MIC 115 Lecture 15: CRISPR-Cas9 and RNAi

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Questions and Answers

What is one of the primary functions of the CRISPR system in bacteria?

  • To produce energy for bacterial growth
  • To enhance bacterial reproduction
  • To facilitate nutrient absorption
  • To remember and destroy invading phages (correct)

Which gene-editing technology was developed first?

  • Zinc Finger Nuclease (correct)
  • RNA interference
  • TALEN
  • CRISPR

Which of the following best describes RNA interference?

  • A way to introduce mutations in plant genes
  • A method to enhance phage survival in bacteria
  • A technique for editing bacterial DNA
  • A gene silencing method utilizing small RNA molecules (correct)

What is a common feature shared by both CRISPR and TALEN technologies?

<p>They both introduce nicks in DNA to create double-strand breaks. (D)</p> Signup and view all the answers

What allows the CRISPR system to incorporate new spacers into its array?

<p>Cas1, Cas2, and Csn2 proteins (B)</p> Signup and view all the answers

What type of crops can benefit from CRISPR technology?

<p>Infection-resistant crops (B)</p> Signup and view all the answers

Which of the following statements regarding TALENs is accurate?

<p>TALENs recognize each base and are very specific. (C)</p> Signup and view all the answers

CRISPR technology was established primarily for what purpose?

<p>Providing adaptive immunity in bacteria (D)</p> Signup and view all the answers

What components does the simplified CRISPR/Cas9 nuclease system consist of?

<p>Cas9 nuclease and single guide RNA (sgRNA) (D)</p> Signup and view all the answers

What is the function of the PAM (protospacer adjacent motif) in the CRISPR-Cas9 system?

<p>To aid in the binding of Cas9 to the target DNA sequence (D)</p> Signup and view all the answers

Which method can be used to introduce plasmids into cultured cells?

<p>Sonoporation (B), Lentiviral transduction (C), Microinjection (D), Electroporation (A)</p> Signup and view all the answers

What is the role of dCas9 in CRISPR applications?

<p>To act as a DNA-binding protein without nuclease activity (A)</p> Signup and view all the answers

What is the effect of siRNA when it binds perfectly to its target mRNA transcript?

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

What structure does siRNA form when it is comprised of two strands?

<p>A duplex 21 bp long with 3' overhangs (A)</p> Signup and view all the answers

What is the purpose of fusing dCas9 with an effector protein?

<p>To control gene expression by activation or repression (D)</p> Signup and view all the answers

What happens during the transcription of plasmids containing Cas9 and sgRNA?

<p>Cas9-sgRNA complex formation occurs after transcription (D)</p> Signup and view all the answers

What are the components transcribed from the CRISPR locus during the processing of precursors?

<p>Pre-crRNA and tracrRNA (D)</p> Signup and view all the answers

Which enzyme processes the RNA duplex formed by tracrRNA and pre-crRNA?

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

What role does the mature crRNA–tracrRNA structure play in CRISPR-mediated immunity?

<p>It directs Cas9 endonuclease to cleave foreign DNA. (A)</p> Signup and view all the answers

How does a new spacer integrate into the CRISPR array?

<p>Through the acquisition machinery including Cas proteins (A)</p> Signup and view all the answers

What does the PAM sequence stand for in the context of CRISPR-Cas9?

<p>Proto-Spacer Adjacent Motif (A)</p> Signup and view all the answers

Which type of RNA is primarily responsible for guiding the Cas9 endonuclease to its target?

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

During the maturation of crRNA, what happens to the length of the guide sequence?

<p>It is reduced to a length of 20 nucleotides. (C)</p> Signup and view all the answers

What is the role of Cas9 protein in the CRISPR system?

<p>To cleave foreign DNA (C)</p> Signup and view all the answers

Flashcards

CRISPR-Cas9

A bacterial defense system that allows bacteria to remember and destroy invading phages.

CRISPR-mediated genome editing

Using the CRISPR system to make precise changes to an organism's DNA.

RNA interference (RNAi)

A gene silencing mechanism that reduces gene expression by interfering with the process of translation.

Spacer

A short piece of phage DNA incorporated into the CRISPR array.

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Cas proteins

Proteins involved in the CRISPR system, including Cas1, Cas2, and Csn2 which are part of the acquisition machinery.

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Phage

A virus that infects bacteria.

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Genome Editing

Precisely altering an organism's DNA.

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Zinc Finger Nucleases (ZFNs)

A gene editing tool, using zinc finger DNA-binding domains.

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CRISPR spacers

Short segments of non-repetitive DNA sequences located between repeating sequences in a CRISPR locus.

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Pre-crRNA

Long precursor CRISPR RNA produced by transcribing the CRISPR locus, which includes spacers and repeats.

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tracrRNA

Trans-activating CRISPR RNA; a unique non-coding RNA that hybridizes with pre-crRNA repeats and helps process it.

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crRNA maturation

The process of producing mature crRNA from pre-crRNA and tracrRNA by enzymes like RNase III.

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crRNA-tracrRNA complex

The functional RNA complex formed when tracrRNA binds to crRNA and guides the Cas9 endonuclease.

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Cas9 endonuclease

A protein enzyme that cuts DNA at specific sites guided by the crRNA-tracrRNA complex.

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PAM sequence

A specific DNA sequence needed near the target site for Cas9 to function.

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CRISPR adaptive immunity

The bacterial system for acquiring and using memory of past viral infections, for defense against those viruses using Cas proteins.

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sgRNA

A single RNA molecule that combines the functions of both crRNA and tracrRNA, simplifying CRISPR experiments.

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Cas9

An enzyme that cuts DNA at a specific location guided by the sgRNA, enabling targeted gene editing.

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dCas9

A modified Cas9 enzyme that lacks nuclease activity (cannot cut DNA), but still binds to DNA sequences guided by sgRNA.

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CRISPRa

Using dCas9 fused with an activator protein to increase gene expression.

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CRISPRi

Using dCas9 fused with a repressor protein to decrease gene expression.

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siRNA

Short, double-stranded RNA molecules that trigger RNA interference, leading to reduced gene expression.

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Study Notes

Lecture 15: CRISPR-Cas9 and RNAi

  • CRISPR-Cas9 is a gene-editing tool used for functional studies
  • RNA interference (RNAi) is a tool for silencing genes
  • Methods are used to study genes, proteins, and cells
  • Gene editing tools can be used to correct mutations in patients

Gene Editing Tools

  • TALENs (Transcription Activator-Like Effector Nucleases): a gene editing tool; recognize each base; nuclease domains introduce nicks; specific but not commonly used now
  • Zinc Finger Nucleases (ZFN): gene editing tool; Zinc Finger domains recognize 3 bases each; nuclease domain introduces nicks, design both sides to make nicks; not commonly used now
  • CRISPR-Cas9: a gene editing tool; clustered regularly interspaced short palindromic repeats
  • CRISPR-Cas9 system consists of sgRNA and Cas9 nuclease

CRISPR Methods

  • CRISPR-mediated "adaptive immunity" in bacteria: a bacterial defense system against invading phages (viruses)
  • CRISPR-mediated genome editing: allows for precise editing of DNA sequences
  • CRISPR is used in experimental models of human diseases, gene function studies on cells, and development of infection-resistant crops
  • History: includes site-directed DSBs, CRISPR-mediated genome editing (comparison with other tools), other CRISPR applications, and RNA interference

RNA Interference (RNAi)

  • RNAi is a technique that silences genes by interfering with mRNA transcripts
  • RNAi uses siRNA (small interfering RNAs) of 21-23 nucleotides in length
  • Uses a protein complex called RNA-induced silencing complex (RISC) to degrade mRNA, Blocking translation
  • RNAi experiments include introducing synthesized siRNA or short hairpin RNA (shRNA) carrying vectors into cultured cells or use microinjection to mouse fertilized eggs

Specific topics

  • Methods for introducing molecules (plasmids, sgRNA) into cells
  • CRISPR experiments in cultured cells: sgRNA and Cas9-containing plasmids can be transfected together to cultured cells.
  • Methods for genome editing
  • Animal Models of Human Diseases
  • Study gene functions in cells
  • Infection-resistant crops
  • Algae to produce fat for biofuels
  • Correct mutations in patients
  • Bacterial adaptive immunity that utilizes CRISPR/Cas9
  • CRISPR/Cas9 is a precise gene-editing tool
  • Methods of incorporating new spacers into the CRISPR array when a new phage enters
  • Comparing different gene-editing techniques (e.g., CRISPR versus TALENs versus RNAi)

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