Editing the genome II Flashcards
1
Q
What is the PAM sequence for spCas9? (2)
A
- 5’-NGG-3’
- Cleavage occurs 3nt downstream of the PAM
2
Q
What is CRISPR immunity? (6)
A
- CRISPR immunity is encoded by loci comprising CRISPR array and neighbouring Cas genes
- CRISPR-Cas belongs to class 2 interference genes which are characterised by RNA-guided effector complexes that only require a single multidomain protein for interference (e.g. Cas9)
- Bacteriophage invades bacterial cell and the bacteriophage genome is chopped up into protospacer motifs which are incorporated into the CRISPR array sequence in the bacterial genome which consists of spacer elements separated by CRISPR repeat elements
- Protospacer selection requires flanking protospacer adjacent motif (PAM) sequences
- This is transcribed into pre-CRISPR RNA with encoded repeat sequences flanked by spacer sequences derived from the bacteriophage protospacer motifs
- pre-CRISPR RNA is processed and cleaved by RNase to form mature CRISPR RNAs which bind back to translated Cas9 enzyme and can target DNA of new invading bacteriophages
3
Q
How is gRNA created? (4)
A
- CRISPR array contains the spacer elements flanked by the CRISPR repeated sequences
- Transcribed into pre-crRNA
- Repeat sequences in the pre-crRNA base pair with tracrRNA = dsRNA complexes which are cleaved out by RNase III
- crRNA-tracrRNA binds to Cas9 to form individual crRNA-tracrRNA-Cas9 RNP complexes
4
Q
What is tracrRNA? (4)
A
- Trans-activating CRISPR RNA
- In type II systems crRNA processing and Cas9 activating requires an additional tracrRNA to create the gRNA
- Small non-coding RNA ~75-110nt
- tracrRNA contains a sequence complementary to the CRISPR repeats that is required for RNP assembly
5
Q
What is crRNA?
A
CRISPR RNA
6
Q
What is an sgRNA?
A
- Synthetic guide RNA (experimental version of gRNA)
- Synthetic fusion of naturally occurring tracrRNA, crRNA and spacer RNA
7
Q
What is the synthetic tetra loop? (3)
A
- Artificial linker that connects the crRNA and tracrRNA
- In sgRNA only
- Means the entire sequence can be encoded from a single plasmid/locus
8
Q
How does Cas9-sgRNA bind to target DNA? (4)
A
- Cas9-sgRNA binds to PAM site and tests the flanking DNA for the target sequence complementary to the guide RNA
- Cas9 rapidly dissociates from DNA that doesn’t contain the appropriate PAM but binds for longer at sites containing a PAM sequence
- The time bound to target sequences is dependent on the degree of complementarity between the guide RNA and the adjacent DNA
- Then strand invasion occurs depending on the amount of base pairing, R loop complex between the spacer and the target strand
9
Q
How does Cas9 bind to the PAM sequence? (5)
A
- PAM (5’NGG-3’) binds in the positive groove on Cas9
- GG-3’ forms hydrogen bonds with R1333 and R1335 in the beta-hairpin PAM interacting motif of Cas9
- PAM-Cas9 interactions trigger local structural changes which destabilise the adjacent DNA duplex base pairing, facilitating binding of spacer RNA bit of gRNA to the target DNA strand
- Phosphate lock loop formed of S1109 and K1107 stabilises the upstream phosphate on the target strand to facilitate guide RNA-target DNA hybridisation
- The first nucleotide of the displaced non-target strand is stacked with the PAM duplex so is flipped out
10
Q
How is the RNA:DNA duplex formed? (5)
A
- Initial PAM recognition is followed by increasing strand invasion of the spacer sequence to form an extended R loop
- Non-target strand DNA sequence extends outside of the Cas9 RNP complex so is accessible (facilitates base editing approach)
- Although the spacer sequence is 20nt, 17-18nt of complementarity is sufficient to maintain binding of Cas9 RNP to target sequence
- Mismatches between spacer and target DNA makes strand invasion inefficient, tests for mismatches during invasion
- If too much mismatch the Cas9 won’t be bound stably enough to cleave and will detach
11
Q
How does strand cleavage occur? (3)
A
- After RNA strand invasion, HNH domain undergoes a conformational change and communicates with the RuvC domain
- HNH and RuvC catalytic domains simultaneously cleave the target and non-target strands
- Cas9 may remain bound to the cut site before returning to pre-target state
12
Q
What is the major issue with CRISPR-Cas9? (3)
A
- Off-target editing
- Specificity is determined by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome
- Ideally the gRNA targeting sequence would have perfect homology but in reality there will be additional sites in the genome where partial homology exists = ‘off-targets’
13
Q
How can you reduce off-target editing? (3)
A
- Truncated gRNA (tru-gRNA)
- Decrease lifetime of Cas9 RNP complex in cells
- Increase specificity of PAM recognition
14
Q
What is tru-gRNA? (4)
A
- Truncated gRNA
- sgRNA is shortened by 2-5nt at 5’ end furthest from the PAM
- Increases sensitivity to mismatches to the shorter complementary gRNA at target sites
- Addition of 2 mismatched guanine nucleotides to the 5’ end can reduce off-target binding but may also reduce on-target binding
15
Q
How can you decrease Cas9 RNP complex lifetime to reduce off-target editing? (2)
A
- Direct transfection of Cas9:sgRNA RNP rather than using plasmids
- Split Cas9: light activated or intein activated Cas9
