Lecture 5 – genome editing

Question 1

Zinc Finger Nucleases (ZFN):

Answer 1

Zinc Finger Motif: Found in some proteins for DNA binding.
Structure: Composed of a loop of amino acids held by a zinc atom.
Function: Normally transcription factors, activating transcription.
ZFN Modification: Linked to FOK1 nuclease for DNA cleavage.
Double-Strand Break: Induces a repair-prone break, leading to mutations.
Repair Mechanism: Non-homologous end joining with high efficiency.
Commercial Design: Companies design ZFNs against target genes for mutation.

Question 2

TALENs (Transcription Activator-Like Effector Nucleases):

Answer 2

Structure: Fusion of FOK1 nuclease with customizable DNA-binding domain.
TALE Motif: 33-35 bp repeat domains, injected by parasitic proteobacteria.
DNA Binding: Repeats stack up to bind single base pairs, determined by hypervariable amino acids.
Mutation Induction: Double-strand break, followed by non-homologous repair.
Adaptability: Used for stable DNA mutations in various organisms and cell types.
Design and Production: Customizable for specific gene mutations.

Question 3

Cas9/CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats):

Answer 3

Cas9 Origin: Derived from Streptococcus bacteria for acquired immunity.
CRISPR Origin: Bacterial immune memory by inserting viral DNA into the genome.
Cas9 Function: Double-stranded DNA cutter, cleaves on both strands.
Guide RNAs: CRISPR-RNA (crRNA) and transactivating CRISPR-RNA (tracrRNA) target Cas9.
Targeting: Guides Cas9 to foreign DNA sequences, inducing cleavage.
PAM (Protospacer Adjacent Motif): Required adjacent to the target site for Cas9.
Versatility: Can cut any genomic DNA adjacent to a PAM sequence.

Question 4

CRISPR Basics:

Answer 4

crRNA (Clustered Regularly Interspaced Short Palindromic Repeats): Complementary to target gene, guides nuclease.
Cas9/CRISPR Complex: Binds to target sequence, induces double-stranded DNA break.
Repair Mechanism: Non-homologous end joining (NHEJ) induces mutations.
Repair Outcome: Ragged endings filled by DNA repair enzymes, causing mutations.
Application: RNA guides Cas9 to specific genes for mutation.

Question 5

CRISPR/Cas9 in Eukaryotic Cells:

Answer 5

Introduction: Cas9, crRNA, and tracrRNA introduced into cells for DNA cleavage.
Repair Process: DNA repair enzymes repair damage, inducing mutations.
Mutation Types: Random base insertions, small deletions/insertions at cut points.
NHEJ (Non-Homologous End Joining): Repair mechanism causing mutations.

Question 6

sgRNA (Single Guide RNA):

Answer 6

Formation: tracrRNA and crRNA encoded next to each other, creating a hairpin structure.
Efficiency: Acts as a single guide RNA (sgRNA) for more efficient targeting.
Mutations in ES Cells: Cas9 and sgRNA introduced through electroporation, efficient mutation in Tet genes.

Question 7

Controlled Mutations - HDR (Homology-Directed Repair):

Answer 7

Process: Electroporation/injection with Cas9, sgRNA, and single-stranded DNA oligonucleotide.
Template for Repair: Oligonucleotide used as a template for DNA repair enzymes.
Outcome: Designed mutation incorporated into the genome at Cas/CRISPR DNA cleavage site.
Wang et al. (2013): Oligonucleotides encoding point mutations introduced alongside Cas9 and sgRNAs.

Question 8

Specific Mutations and Reporter Tags:

Answer 8

Yang et al. (2013): CRISPR/Cas system used to eliminate STOP codon, add reporter tags (e.g., Red fluorescent protein).
Application: Introduce LoxP sites into DNA using CRISPR/Cas system.
Gene Example: Mecp2 gene targeted in Rett syndrome using Cas9, sgRNA, and DNA oligonucleotides.