L4 - Crispr Technology Flashcards
Where was crispr first identified / how
Bacterial defence against bacteriophage dna upon reinfection
What are the 3 components
Cas9 nuclease
Crispr rna - has comp sequence/ protospacers and binds tracr rna
Tracr rna- binds crrna and is a hairpin
Both form the grna
What are the steps during first infection with Phage dna
Dna is cut by cas1 and 2 into small fragments called protospacers which are then stored in the crispr locus amongst identical repeat arrays (to form crrna)
What happens during reinfection
Crispr locus txn so the complementary protospacers will be transcribed and tracr/crrna with the spacers will bind cas9 and direct it to the comp sites of phage invading dna
Ds dna breaks occur preventing infection
What is the cas operon in crispr locus
Operon transcribing diff cas proteins eg cas1,2 and 9
What’s the diff between cas1/2 and 9
Cas1/2 are multi protein complexes whereas cas9 is a single protein but with multidomains
Explain what happens to form mature cr rna
Protospacers sequences amongst repeat arrays will be joined
Protospacers will be the point which the rna will be complementary to invading dna whereas the repeat array part will bind tracrrna hairpin
Which domains in cas9 are required to recognise and guide cas9 to the grna/dna duplex
Rec1 and 2
Which lobe is the hnh nuclease domain which cleaves comp dna on
The nuc lobe (not rec lobe)
There is another domain which cleaves non comp dna sequence for it to be a double strand break. What’s it called
RuvC
Which domain recognises and interacts with the Pam sequence downstream of cleavage site
PI domain
What is the Pam sequence and why important
It’s a small bp sequence about 3-4 bp adjacent to the cut site usually downstream
Required to be present on the invading dna for cas9 to cause cleavage
This is because this distinguishes bacterial self dna from invading dna as upon first infection the Pam sequence is removed from crispr locus after protospacers insertion by cas1/2
So Pam is not present in bacterial crispr locus and prevents cleavage of this by own cas9
Which sequence commonly used in research as a Pam site
NGG strep pyogenes
What have they done to grna for actual genome editing in research which makes it easier
Linked crrna with tracrrna by a linker loop to get a single grna
What are the 2 ways you can deliver cas9/grna into cells for research
Either through transfecrion with plasmids 1 with cas9 and one with the grna of interest which will then be translated
Or can make a grna/cas9 complex in test tube and deliver it into cell that way
What is crucial when designing grna
Has a protospacers sequence specific for locus of interest and that it is upstream of a Pam site (NGG)
Which 2 dna repair mechanisms are used for ko/ki genomic editing
Homology directed repair in s phase
Or nhej in interphase
Which one is used for knock outs and explain why
NHEJ
Very error prone as it randomly with ligate the dna strands as no template present. This causes insertions or deletion called indels which produce framshifts or ptc meaning no functional protein
After txn, what scans for ptc and will degrade transcripts causing ko
Non-sense mediated decay
How does HDR work for knock ins
Very precise mechanism as it uses sister chromatids after replication in s phase as a template usually
So we can in research make donor templates which have a desired mutation for example which you want to study and this will be inserted as long as the sites next to it are homologous
What needs to be removed from templates and why
Pam sites to prevent re-targeting of the region back
What kb needed to be homologous for this to work
More than 60bp
Prostate cancer patient resistance to hormone therapy is due to splicing variants in the androgen receptor which have lost exon4-8. How can crispr be used to form a cell line which only expresses variants and not full length for research (knock in)
Can design a grna complementary to exon 5 which and delivered to cell line
A hdr template encoding stop codon but has the rest homology to exon 5
Causes a truncated AR with only 4 exons to be made/expressed
How did they screen many different splice factors in AR-V only cancer cell lines using crispr knock out strategies
Used a library of splice factors and chose 3
Designed grna for these 3 and then transfected cancer cell lines with cas9 plasmid and then also later a specific grna
Cell imaging was used to detect whether AR-V were being expressed via anti-AR antibodies
This helped determine which splice factor was important for production of splice variants as the grna ko of this factor would prevent any AR-Vs in cell lines or reduce their expression
How did they validate that the splice factor ko actually reduced AR-Vs
Through western blot detect if ar-v had reduced compared to scrambled rna control
Also can use RTqpcr to see if ar-v mrna levels changed (reduced for 2 of the splice factors)
Also can validate using siRna technology which degrades the splice factors too
In vivo uses and in humans
What do you need to consider with cell therapy
Ethical guidelines
Mosaicism
Off target effects?
Delivery strategy and efficacy
How could you deliver / genetically modify humans ex vivo
Extract cells and modify them using crispr
Validate cells by screening before engraftment back into patients
What is the alternative delivery method
Package crispr cas in a vehicle and deliver to target organ or tissue
Accidentally found that when a hiv patient with aml received a hsc transplant , the donor had a ccr5 mutation which meant the recipient now was hiv resistant. Was this same concept using crispr hsc editing confirmed in mouse models
Yes
What about in a hiv human patient?
No, although there was some resistance in few months, not enough hsc were modified to provide hiv resistance
= issue of mosaicism for genome editing
What unethical case study in China using this happened recently
Embryo had inserted crispr technology causing indels in its ccr5
Very unethical as no informed consent, also don’t know if any off target effects etc
