Genetic manipulation 5 Flashcards
List 3 types of genome editing
zinc finger nucleases
TALENS
cas9/CRISPR
what is the general action of all genome editing techniques?
direct DNA cleaving enzymes to specific sites in any genome where you want to make mutations
what is the zinc finger?
protein motif that binds 3bp of DNA
normal function of zinc finger
bind specific DNA sequences and activate transcription
what DNA nuclease is used?
fok1
briefly explain ZFN action
2 ZFN bring 2 fok1 cleavage domains together in gene target
double stranded break - repaired, error prone
random bases inserted, small deletions/insertions
what is the error prone repair known as?
non homologous end joining
Why are ZFN convenient?
commercial companies design them against target gene
work in vitro and in vivo
good specificity
work on any animal
What do TALENs comprise of?
non specific fok1 nuclease domain fused to a customisable DNA binding domain
what does TALENs stand for?
transcription activator like effector nucleases
what are TALEs?
highly conserved 33-35 bp repeat domains
what are TALEs encoded by?
xanthomonas spp proteobacteria
what happens to TALEs in the wild?
injected into host plant cells by the bacteria and bind to genomic DNA to alter transcription in host cells
What determines the identity of the single base of DNA TALE binds to?
2 hypervariable residues typically found at positions 12 and 13 of the repeat
what is cas9?
endonuclease from bacteria - acquired immunity against viruses
what bacteria is cas9 from?
streptococcus
CRISPR?
clustered regularly interspaced short palindromic repears
how is cas9 targeted to cleave DNA?
short RNAs - crRNA and tracr RNA
cas/CRISPS system can cut any piece of genomic DNA adjacent to what?
PAM (protospacer adjacent motif)
what are PAM’s?
2-5bp sequences that differ between bacterial species
what is the PAM for cas9?
GGG
the first CRISPR crRNA has a region complementary to what?
part of the target gene, upstream to PAM
what does the tracr RNA have a region complementary to?
rest of the crRNA
disadvantage of cas9/CRISPR
mutations are uncontrolled and variable
how to induce controlled specific mutations in gene of interest using cas9/CRISPR?
catalyse some homology directed repair - electroporate/inject with cas9, sgRNA and a single stranded DNA oligonucleotide homologous to area around breakpoint
