Module 02 - Section 06 Flashcards
What is Site-Specific Recombination?
Precise and predicable process in which DNA is rearranged between 2 specific sequences.
Involves the movement of specialized nucleotide sequences called mobile genetic elements between non-homologous sites
What are mobile genetic elements
DNA sequences that can move around the genome changing either their number of copies or location. They include transposable elements, plasmids, and bacteriophage elements
What are the effects of Site-Specific recombination?
- Can alter gene order, this process is used to regulate gene expression and to increase the organisms’s genetic repertoire
- Can give rise to spontaneous mutations in organisms
What are some biotechnological applications of Site-Specific Recombination?
Activate a particular gene, insert a new gene into a cell at a chosen location, and replace one gene with another gene or an altered version of the same gene, delete a gene or alter the linear structure of an entire chromosomes
- Know-out mice and knock-in mice (deletion or adding of a gene to study its effect)
What are the two systems that use site-specific recombination for genome editing?
Cre-Lox
Flp-FRT
Cre-LoxtP and Flp-FRT systems; what are LoxP and FRT?
Specific directional sequences that are placed in the genome – but are NOT naturally occurring in the genome
Cre-LoxtP and Flp-FRT systems; what are Cre and Flp?
Recombinase enzymes that recognize the LoxP and FRT sites – also NOT naturally occurring in eukaryotes
What is the importance of the orientation of the LoxP or FRT sites?
(1) Inverted (facing towards each other); recombinase will invert the intervening sequence, changing its orientation on the DNA
(2) If sites are oriented in the same direction; recombinase can cleave out the intervening sequence, leaving behind one perfectly reformed LoxP or FRT site
Name an application of the Cre-LoxP system?
Brainbow technology: visualization of using fluorescent proteins to mal all then neurons in a mouse brain
Describe the method of the brainbow technique
(1) Gene cassette with several copies of GFP variants that encode proteins fluorescing with different colors
(2) Variants of the LoxP site for the Cre recombinase are engineered between the genes - different lox sites differ in their core sequences so lox1 only react with lox1, lox2 with lox2 ,etc..
(3) Cassette utilizes 3 or more different lox sites so that Cre-mediated recombination results in different patterns of GFP varian expression and thus a different colour in each neuron
(4) Cassette also includes a promoter that directs gene expression only in neurons
What is the outcome of Brainbow?
A mouse with GFP cassettes mates with a mouse expressing Cre recombinase, their progeny are heterozygous. Recombination events, occur, 1 type per cel= particular colour. Result= rainbow like array of fluorescent colours in the neural network to trace paths of axons
How are the different lox sites prevented from recombining with each other?
Core sequence must be modified to prevent recombination between the different loxP sites
What is one of the main methods molecular biologists use to understand gene function?
Delete it – then examine effects of an absence of a protein on the cell function and infer the gene product’s function
What are the 3 technologies that have been developed to cut genes at particular sites in vivo?
(1) Zinc Finger nucleases
(2) TALENs
(3) CRISPR/Cas systems
What is a zinc finger?
protein domain characterized by a single atom of zinc coordinated to four Cys residues or 2 His and 2 Cyz residues. It’s about 30 amino acids and folds in a beta sheet-alpha helix-beta-sheet structure aided by a Zn2+ ion
What gives Zinc Finger nucleases selectivity?
alpha helix structure of the zinc finger are able to contact 3 consecutive nucleotides within the major groove of DNA
What are some applications of ZFN
They can stitch together in tandem which creates structure that allow the specific recognition of almost any DNA sequence 9 to 18 BPs long
Describe the mechanism of action of ZFNs
When Zinc fingers are fused to a nonspecific nuclease domain, the DNA sequence is cleaved at a site adjacent to the recognition sequence bound to ZFNs
What direct the binding of TALENs?
Transcription activator-like effector (TALE)
Which technology(ies) recognize single base pairs?
TALENs
Which technology(ies) can be linked together to recognize non specific nuclease domain?
ZFNs & TALENs
What technology(ies) can be designed to inactivate genes and even to inactivate viral DNA that is integrated into a genome
TALENs
What technology(ies) can fuse to a nonspecific nuclease domain to yield a nuclease?
ZFNs & TALENs
What technology(ies) require modularity to recognize different DNA Sequence (needa different designer enzyme for every DNA site you want to recognize)
ZFNs & TALENs
What is the major limitation of ZFNs and TALENs?
They require modularity (different designed enzy for every DNA site that needs recognition) which can take weeks or months and require a lot of protein engineering
Which technology(ies) has been derived from a kind of bacterial immune system?
CRISPR/Cas
Describe the mechanism of CRISPR/Cas
(1) Viral DNA is incorporated into the CRISPR regions
(2) CRISPR region + Viral DNA sequences are then expressed in the form of short RNA (Guide RNAs)
(3) Guide RNAs + Cas 9 nuclease can recognize sequence and cleave it out
What are the roles of the 2 domains of Cas9 proteins
One domain cleaves the DNA strand paires with the sgRNA and the other cleaves the opposite DNA strand
What manipulation can be done to the 2 different domains to achieve different outcomes
(1) Inactivating 1 domain, creates an enzyme that cleaves just 1 strand – Homology- directed repair which makes sure the template is precisely integrated
(2) both are activated which create a DSB repaired by non-homologous end joing
How are components of CRISPR/Cas9 introduced into cells?
electroporation
