34 Gene Editing Flashcards
(18 cards)
What is genomics?
Branch of biology dealing with aspects of the genome (entire collection of genetic info in an organism)
Mainly descriptive
What is GWAS?
Genome-wide association studies
- can be used to identify disease-associated genes
What is predictive modelling?
Predict antibiotic resistance, susceptibility to deisease, rxns to drugs (pharmacogenomics)
What are zinc finger domains?
Ubiquitous small protein motifs which bind to a specific DNA triplet sequence (eg 5’GAA’3)
- typically part of transcription factors
- Each zinc finger domain binds to 3 bases of DNA
- can be linked linearly to target larger sequences
What is a Zing finger nuclease? (ZFN)
- Two zinc finger arrays linked to subunits of the heterodimeric FokI endonuclease
- enzyme is split into 2 different pieces; they must come together to cut DNA
- Individual FokI units are inactive but when heterodimer is formed they cleave DNA
- The ZFNs bind to the left and right sequences surrounding the spacer where the cut occurs
What are TALEs?
Transcription Activator Like Effector
- Produced by plant pathogen
- Each TALE domain binds 1bp of DNA
- can be linked to form arrays that target longer sequences
- Within each repeat domain, 2 aa at positions 12 and 13 dictate which specific base is bound
What are TALE nucleases? (TALEN)
TALEN = two TALE arrays linked to subunits of the heterodimeric FokI endonuclease
- no protein engineering required since each base can be targeted by a particular domain and domains can be independently linked together to form arrays
What are two main mechanisms in which DNA double-strand breaks can be repaired?
- Non-homologous end joining (NHEJ)
- inactivation/knockout
- Homologous recombination (HR)
- knock-in or gene editing by supplying repair DNA
How does NHEJ repair DNA ds breaks?
Non-homologous end-joining
- induces insertions or deletions that inactivate a gene
How does Homologous recombination repair DNA ds breaks?
HDR repairs the DNA break using information contained in a repair template
What are the four steps of CRISPR/Cas9
- Invasion
- Adaptation
- Production
- Targeting
What are the three components of the CRISPR/Cas9 mechanism?
- Cas9 (endonuclease)
- crRNA - contains a 20bp sequence used to target
- Trans-activating crRNA (tracrRNA)
Cas9 displays inherent specificity for a ______ sequence
Cas9 displays inherent specificity for a PAM sequence
PAM = Protospacer Adjacent Motif
What is the sequence of events for the mechanism of CRISPR/Cas9 DNA cleavage?
- Cas9 binds PAM (5’NGG-3’) N=A,G,C,T
- After PAM binding, cas9 tries to pair guide RNA with DNA
- Cas9 contains two nuclease domains and each of these cuts one strand of DNA = ds break repaired via NHEJ or HDR
How are gene editing tools delivered to cells? (3)
- Viruses
- Physical disruption of the membrane
- microinjection
- electroporation
- chemical coating/tethering methods
- Cas9-CPP/NLS
- Lipid nanoparticle (LNP)
- AuNP (gold nanoparticle)
What is the difference between Somatic cells and Germline cells?
- Somatic cell:
- Any cell in a living organisms other than reproductive cell
- could involve many tissues
- Germline cell:
- involved in the reproductive process that are passed on to progeny (eggs, sperm)
What is the difference between Ex-vivo therapy and In vivo therapy?
- Ex-vivo
- performed outside of the body
- useful for blood diseases
- performed outside of the body
- In vivo
- directly treats
- involve injection of gene editing agents directly into the body
Somatic therapy vs Germ Line therapy?
- Somatic Therapy:
- Gene inserted into body cells
- Modification restricted to individual patient
- Considered safe and ethical
- Germ Line Therapy:
- Gene inserted into germ line cells (become gametes)
- Modification will be inherited by offspring
- Not considered safe or ethically sound
