Crispr Flashcards
What is the definition of CRISPR?
Clustered Regularly Interspered Palindromic Repeats
What is CRISPR?
-Technology that can be used to edit genes in plants and animal genomes, derived from a bacterial immune system
How does CRISPR work?
You get a spacer from dna from infection, incorporate it into CRISPR region in bacterial cell
When the virus tries to infect the bacteria cell, the crispr region gets transcribed into RNA, and the different pieces become guide RNAS, and each guide rna gets encoded with cas9 proteins, so when a new infection comes in the guide rna chops it off as it has it already so it recognizes it. Cas9 is enzyme that makes cut in invading virus
Is CRISPR part of genome, what are the implications of this?
Yes, it gets passed on as the genome replicates
Does cas9 make a double stranded or single stranded cut?
double stranded
How do we do CRISPR in animal cells?
Express the cas9 enzyme in our cell, we design guide rna to direct cas9 to make a double stranded cut
Where do we have to design guide rna?
Has to be 3 nucelotides upstream of the PAM sequence in the genome (NGG)
What is the PAM sequence?
protospacer adjacent motif
What are the two ways the double stranded cut gets repaired?
Homologous recombination - do this by introducing donor DNA, used as a template to repair the double stranded break (gets inserted in middle) Can be anything we want to introduce
Nonhomlogous end joining- messey repair mechnanism, introduces mutations and deletions
What do we need for Homologous recombinaton to work?
We need the sequence on the ends of the cuts to be the same as the ends of the donor dna
Genome editing is limited to ____
somatic cells
in humans, genome editing is currently not approved for ______
germline cells
Why is germline editing not approved for CRISPR but somatic cells are?
because germline cells are inheritable
