VL 10: Zebrafish II: Genetic manipulation, Morphogenesis & Organogenesis Flashcards
“Daily” practice in a zebrafish lab: microinjection
- 1-cell-stage embryo
- injection under microscope into the embryocell (not yolk?)
- Needed: pressure controller, micro needle, needle holder
- Microinjection allows, e.g., the production of transgenic cell lines or animals. It also offers the direct administration of supporting or harmful substances into cells to investigate their mode of action or toxic potential
Central Dogma
DNA (Replication) → RNA (Transcription) → Protein (Translation)
- Regulatory element: enhancers, promoters, 5’ UTR, 3’ UTR
- Coding sequence: is the portion of the gene that directly codes for the amino acid sequence of a protein.
UTR- Untranslated regio, located at the end of the mRNA, influencing mRNA stability….
Principle of mutagenesis in living organisms
Genome editing technologies efficiently produce site-specific DSBs that are usually repaired by non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ) and homologous recombination (HR).
Targeted genomic modifications using genome editing technologies.
* DNA double-strand breaks (DSBs) induced by genome editing technologies are repaired by non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ) and homologous recombination (HR).
* NHEJ repair, which connects the ends of the broken strands, leads to unpredictable insertion and/or deletion mutations (green bar),
* while MMEJ repair uses microhomology sequences (yellow box) and often causes a predictable small deletion.
* HR repair requires long double-strand DNA fragments (blue bar) that possess homology to the targeted genomic locus.
Site-specific integrations of donor DNA are mediated by these DNA repair mechanisms.
Forward vs. Reverse genetics
Forward and reverse genetics has enabled the systematic linkage of genotype to phenotype with a potential to mutagenize all the genes of the zebrafish genome.
Forward Genetics:
* Begins with a phenotype and seeks to identify the underlying genetic factors.
* Mutagenisis: Involves inducing random mutations across the entire genome.
* Used for gene discovery and identifying novel pathways
* Classical mutagenesis, chemical mutagenesis, insertional mutagenesis.
Reverse Genetics:
* Begins with a known gene or DNA sequence and investigates its phenotypic effects.
* Mutagenisis: Involves introducing targeted mutations to specific genes of interest.
* Used for confirming gene function, studying specific gene roles, and understanding molecular mechanisms.
* CRISPR-Cas9 gene editing, ZFNs, TALEN
Zinc-finger nucleases (ZFNs) mediated knock-out
Zinc-finger nucleases (ZFNs) is used to introduce targeted mutations in the zebrafish germ line.
* To engineer the ZFNs, the researchers designed them to recognize specific sequences in the zebrafish ortholog of the (kdr)
* These ZFNs were then injected as mRNA into one-cell-stage zebrafish embryos.
* This resulted in the introduction of mutagenic lesions at the target site, which were transmitted through the germ line with high frequency.
TALENs
targeted gene editing
& TAL effectors are introduced by microinjection into embryos.
- DNA recognition occurs via two repeat-variable di-residues (RVD)
- TAL effectors can be designed to target any locus in the genome
- Fused to the Fok1 nuclease, DNA double-strand breaks can be generated at your specific genomic locus. Using a pair of TAL proteins for targeting reduces off-target effects.
- The breaks induced by the Fok1 nuclease domain are subsequently repaired by one of two endogenous cellular mechanisms: non-homologous end joining (NHEJ) or homologous recombination (HDR).
*NHEJ is error-prone and often results in a frameshift mutation when it occurs within the coding sequence of a protein-coding gene, resulting in gene silencing.
In HDR, homologous DNA “donor sequences” can be used to introduce a specific new DNA sequence.
-> a protein fused to a Fok1 endonuclease can be used to knock out a gene or to insert an engineered DNA fragment at a precise location in the genome.
CRISPR/Cas9 mediated knock-out
- Design of Guide RNA (gRNA):
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Cas9 Nuclease Introduction:
The Cas9 enzyme is introduced into zebrafish embryos, either as purified protein or as mRNA encoding Cas9. Additionally, the designed gRNA is delivered along with Cas9. - gRNA Binding and Recognition:
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DNA Repair Mechanism:
The cell’s natural DNA repair mechanisms, primarily non-homologous end joining (NHEJ), come into play to repair the DSBs.
*NHEJ repair can result in insertions or deletions (indels) at the target site, leading to frameshift mutations and disrupting the open reading frame of the gene.
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Analysis of Knock-Out Efficiency:
. Common techniques include PCR, DNA sequencing, and assays that detect mutations. -
Phenotypic Analysis:
The phenotypic consequences of the gene knock-out are studied by observing the morphological, developmental, or behavioral changes in the zebrafish embryos or adults.
(Establishment of Stable Lines:
If heritable mutations are desired, zebrafish with successful knock-out can be bred to establish stable lines with the disrupted gene.)
CRISPR/Cas9-mediated knock-out has revolutionized genetic research due to its simplicity, efficiency, and versatility. It allows researchers to study gene function, model diseases, and explore various biological processes by precisely manipulating the genomic DNA of organisms like zebrafish.
only one customized sgRNA is required to target a specific sequence <-> in TALEN and ZNFs the design and assembly of two necleases for each target site i needed
Outline of how to create germline zebrafish CRISPR-Cas9 mutants and CRISPants.
To create a germline zebrafish CRISPR-Cas9 mutant (follow path of solid lines), guide RNAs (gRNAs) targeting a gene of interest along with Cas9 mRNA or protein are injected into newly fertilized zebrafish embryos.
Mutant:
* G0 adult founders are crossed to wildtype adults, and the G1 progeny screened for mutations.
* G1 with mutations are then incrossed and screened for phenotypes.
CRISPant larvae:
* Injected CRISPant G0 larvae are then screened for phenotypes days after the injection.
* CRISPant phenotypes can be verified by generating a stable, germline mutant
Genetic compensation
Genetic compensation= organism compensates for the loss or reduction of gene function by upregulating the expression or activity of related genes.
- the analysis of zebrafish and mouse models reveals a requirement for mutant mRNA degradation in achieving transcriptional adaptation. Mutations that result in the failure to transcribe the mutated gene do not exhibit transcriptional adaptation.
- Alleles that fail to transcribe the mutated gene and, consequently, lack transcriptional adaptation give rise to more severe phenotypes compared to alleles that display mutant mRNA decay. This implies that transcriptional adaptation contributes to genetic robustness and less severe phenotypic consequences.
CRISPR/Cas9 mediated knock-in (exon targeted)
1) Create a guide RNA (gRNA) targeting the desired exon of the gene.
2) Design a donor DNA template with the desired sequence flanked by homology arms.
3) Introduce gRNA and Cas9 to induce a double-strand break (DSB) at the targeted exon.
4) Homologous Recombination
5) The desired sequence integrates into the genomic locus, replacing the targeted exon.
6) Select and screen for successfully modified cells using markers or specific assays.
CRISPR/Cas9 mediated knock-in (intron targeted)
Intron targeting is often chosen to study gene regulation, alternative splicing, or the impact of intronic elements on gene expression.
-> investigate the functional consequences of modifying non-coding regions within genes, contributing to a deeper understanding of gene regulation and expression.
Knockdown by Cas13d
using RNA-targeting CRISPR effector Cas13d to specifically degrade target RNA molecules
* design crRNAs specific to the target RNA sequence.
* Introduce Cas13d and crRNAs into target cells through transfection or other methods.
* Cas13d, guided by crRNAs, binds to target RNA through sequence complementarity.
* Cas13d induces cleavage and degradation of the target RNA, reducing gene expression.
* Cas13d offers high specificity, minimizing off-target effects & Cas13d-mediated knockdown is transient, allowing reversible modulation of gene expression.
Validate knockdown efficiency by assessing target gene expression reduction at the RNA or protein level.
Morphogenisis & Organogenisis def.
Morphogenesis:
* understanding shape changes in the embryo (examples: optic cup; neurovascular and cardiovascular system).
* early events during embryonic development
* A system attains its form (or shape) by integrating chemical and mechanical signals with the spatiotemporal information provided to it via patterning.
Organogenesis:
* understanding the mechanisms of organ formation, and the cellular and molecular events influencing the generation of different cell types which compose that organ
* is the next step, by which the embryo finally becomes a fully functioning organism. Organogenesis is a subset of morphogenesis, specifically focusing on the development of organs.
