DNA Manipulation CH4

CRISPR-Cas9 Defence system

Done to create bacterias own adaptive immune system similar to human memory cells

Steals a bit of viral DNA to remember it for next time

Bacteriaphage

a virus that infects prokaryotic organism

(the diamond thing with legs)

CRISPR

A naturally occurring sequence of DNA found in bacteria that plays a role in defence against viral attacks

Short clustered repeats of DNA in prokaryotes

PAM (Protospacer adjacent motif)

sequence of two-six nucleotides that is found

signals for the viral DNA to cut and where to cut

bacteria uses to protect itself

similar to a humans MHC1 marker

Endonuclease

Enzyme

breaks the phosphodiester bond between two nucleotides in a polynucleotide chain

‘Molecular scissors’

CRISPR-Cas9 Defence system STAGE 1

Exposure

bacteriophage injects its DNA into bacterium which identifies the foreign substance

PAM signals to cut the viral DNA and where to cut

Cas1 and Cas2 cut out a sequence part of the viral DNA called a protospacer

The protospacer is introduced as a spacer to the bacterium’s CRISPR gene

Sticky end

Result of a staggered cut through double stranded DNA by an Endonuclease

Resulting in overhanging nucleotides

Blunt end

Result of straight cut across double stranded dna by an endonuclease

Results in no over hanging nucleotides

Overhanging nucleotides

Unbonded nucleotides on the ends of the dna strand resulting from a staggered cut

Primer

Short single strand of nucleus acid rhat acts as a starting point for polymerase enzymes to attach

Ligase

Enzyme

Joins molecules including dna or rna together by catalysing the formation of phosphodiester bobds

Polymerase

Enzyme

Synthesises a polymer from monomers such as forming a dna strand from nucleic acids

Spacer

Short sequences of Ena obtained from invading bacteriophages that are added into the CRISPR sequence

CRISPR-Cas9

Complex formed between gRNA and Cas9 which can cut a target sequence of DNA.

Bacteria use this complex for protection from viruses and scientists have modified it to edit genomes

Cas 1 and Cas 2 / cas1-cas2 complex

Cut a short section of the viral dna known as a protospacer

guide RNA (gRNA)

RNA which has a specific sequence determined by CRISOR to guide Cas9 to a specific site

Gel electrophoresis

Technique that seperate a dna fragments based on their molecular size

Agarose gel

Sponge like gel used in gel electrophoresis that contains pores for dna fragments to move through

Is immersed in a buffer solution which helps carry an electric current

Gel electrophoresis process STEP 1

dna samples are placed in wells at one end of the gel

Standard ladder of dna fragments with known sizes is typically loaded into one well

The agarose gel is immersed in a buffer solution which helps carry an electric current

Gel electrophoresis process STEP 2

An electric current is passed through the gel using two electrodes - one positive one negative

Negative electrode is put by the wells and the positive electrode on the other side

Since dna is negatively charged it’s attracted to the positive electrode

When electric current is applied, dna will move towards the positive electrode

Gel electrophoresis process STEP 3

Smaller dna fragments move faster through the gel than larger ones

After a few hours fhe current is switched off and the dna fragments stop moving and settle into bands and are separated based off size

Gel electrophoresis process STEP 4

Gel is stained with a fluorescent dye so the bands of dna can be seen under a UV lamp that the naked eye can see

Cas9

endonuclease

creates a blunt end cut at a site specified by guide gRNA

contains two active sites to cut both strands of DNA and create blunt ends

gRNA

Guide RNA

RNA that has a specific sequence determined by CRISPR to guide Cas9 to a specific site

CRISPR-Cas9 Defence system STAGE 2

Expression

the CRISPR spacers are transcribed and converted into gRNA

gRNA binds to Cas9 to create a CRISPR-Cas9 complex that is directed to any viral DNA inside the cell that’s complementary to the gRNA

gRNA forms a hairpin loop-like structure from the transcribed palindromic repeats either side of the spacer

CRISPR-Cas9 Defence system STAGE 3

Extermination

The CRISPR-Cas9 complex flies around in the cell scanning for any viral DNA that matches/is complementary to the one it has

when it does find some, the Cas9 cleaves the phosphate sugar backbone to inactivate the virus.

When the viral DNA is cut, enzymes in the bacterium will act to repair it. The gRNA will fine the gene again and repeat the process of cutting it until the DNA repair system make an error and induce an mutation that inactivates the virus.

Limitations of CRISPR-Cas9 (3)

Has not yet worked on humans but has in other animals

Sometimes the injected nucleotide sequence isn’t successful in being taken into the DNA and is difficult to achieve precision in adding it in

Ethetical concerns as to alter an organisms genome they must treat an embyro prior to the cell differentiating. AKA genetically modifying an embryo - concerned over the mothers and unborn babies health n stuff.

CRISPR-Cas9 for gene editing STEPSSS (8)

1. Synthetic gRNA is created in a lab that has a complementary spacer to the target DNA that scientists wish to cut

2. A Cas9 enzyme is obtained by an appropriate target PAM Sequence

3. Cas9 and gRNA are added together in a mixture and bind together to create the CRISPR-Cas9 compelx

4. The gRNA-Cas9 mixture is then injected into a specific cell, such as a zygote

5. Cas9 finds the target PAM sequence and check whether the gRNA aligns with the DNA

6. Cas9 cuts the selected sequence of DNA

7. The DNA has a blunt end cut the cell attempts to repair

8. When repairing the DNA the cell may introduce new nucleotides into the DNA at the site - scientists may inject particular nucleotides sequences into the cell with the hope that it will ligate the gap

Trp operon

Controls the amount of tryptophan in a cell

When tryptophan lvls are high

Tryptophan binds to the repressor which causes the repressor to bind to the operator

To block RNA polymerase and transcription

Attenuation

When tryptophan lvls are low

Repressor releases from operator to allow transcription to occur

Repression

Polymerase Chain Reaction (PCR)

Technique to make many copies of specific DNA fragments

easy and quick

3 stages: D.A.E

Denaturation 95

Annealing 50-60

Elongation 72

PCR Stage1

Denaturation

DNA is heated to 95.C to separate the 2 strands

PCR Stage 2

Annealing

DNA fragment is cooled to 50-60.C to allow the primers to anneal to each strand

PCR Stage 3

Elongation

DNA fragment is heated to exactly 72.C to allow taq polymerase to elongate the strands

then the entire pcr process repeats and repeats!!

Requirements for PCR (5)

DNA w region of interest

Free nucleotides

Taq polymerase

DNA Primers

Buffer