Topic 14 CRISPR Flashcards
What does crisper stand for
Where is it discovered
What is it used for
Clustered regularly interspersed palindromic repeats
Found in bacteria, was a defence system for them do develop an immune system
Edits genes in plants and animals
What are steps 1 and 2 in crispr in bacteria
Step 1: Cas1 and Cas2 complex cut out the spacer dna from the viral infection
Step 2: the spacer is incorporated into the the separate genomic crispr region
What are steps 3 and 4 in crispr in bacteria
- The crispr region with the spacers gets transcribed and cleaved to give guide rnas (gRNA)
- The gRNA connects with the cas9 protien to guide it to the corresponding infection for cleavage
This immune system gets passed on to generations
How is crispr done in non bacteria
Get the cas9 enzyme and the gRNA into our cell of interest
The gRNA tells cas9 to cut (double stranded) and a specific location
This location has to be 3 nucleotides upstream of the PAM (5’ NGG), meaning the guide has to be designed to cut 3 upstream of Pam
N means any nucleotide
What are the two ways of repair of the double stranded cas9 break
Homologous recombination (HR)
Nonhomologous recombination (NHEJ)
What is Homologous recombination (HR)
We introduce a donor dna to the cell which is used as a template to repair the double stranded break
Need sequence similarly on either side of donor dna to recognize either side of the double stranded cut
With the donor dna we Can introduce mutations restore a wt sequence, inactivate a gene, etc.
What is nonhomologous end joining (NHEJ)
We don’t add in donor dna and the dna fixes itself by adding mutations or deletions
Messy way
What are the ethical considerations of crispr
Gene editing is limited to somatic cells (tissue types), fixes blood disorders, retinal disorders
But not approved for Germline cells because those changes are heritable
