Genome editing Flashcards
What are the 2 double strand break repair mechanisms?
Non-homologous end joining: not perfect, tends to generate indels
Homology directed repair: if we provide the cell with some homologous sequences around the break, the cell will repair the break to look like the provided homologous sequences
What is the problem with ends-out and ends-in recombination?
They happen at low efficiency and random integration is still an issue
Why can’t we use restriction enzymes for genome editing?
They cut too many times in the genome
What are 3 methods of genome editing?
Zinc Finger Nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), CRISPR-Cas9
What is a zinc finger domain?
Common DNA binding motif in zinc finger transcription factors
Do zinc finger domains cut DNA?
No, they act as homing beacons but something else is the scissors
What sequences will zinc fingers bind to?
Each one makes contact with 3 specific nucleotides in the major groove in DNA, and we can customize them to bind to whatever sequence we want. We can also link ZFs together to recognize a longer, more specific sequence
If ZFs are only the homing beacons, what is the scissors?
Fok1 nuclease domain. Comes from a restriction enzyme but is modular and can act on its own
How do we do ZFN genome editing?
Design as many zinc fingers as we need for specific targeting, then link them together. Then attach the Fok1 nuclease domain to the ZFs, and it will cut where the ZFs direct it to
What is a TALE domain?
Very specific domain from a bacterial plant pathogen that binds to a single nucleotide
What dictates which nucleotide a TALE binds to?
2 particular amino acid residues: the repeat variable di-residues (RVDs)
Which 2 amino acids causes a TALE to bind to a C?
HD
Which 2 amino acids causes a TALE to bind to a A?
NI
Which 2 amino acids causes a TALE to bind to a T?
NG
Which 2 amino acids causes a TALE to bind to a G?
NN
How do we use TALENs for genome editing?
We can link together specific TALEs to bind to a particular nucleotide sequence, then attach Fok1 nuclease to it to have the TALEs target a specific nucleotide sequence and the nuclease to cleave it
What is the CRISPR locus? Where is it?
A string of repeating sequences with spacers of foreign DNA in between the repeats. Found near the Cas genes and the tracrRNA
What happens with CRISPR when a bacterial cell survives a viral infection?
Cas proteins will fragment phage DNA near protospacer-adjacent motifs (PAM) into 30 bp fragments, then they get integrated into the 5’ end of the CRISPR locus as spacers once the PAM sequence is cleaved off
What happens when the same phage from before tries to infect a bacterial cell again, when the bacteria has a complementary spacer?
The entire CRISPR locus gets transcribed into pre-CRISPR RNA, and the tracrRNA anneals to the repeats. Endogenous RNase III cleaves the pre-CRISPR RNA/tracrRNA into small units with one spacer, one crRNA, and a piece of tracrRNA. Mature crRNA attach to Cas9, and the tracrRNA and the spacer homes in on the complementary piece of invading phage DNA and Cas9 cleaves it
