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?

They both introduce nicks in DNA to create double-strand breaks. Signup and view all the answers

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

Cas1, Cas2, and Csn2 proteins Signup and view all the answers

What type of crops can benefit from CRISPR technology?

Infection-resistant crops Signup and view all the answers

Which of the following statements regarding TALENs is accurate?

TALENs recognize each base and are very specific. Signup and view all the answers

CRISPR technology was established primarily for what purpose?

Providing adaptive immunity in bacteria Signup and view all the answers

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

Cas9 nuclease and single guide RNA (sgRNA) Signup and view all the answers

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

To aid in the binding of Cas9 to the target DNA sequence Signup and view all the answers

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

Sonoporation Signup and view all the answers

What is the role of dCas9 in CRISPR applications?

To act as a DNA-binding protein without nuclease activity Signup and view all the answers

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

Degrades the mRNA transcript Signup and view all the answers

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

A duplex 21 bp long with 3' overhangs Signup and view all the answers

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

To control gene expression by activation or repression Signup and view all the answers

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

Cas9-sgRNA complex formation occurs after transcription Signup and view all the answers

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

Pre-crRNA and tracrRNA Signup and view all the answers

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

RNase III Signup and view all the answers

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

It directs Cas9 endonuclease to cleave foreign DNA. Signup and view all the answers

How does a new spacer integrate into the CRISPR array?

Through the acquisition machinery including Cas proteins Signup and view all the answers

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

Proto-Spacer Adjacent Motif Signup and view all the answers

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

Mature crRNA Signup and view all the answers

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

It is reduced to a length of 20 nucleotides. Signup and view all the answers

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

To cleave foreign DNA Signup and view all the answers

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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Description

This quiz covers key concepts around CRISPR-Cas9 and RNA interference (RNAi) as gene-editing tools. It explores their mechanisms, applications in gene correction, and comparison with other tools like TALENs and ZFN. Test your knowledge on these revolutionary technologies shaping genetic research.