Week 6 Tuesday Flashcards
Why is expressing genes and pathways on the genome vs the plasmid?
no need for selective media, less concerns about the strain losing the genes, better growth of strains
How is genome editing repair activated?
DNA cleavage
How do we do the double-stranded break?
A nuclease e.g. restriction enzymes
What is the disadvantage of using restriction enzymes?
sites are too common in the genome (hard to use the restriction enzymes in a really large genome because it will cut hundreds of time)
CRISPR best for genome editing
What is a mega nuclease?
combination of 2 proteins
How do zinc fingers work?
Zinc fingers are proteins that recognize 3 nucleotide sequences. If you have a right zinc finger and left zinc finger, it will bind the DNA on both sides. The enzyme with them come together and form a dimer.
What are the disadvantages of zinc fingers?
It’s not robust if you need to reprogram it. If you want to change the sequence that it is recognizing, you have to do complete protein engineering.
What are TALENs?
Transcription activator like effectors consist of 2-aa domains recognizing single nucleotides (gives more leeway to make something that will bind to any DNA sequence)
How is CRISPR different from previous methods?
- Before you have protein-DNA interactions for CRISPR it is RNA and DNA interacting. Aka CRISPR RNA.
- Nature enzymes (where before they were a synthetic combination of natural enzymes; taking 2 enzymes and linking them together)
CRISPR systems naturally exists and we are repurposing them.
How does Cas9 work in CRISPR?
The CAS9 protein will recognize a really short sequence, aka the PAM, and bind to everyone. It then unzips the sequence, and it will take the CRISPR RNA and see if it base pairs with whatever sequence is next to the pam. If not, it will rezip and move on to the next PAM.
Explain how CRISPR works briefly.
Deliver a plasmid that has CAS9 and crRNA, it will bind, CAS9 will do the double-stranded break. Give it a homology template to fix the DNA. When it does that, it forgets that it has the big chunk, only fixes the upstream and downstream. Gets rid of middle part.
