12.2 Flashcards
what is CRISPR-CAS?
bacterial defence against foreign DNA that is used as a genetic engineering tool
what does CRISPR-CAS target?
specific DNA molecules, comparable to adaptive immune system
what are the three steps of CRISPR immunity?
spacer acquisition
expression of crRNAs
Interference
what are the three components of interference?
Cas9 enzyme
crRNA
tracr RNA
what is PAM?
protospacer adjacent motif
not found in crispr dna array
simple and common elsewhere
what does sgRNA do?
(s)gRNA designed to target a specific sequence in genome
how does genome editing with CRISPR-Cas work?
sgRNA assembles with Cas9 to form effector complex
effector complex unwinds PAM then Cas9 undwinds DNA
Cas9 makes ds cut in genome
what are the possibilities of cellular DNA repairing mechanism?
broken ends can be rejoined without any template (non-homologous end joining)
broken ends rejoined using template (homology directed repair)
what happens during non-homologous end-joining?
most common
no template
nucleotides are randomly inserted or deleted as cleaved ends of chromosome are rejoined
often results in INDELS
if no INDEL mutation, Cas9 keeps cutting until mutation occurs
frameshift lead to non-functional alleles
what happens during homology directed repair?
another way to repair enzymes
can use homologous chromosomes as template
can inject donor DNA at same time
what are the advantages of CRISPR-Cas9?
cheap and easy
can target almost any sequence
relatively specific
indels created by non-homologous end-joining can create gene knowouts
can be introduced to intact living cells
can introduce Cas9 with donor DNA to stimulate HDR
what are the challenges of CRISPR-Cas9?
off-target effects (cleavage isnt always specific)
modified Cas9 structure uses longer target sequence but is slower
can be hard to control which is used
mosaicism
what is Cas9 induced mosaicism?
not all cells are edited
what are potential uses of CRISPR?
gene knockouts
editing genomes
gene drives
