Lecture 6

Question 1

What does CRISP stand for?

Answer 1

Clustered Regularly Interspaced Short Palindromic Repeats

Question 2

What is the normal infectious cycle of a phage infecting a bacterium?

Answer 2

• The phage (eg phage t4) attaches to and punches a hole in the membrane of the bacterium
• The DNA of the phage is then injected into the bacterium’s cytoplasm
• The DNA from the phage is then translated into protein
• Host cells DNA nucleotides are used to synthesis a new phage genome, lots are assembled
• The phage enzyme lyse the membrane of the bacterium and hundred of these new phages are released

Question 3

What does CRISPR Cas9 do?

Answer 3

• CRISPR is a genomic locus in bacteria and archaea that’s part of the adaptive immune system and used as a defence against viral infections
• CRISPR Cas9 is like a memory card that stores phage DNA from a previous infection and compares it to future infections to recognise and destroy it before it can be replicated

Question 4

How does CRISPR Cas9 work?

Answer 4

• During the infection Cas1 and Cas2 (endonucleases) acquire a sample of the phages DNA (the sample of phage DNA is called a protospacer)
• This protospacer is then integrated into the CRISPR locus in the bacteria chromosome and is now called a spacer
• Multiple spacers from multiple phages can be stored in this chromosome
• When reinfected the spacer sequences are transcribed as RNA and a Cas9 protein is synthesised
• The RNA is integrated into the Cas9 protein which acts as a surveillance mechanism as the stored spacer sequences are comapared to the new DNA coming into the cell
• If there is a match between the DNA and the spacers then Cas9 can cleave and destroy the phage DNA avoiding reinfection from the same phage

Question 5

What are the important components of the CRISPR locus?

Answer 5

• There is a Cas Operon - transcribed and translated into the Cas9 protein (nuclease enzyme that cleaves DNA and cuts at specific locations in the genome)
• Trans-activating crRNA (tracrRNA) genes - tells Cas9 where in the genome to cut
• There are many integrated spacers and palindromic repeats within the CRISPR locus

Question 6

What forms single guide RNA (sgRNA)?

Answer 6

tracrRNA and crRNA

Question 7

How long is sgRNA?

Answer 7

It is 20 nucleotides long

Question 8

What shape is sgRNA and what does it do?

Answer 8

It is a 3 loop cluster that is recognised by Cas9 and directs Cas 9 to the correct location

Question 9

What do Cas9 and sgRNA work together to do?

Answer 9

• They scan the DNA for protospacer adjacent motifs (PAM)s which are a sequence 3 nucleotides long consisting of any nucleotide and then two guanine bases (NGG) they are very common throughout the genome meaning the majority of genes can be processed through CRISPR Cas9 function
• CRISPR Cas9 will only bind to dsDNA at the PAM
• Cas9 then unwinds the dsDNA next to the PAM sequence
• It then compares the sgRNA to the dsDNA unwound region
• If the nucleotides match then Cas9 nuclease domain is activated and the DNA is cleaved just next to the PAM sequence

Question 10

What happens after Cas9 has cleaved the DNA next to the PAM sequence?

Answer 10

• The cleaving results in a double stranded break in the DNA which triggers the cellular DNA repair mechanism
• Because of the PAM site the DNA repair mechanism only works at that precise location and so this can be used to integrate specific products into specific sites in DNA

Question 11

What are the 2 types of repair when dsDNA breaks?

Answer 11

• Non-homologous end joining (NHEJ)

- Homology directed repair (HDR)

Question 12

What happens during NHEJ?

Answer 12

• The reannealing of 2 blunt ends of DNA happens
• This can resolve the problem however base deletions and insertions can cause mutations in the gene sometimes knocking out the gene expression or activity

Question 13

What happens during HDR?

Answer 13

• Uses the DNA template to add exogenous DNA into a target sequence, modified the gene without disrupting it

Question 14

How do we exploit this to become beneficial?

Answer 14

• We could replace the sgRNA within the bacterial Cas9 complex with RNA which matches a mutation responsible for human disease. NHEJ could then knock the gene out all together \
• We could also include the DNA that contained the correct DNA sequence with the healthy gene and a modified version of HDR could replace the faulty gene that causes a specific disease

Question 15

What are the ethical implications of CRISPR Cas9?

Answer 15

• Many of the investigations have been done using somatic cells however there are ethical concerns about the potential to edit germ line cells
• This germ line cell modification would be passed on from generation to generation and you cant determine the long term effects of editing the germ line cells (designer babies)
• This is illegal in the UK and most other countries

Question 16

Why use CRISPR Cas9?

Answer 16

• Fast
• Cheap
• Accurate
• Efficient
• Relatively easy to use

Question 17

What is pGLO?

Answer 17

A genetically modified plasmid which contains a specific reporter gene called green fluorescent protein (GFP)

Question 18

What is GFP?

Answer 18

Green fluorescent protein that came from Aequorea Victoria (jellyfish)

Question 19

What was GFP used for in 1987?

Answer 19

Used as a biological tracer, it was linked to the haemoglobin molecule

Question 20

What else can GFP be used for?

Answer 20

• Used as a reporter molecule to show when and where a gene is switched on
• 2007 a red protein was found that has similar properties to GFP
• Mutants of GFP that have been created that are faster at fluorescing and brighter in a whole array of colours
• Some proteins have been found that work like GFP however don’t use a uv light they use infrared light

Question 21

What are the key features of the pGLO plasmid?

Answer 21

• It is 5371 bp long
• It has the beta lactamase gene which shows ampicillin resistance
• It has the GFP gene which encodes for the GFP (when successfully transformed the e.coli colonies will fluoresce
• It has an araC regulator protein which regulates the transcription of GFP

Question 22

What is the general structure of an operon?

Answer 22

Promoter - recognised by RNA polymerase which initiates transcription
Operator - a segment of DNA that a regulator (depressor) can bind to and halt transcription (acts as a switch)
Genes - genes which are encoded by the operon

Question 23

What is the difference between an inducible operon and a repressible operon?

Answer 23

R - Transcription is always occurring but it can be inhibited or repressed (eg. Tryptophan operon)
I - Transcription doesn’t occur unless induced, when a specific small molecule interacts with a regulatory protein (lac operon)

Question 24

How does transcriptional regulation occur with the ara operon?

Answer 24

• Requires an activator for expression
• Arabinose B,A and D genes code for 3 digestive enzymes involved in the breakdown of arabinose
• Metabolise the pentode sugar L-arabinose which can be used as an energy source for E.coli
• Has a Pbad promoter where RNA polymerase binds to transcribe the BAD genes
• Transcription is dependant on the presence of the promoter, RNA polymerase and arabinose

Question 25

How does the araC gene affect the transcriptional regulation of the ara Operon?

Answer 25

• araC gene binds to DNA upstream of the transcriptional start site
• When arabinose is absent ara BA and D aren’t transcribed, but when present the operon becomes induced and starts the transcription
• The arabinose binds to the araC gene causing a conformational change to the protein subunit allowing RNA polymerase to bind to the Pbad promoter
• When arabinose is broken down and removed from araC then the transcription once again halts

Question 26

Which genes are present in pGLO that help with its regulation?

Answer 26

• araC and Pbad
• However instead of having the BAD genes it is replaced by the GFP gene
• The GFP gene is then only transcribed and expressed in the presence of arabinose