas9 binding, similar to a 'key' that unlocks access to the target gene.

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Tunability of CRISPRi

The ability to control the level of gene silencing by adjusting the sgRNA sequence or the PAM strength. This enables fine-tuning of gene expression for studying gene function.

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Inducible CRISPRi

The use of a chemical inducer like doxycycline to control the activity of dCas9. This allows researchers to turn gene silencing on or off at will.

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Hypomorphic silencing

A method for studying the function of essential genes by partially silencing them, allowing researchers to create a range of phenotypes.

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Array-based sgRNA synthesis

A method for creating a large number of unique sgRNAs with specific targeting sequences. This allows for high-throughput screening of genes.

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Transcriptional interference

The process of blocking RNA polymerase from accessing the target gene, thereby inhibiting transcription.

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Study Notes

CRISPR Interference (CRISPRi) in Mycobacteria

  • CRISPRi is a robust platform for programmable gene silencing in Mycobacterium tuberculosis
  • CRISPRi offers advantages over traditional genetic approaches, allowing for easier and faster gene manipulation
  • The method can target multiple genes simultaneously, unlike existing methods which focus on one gene at a time
  • CRISPRi system uses dCas9 (dead Cas9) which is guided to the target gene by sgRNA, preventing cleavage of DNA
  • Using S. thermophilus Cas9 (Sth1Cas9) offers superior performance compared to other Cas9 enzymes
  • Specificity is determined by base pairing with target DNA and presence of PAM sequence next to the target sequence
  • The system is tunable - magnitude of gene silencing can be adjusted by varying PAM strength or sgRNA length
  • CRISPRi is adaptable to different experimental methods: in vitro culture, infected macrophages and animal models
  • The approach is scalable and cost-effective, allowing for large-scale analysis of gene interactions

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CRISPR Interference for Targeted Gene Silencing in Mycobacteria PDF

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