L4 - Crispr Technology

Question 1

Where was crispr first identified / how

Answer 1

Bacterial defence against bacteriophage dna upon reinfection

Question 2

What are the 3 components

Answer 2

Cas9 nuclease
Crispr rna - has comp sequence/ protospacers and binds tracr rna
Tracr rna- binds crrna and is a hairpin

Both form the grna

Question 3

What are the steps during first infection with Phage dna

Answer 3

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)

Question 4

What happens during reinfection

Answer 4

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

Question 5

What is the cas operon in crispr locus

Answer 5

Operon transcribing diff cas proteins eg cas1,2 and 9

Question 6

What’s the diff between cas1/2 and 9

Answer 6

Cas1/2 are multi protein complexes whereas cas9 is a single protein but with multidomains

Question 7

Explain what happens to form mature cr rna

Answer 7

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

Question 8

Which domains in cas9 are required to recognise and guide cas9 to the grna/dna duplex

Answer 8

Rec1 and 2

Question 9

Which lobe is the hnh nuclease domain which cleaves comp dna on

Answer 9

The nuc lobe (not rec lobe)

Question 10

There is another domain which cleaves non comp dna sequence for it to be a double strand break. What’s it called

Answer 10

RuvC

Question 11

Which domain recognises and interacts with the Pam sequence downstream of cleavage site

Answer 11

PI domain

Question 12

What is the Pam sequence and why important

Answer 12

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

Question 13

Which sequence commonly used in research as a Pam site

Answer 13

NGG strep pyogenes

Question 14

What have they done to grna for actual genome editing in research which makes it easier

Answer 14

Linked crrna with tracrrna by a linker loop to get a single grna

Question 15

What are the 2 ways you can deliver cas9/grna into cells for research

Answer 15

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

Question 16

What is crucial when designing grna

Answer 16

Has a protospacers sequence specific for locus of interest and that it is upstream of a Pam site (NGG)

Question 17

Which 2 dna repair mechanisms are used for ko/ki genomic editing

Answer 17

Homology directed repair in s phase

Or nhej in interphase

Question 18

Which one is used for knock outs and explain why

Answer 18

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

Question 19

After txn, what scans for ptc and will degrade transcripts causing ko

Answer 19

Non-sense mediated decay

Question 20

How does HDR work for knock ins

Answer 20

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

Question 21

What needs to be removed from templates and why

Answer 21

Pam sites to prevent re-targeting of the region back

Question 22

What kb needed to be homologous for this to work

Answer 22

More than 60bp

Question 23

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)

Answer 23

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

Question 24

How did they screen many different splice factors in AR-V only cancer cell lines using crispr knock out strategies

Answer 24

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

Question 25

How did they validate that the splice factor ko actually reduced AR-Vs

Answer 25

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

Question 26

In vivo uses and in humans

Question 27

What do you need to consider with cell therapy

Answer 27

Ethical guidelines
Mosaicism
Off target effects?
Delivery strategy and efficacy

Question 28

How could you deliver / genetically modify humans ex vivo

Answer 28

Extract cells and modify them using crispr
Validate cells by screening before engraftment back into patients

Question 29

What is the alternative delivery method

Answer 29

Package crispr cas in a vehicle and deliver to target organ or tissue

Question 30

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

Answer 30

Yes

Question 31

What about in a hiv human patient?

Answer 31

No, although there was some resistance in few months, not enough hsc were modified to provide hiv resistance

= issue of mosaicism for genome editing

Question 32

What unethical case study in China using this happened recently

Answer 32

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