RNA analysis and CRISPR

Question 1

What is RNAseq

Answer 1

RNA sequencing technology. Quantitative. Does not detect unprocessed lncRNAs

Question 2

How does GROseq work?

Answer 2

Restarts transcription and maps sites of transcriptionally active RNAPII

Question 3

What does PROseq do?

Answer 3

Uses Bromo-dNTPs for base-pair resolution

Question 4

What does NETseq do?

Answer 4

Maps at nucleotide level by immunoprecipitating DNA transcripts by RNA polymerase

Question 5

What does TTseq do?

Answer 5

Labels nascent RNA and purifies it to measure transcription levels in cells

Question 6

How can lncRNA function be tested?

Answer 6

Knocking them out/down to see changes in DNA function

Question 7

What features of lncRNA have been discovered by knockout/down experiments?

Answer 7

They are not deactivated by framshifts or premature stop codons
Often present in functionally important regions and introns
Not targetted by RNAi in the nucleus

Question 8

What sort of nucleic acids can be used to test RNA function?

Answer 8

Bromo-dNTPs are used in PROseq for base pair resolution
Antisense oligonucleotides (ASO) are complimentary to the RNA sequence and can be depleted by RNase H
Locked nucleic acids (LNA)- modified DNA sequences that protect from nucleases. Depleted with RNase H

Question 9

What are CRAC and iCLIP used to do?

Answer 9

Identify positions of a protein on a specific RNA
Process rRNA with U3 snoRNA (ncRNA which modifies the ribosome)

Question 10

Why are 2 steps of purification done in CRAC?

Answer 10

Increase signal to noise and allow modifications

Question 11

Outline the steps of CRAC

Answer 11

1. The POI is labelled at the C-terminus with 6xHis-TEV-bait protein
2. Cell culture
3. IgG purification under high salt conditions and TEV is cleaved
4. 6xHis-TEV is partially RNase treated
5. Purification with Ni coated beads
6. RNA on the bait RNP has 5’ and 3’ linker ligated. %’ is labelled with 32P
7. Elution from Ni beads and SDS page is done
8. Proteinase K extracts RNA and it is sequenced by illumina

Question 12

How is iCLIP done?

Answer 12

1. The extract undergoes partial RNase treatment
2. Immunoprecipitated using an antibody against the bait protein
3. 5’ 32P labelling and 3’ linker ligation
4. SDS page and transfer
5. Reverse transcription and ligation of cDNA into a circle due to 3’ linker
6. Oligo is cleaved which linearizes the product giving cDNA. PCR is done

Question 13

What can hnRNP C be used to recognise?

Answer 13

They assemble on introns and exons but are not on splice sites
Recognise uridine tracts

Question 14

Explain split CRAC

Answer 14

CRAC but maps binding patterns of different domains of the same protein
Done with the same method as CRAC except labelling the protein between the domains or the C-terminus

Question 15

What is PAR-CLIP used for?

Answer 15

Finding low abundance interactions by labelling DNA with 4 Thiouridine

Question 16

What is Pulse-chase labelling?

Answer 16

Used for studying pre-rRNA processing in growing cells as this is associated with transcription
No mRNA detected as it is of different sizes so gives a diffused signal
Pre-mRNA is unstable so degraded before detection

Question 17

What is hnRNA

Answer 17

Short lived
Fast sedimenting
Detected by pulse-chase labelling if non-ribosomal
Carries genes

Question 18

What is gRNA made up of?

Answer 18

Scaffold sequence and spacer (which defines 20nt region of genome to be targetted)

Question 19

Explain CRISPR

Answer 19

1. The Cas9 enzyme recognises gRNA at its PAM site
2. HNH cuts the target strand, RuvC cuts the non-target strand, 3 bases after PAM
3. After cleavage, gRNA detaches and the DNA is edited
4. Double strand breaks are the repaired

Question 20

Explain NHEJ

Answer 20

The Ku70/80 dimer binds to broken DNA strands and DNA PKes join them together.
DNA ligase can then ligate the strands together

Question 21

Explain HDR

Answer 21

DNA is resected to create sticky ends
Rad51 guides strand invasion and DNA from a sister chromatid is inserted into the gap. DNA ligase seals nicks

Question 22

How can mutations be found with T7 endonuclease I?

Answer 22

1. Amplify the mutated region with PCR
2. Denature and rehybridise to create a larger pool of mutants
3. Mismatches are cleaved by SURVEYOR and T7 endonuclease I

Question 23

How can CRISPR efficiency be improved?

Answer 23

Increasing rate of plasmid transfection
HDR happens in <10% of modified alleles, so NHEJ machinery can be suppressed and cell cycles can be synchronised where HDR is most active (G2 and S)
Small molecule enhancers of HDR
Degrading DNA ligase IV

Question 24

Why is it favourable to have Cas9 and gRNA from different vectors in CRISPR?

Answer 24

Increases stability

Question 25

Why is dCas9 used?

Answer 25

Epigenome editing
It can still target DNA but does not cleave the strands due to a double mutation
SunTag can target it to the transcriptional activator VP64 to bind the promotor and block gene expression
Can block RNA pol and fuse to transcriptional repressors

Question 26

Why is nickase Cas9 (nCas9) used?

Answer 26

Creates DSBs with an overhang, which can be used to produce indels
Less off target effects as SSBs are repaired efficiently

Question 27

What is Cas13d used for?

Answer 27

Allows RNA directed RNA targetting
tracrRNA hybridises with repeat sequence RNA
More specific than Cas9 but less functional
Uses a protospacer flanking site (PFS) instead of PAM site

Question 28

How can the epigenome be edited with dCas9?

Answer 28

Used to increase H3K27ac on enhancers by fusing to a p300 histone acetyltransferase domain

Question 29

How is prime editing done with nCas9?

Answer 29

nCas9 is fused with reverse transcriptase
sgRNA is extended to encode a gRNA and repair template PEG RNA which contains the mutation

Question 30

How can base editing be undertaken in CRISPR

Answer 30

A 9nt non-target ssDNA strand outside the R-loop complex is targetted
Cytosine deaminases convert C -> U
During replication, U is converted to T
Base pair excision is also used

Question 31

What are protospacer adjacent motifs?

Answer 31

PAM sequences are what tracrRNA in gRNA binds to on the target site
Tests flanking DNA for anything complimentary to gRNA
The time PAM is bound to a target depends how complimentary gRNA is to it

Question 32

How is an accessible ssRNA loop created in CRISPR for editing?

Answer 32

Spacer and target sit in a positively charged groove which forms an R-loop
The ssDNA protrides out of Cas9

Question 33

How does off-target CRISPR editing come about?

Answer 33

If sgRNA and the spacer are not complimentary

Question 34

How can off-target CRISPR be reduced?

Answer 34

Using truncated RNA
sgRNA is shortened 2-5 nucleotides at the 5’ end.
Increases sensitivity to mismatches
Increases specificity of PAM
Decreases Cas9-RNA complex lifetime

Question 35

What is Hi-Fidelity Cas9?

Answer 35

Where spacer has higher affinity for DNA than what is required for recognition
Mutations can be used to reduce affinity for off-targets
Not compatible with tru-RNA

Question 36

How can Cas9 be co-expressed using lentiviral transduction for CRISPR screening?

Answer 36

LentiCRISPR can be used which coexpresses Cas9 and sgRNA
LentiCas9 and sgRNA are coexpressed from different vecotrs for more stable Cas9 expression

Question 37

What aptamer binds to telomeres?

Answer 37

Spinach binds to flurophores and gives bright signals when bound to telomeres