CRISPR Mechanisms and Applications Quiz

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Questions and Answers

What is the role of the egg cell-specific promoter (EC1.2p) in the expression cassette for Cas9?

It enhances the efficiency of generating homozygous mutants in transgenic plants. (correct)

It provides termination signals for the Cas9 gene.

It increases the expression of gRNAs only.

It functions to suppress Cas9 expression in non-egg cells.

What is necessary for designing the sgRNA primers for the CBF4 gene?

Using standard primers without any gRNA sequences.

Designing primers that include the two selected gRNAs. (correct)

Employing gRNA sequences from unrelated genes.

Selecting only one gRNA for amplification.

What is the function of BsaI sites in the context of cloning the sgRNA?

To enable Golden Gate assembly for cloning the sgRNA into the expression vector. (correct)

To terminate the transcription of the sgRNA.

To aid in the amplification of the CRISPR cassette.

To restrict the cloning of the sgRNA into any plasmid.

Which vector is used solely for assembling a second CRISPR expression cassette?

pCBC-DT1T2 Signup and view all the answers

What is the primary goal of selecting two gRNAs for the CBF4 gene?

To delete a large region of the CBF4 gene. Signup and view all the answers

What is the primary function of crRNA during the interference phase?

To guide Cas9 endonuclease for DNA cleavage Signup and view all the answers

Which two domains are crucial for the function of Cas9?

HNH-like nuclease domain and RuvC-like nuclease domain Signup and view all the answers

Which component of the CRISPR system is responsible for cleaving foreign DNA?

Cas9 Signup and view all the answers

What does PAM stand for in the context of Cas9 activity?

Protospacer-associated motif Signup and view all the answers

Which sequence is required immediately downstream of the crRNA complementary sequence for Cas9 to function?

NGG Signup and view all the answers

What is the function of the PAM sequence in CRISPR systems?

To provide a docking site for Cas9 Signup and view all the answers

Which variant of Cas9 only exhibits nickase activity?

Cas9 D10A Signup and view all the answers

How are fragments of invading DNA integrated into the bacterial genome during the acquisition phase?

They are cut into small fragments and incorporated Signup and view all the answers

What is the role of tracrRNA in the Cas9 complex?

To provide sequence complementarity to crRNA Signup and view all the answers

What does dCas9 refer to in CRISPR technology?

A nuclease-deficient variant of Cas9 Signup and view all the answers

What is the significance of the Nobel Prize in Chemistry 2020 concerning CRISPR?

It highlighted the method for genome editing through CRISPR technology. Signup and view all the answers

What happens to the double-stranded DNA during cleavage by Cas9?

It results in double-stranded breaks (DSBs) Signup and view all the answers

Which factor is critical in reducing off-target mutations during CRISPR editing?

Increased PAM specificity Signup and view all the answers

What is the consequence of not having a PAM sequence present during Cas9-mediated cleavage?

The foreign DNA is ignored by Cas9-RNA Signup and view all the answers

What role does sgRNA play in CRISPR systems?

It facilitates the binding of Cas9 to the target DNA. Signup and view all the answers

What is the primary goal of generating a cbf4 knock-out mutant in Arabidopsis using CRISPR-Cas9 technology?

To analyze the mutant phenotype Signup and view all the answers

What is a common application of CRISPR technology in molecular biology?

Genome editing in plants and animals Signup and view all the answers

In the process of designing sgRNA to target CBF4, what must be accomplished first?

Clone sgRNA in the CRISPR expression vector Signup and view all the answers

What technique is used to visualize the localization of the CBF4-YFP fusion protein?

Confocal microscopy Signup and view all the answers

Which step is NOT part of transforming plants with the cbf4 CRISPR mutants?

Clone CBF4 into a plant expression vector Signup and view all the answers

What is the purpose of using Gateway recombination technology in this study?

To determine protein localization and overexpression phenotype Signup and view all the answers

Which organism is involved in the transformation process for generating the cbf4 knock-out mutant?

Agrobacterium tumefaciens Signup and view all the answers

What does the AP2/ERF family of transcription factors primarily influence?

Gene expression related to stress responses Signup and view all the answers

What does the successful transformation of the CBF4 gene into plants contribute to?

Insight into gene function and expression Signup and view all the answers

What is the sequence that will be used as the target for sgRNA?

The 20 nucleotides upstream of the PAM sequence Signup and view all the answers

What role does the PAM sequence play in the cleavage process?

It is required for the Cas9 cleavage mechanism Signup and view all the answers

Where does the Cas9 nuclease cleave in relation to the PAM sequence?

Approximately 3 bases upstream of the PAM Signup and view all the answers

Which web-based tool is mentioned for designing sgRNAs?

CHOPCHOP Signup and view all the answers

What is the function of the U6 promoter in the expression cassette for sgRNA?

To direct high expression of sgRNA in the nucleus Signup and view all the answers

What is the primary goal of web-based tools for designing sgRNAs?

To identify potential CRISPR target sites and assess their specificity Signup and view all the answers

What is one characteristic of the Cas9-sgRNA complex?

It triggers a double strand break when near a PAM Signup and view all the answers

In which type of organism will the U6 promoter be used for expressing sgRNA?

Plants Signup and view all the answers

What is the primary benefit of using a synthetic single guide RNA (sgRNA) with Cas9?

It simplifies the system to two components for genome editing. Signup and view all the answers

What is the result of the wild-type Cas9 cleaving double-stranded DNA?

It activates the double strand break repair machinery. Signup and view all the answers

What distinguishes the Cas9 D10A variant from the wild-type Cas9?

It has only nickase activity and does not activate NHEJ. Signup and view all the answers

Which statement about the dCas9 variant is correct?

It can bind DNA but cannot cleave it. Signup and view all the answers

What happens when a donor template is supplied during the repair of a DSB?

Precise replacement mutations may be achieved through HDR. Signup and view all the answers

What is a potential application of dCas9 fused to GFP?

To visualize repetitive DNA sequences. Signup and view all the answers

Why is reducing indel mutations significant when using Cas9 D10A?

It enhances the reliability of gene replacements. Signup and view all the answers

What mechanism is engaged by wild-type Cas9 during DNA repair?

NHEJ pathway for random insertions. Signup and view all the answers

Study Notes

Genome Editing by CRISPR-Cas

Staphylococcus aureus Cas9 complex (SaCas9) crystal structure is shown
3D printed model of the Cas9 complex is also displayed
Source: Innovative Genomics Institute

Arabidopsis CBF4 Gene Function Study

Arabidopsis CBF4 gene encodes a transcription factor in the AP2/ERF family
Designing sgRNA to target CBF4 in Arabidopsis is a part of the project
Cloning sgRNA in CRISPR expression vector using Golden Gate cloning is required
Transforming E. coli, isolating the plasmid and transforming Agrobacterium tumefaciens is part of the process
Transforming Arabidopsis plants for stable expression
Genotyping and phenotyping of cbf4-crispr mutants are needed (weeks 8-9)

CBF4-YFP Fusion Protein Generation

Generating CBF4-YFP fusion protein by Gateway recombination cloning technology
Transforming LR reaction into E. coli, colony PCR, plasmid isolation, and sequencing (Week 10) is required
Transforming plasmid into Agrobacterium and infiltrating plant leaves (N. bentamiana) for transient expression
Localizing protein through confocal microscopy (Week 11) is part of the process

CRISPR-Cas System Details

CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA or trRNA) are components
CRISPR-Cas9 used as in molecular biology tools
Cas9 D10A variant has only nickase activity
dCas9 variant lacks nuclease activity
Applications include genome editing and genome targeting

Nobel Prize in Chemistry 2020

Emmanuelle Charpentier and Jennifer Doudna jointly awarded for the development of a method for genome editing

CRISPR/Cas Function

CRISPR (Cluster Regularly Interspaced Short Palindromic Repeats) and Cas genes essential for adaptive immunity in bacteria and archaea
Enable these organisms to respond to and eliminate invading genetic material
Foreign DNA incorporated into bacterial genome at CRISPR loci
CRISPR loci transcribed into crRNA during crRNA biogenesis
Cas9 endonuclease complexed with crRNA and tracrRNA cleaves foreign DNA with complementary sequence adjacent to PAM sequence

Type II CRISPR Mechanism

One Cas protein (Cas9) required for gene silencing
Three CRISPR mechanisms identified, type II most studied

Acquisition Phase

Invading DNA, from viruses or plasmids, cut into small fragments, incorporated into CRISPR locus amidst repeats (around 20 bps) in bacteria genome

Interference Phase

CRISPR loci transcribed and processed to generate small RNAs (e.g., CRISPR RNA or crRNA)
crRNA guide Cas9 endonuclease, which cleaves invading DNA by sequence complementarity
Cas9 must be complexed with crRNA and tracrRNA (or trRNA) to recognize and cleave DNA. tracrRNA partially complementary to crRNA
Cas9 function depends on nuclease domains (RuvC-like and HNH-like) and PAM-interacting domain

Protospacer Associated Motif (PAM)

Cas9 requires a short conserved sequence (NGG) to function as a protospacer-associated motif (PAM)
PAM follows the crRNA sequence immediately
Cas9-RNA ignores fully complementary sequences without the PAM sequence
HNH and RuvC-like nuclease domains cut both DNA strands during cleavage, generating double-stranded breaks (DSBs) at defined sites in the target

CRISPR-Cas9 as Molecular Biology Tools

The simplicity of the type II CRISPR nuclease, with only three components (Cas9, crRNA, and tracrRNA), allows for adaptation to genome editing
Doudna and Charpentier labs simplified system, combining trRNA and crRNA into synthetic single guide RNA (sgRNA)
sgRNA-programmed Cas9 effective in guiding targeted gene alterations
Three Cas9 variants are used in genome editing protocols

Wild-Type Cas9

Cleaves double-stranded DNA (dsDNA) site-specifically
Cellular Non-Homologous End Joining (NHEJ) pathway repairs DSBs, leading to insertions and/or deletions (indels) impacting target gene
Homology-directed repair (HDR) pathway allows precise replacement mutations if a donor DNA template is provided

Cas9 D10A Variant

Mutant form (Cas9 D10A) has only nickase activity
Cleaves only one DNA strand and does not activate Non-homologous end joining (NHEJ)
High-fidelity homology-directed repair (HDR) pathway leads to reduced indel mutations

dCas9 Variant

Nuclease-deficient (dCas9) variant lacking HNH and RuvC domains cannot cleave DNA; however, prevents DNA binding
Used for gene silencing or activation, as well as visualization tools by fusing with effector domains (e.g., GFP)

Targeting Efficiency and Off-Target Mutations

Cas9 system high efficiency (up to >70%) in zebrafish and plants
Off-target mutations occur in sites that have slight nucleotide differences compared to original sequence, often adjacent to PAM sequence
Difficult to detect off-target mutations; whole-genome sequencing required

Applications of CRISPR/Cas9

Target important genes in various organisms (human, bacteria, zebrafish, etc.)
Introducing single-point mutations, deletions, or insertions using single or multiple gRNAs
Enabling rapid genome-wide interrogation of gene function through gRNA libraries

Week 6-9 Labs: Generating a cbf4-CRISPR Vector

Designing single guide RNAs (gRNAs) targeting CBF4
Cloning sgRNA in a CRISPR vector suitable for plant expression, containing Cas9
Transforming bacteria with the vector, isolating plasmid, and transforming Arabidopsis plants
Confirming deletion in CBF4 gene in cbf4-crispr mutants

Bioinformatics Lab 4

Designing sgRNA using Chopchop
Designing primers containing sgRNA and restriction sites

sgRNA Design Principles

CRISPR/Cas9 targeting requires a custom sgRNA with a targeting sequence (crRNA) and Cas9 nuclease-recruiting sequence (tracrRNA)
Targeting sequence (crRNA) is a 20-nucleotide sequence homologous to the region of interest
Targeting sequence directs Cas9 nuclease activity

Guidelines for sgRNA design

Identify PAM (NGG) sequence in target DNA region
Determine 5' start of the 20 nucleotide sgRNA targeting sequence based on PAM (NGG)
Determine the actual sgRNA targeting sequence
Identify the actual sgRNA targeting sequence that does not include the PAM sequence

Cloning gRNA: CRISPR Vector & Expression Cassettes

Cas9-sgRNA complex binds to DNA target and triggers double-strand break (DSB) when next to short PAM (NGG)
U6 promoter (U6-prom) of RNA Pol III directs high sgRNA expression in the nucleus; repeated components (gRNA scaffold, and terminators) present
Cas9 plant codon-optimized expressed using egg cell-specific promoter (EC1.2p) and terminated by rbcS-E9 terminator

Cloning 2 gRNA in plant expression vectors

Selecting two gRNAs to create cuts in the target CBF4 gene
Designing sgRNA primers for gRNAs (e.g., CBF4-sg11_For and CBF4-sg1_Rev)
Amplifying a CRISPR cassette that includes the two gRNAs from pCBC-DT1T2 vector
Cloning PCR product into a plant expression vector such as pHEE401E
pCBC-DT1T2 plasmid serves as a template

Primer Design

Primer CBF4-sg11_For and CBF4-sg1_Rev for cbf4 gRNAs
Include overhang with Bsal sites (Bsal restriction sites) in the primers
sgRNA sequences exclude the PAM sequence

Final CRISPR Plant Expression Vector

PCR product cloned into a CRISPR-plant expression vector
gRNA expression controlled by U6 RNA Pol III promoters and terminators
Cas9 expression driven by egg cell-specific promoter and terminated
Final cbf4-CRISPR vector resistant to Kanamycin but not Spectinomycin.

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Description

Test your knowledge on CRISPR technology, focusing on the roles of egg cell-specific promoters, sgRNA design, and Cas9 functionality. This quiz covers essential concepts such as PAM sequences, crRNA functions, and cloning techniques, making it perfect for students of molecular biology.