CRISPR LAB - INTRODUCTION Flashcards
In lab we are working with:…..
because…
‘Saccharomyces cerevisiae’
- easy to GROW
- easy to TRANSFORM with DNA
- easy to SELECT FOR GENETIC VARIANTS
We are working in this practical with ‘Saccharomyces cerevisiae’ as it is easy to grow, transform with DNA, and select for genetic variants.
WE SELECTED ONE GENE TO EDIT IN THIS STRAIN: what is the strain?
Adenine biosynthetic gene 2 (ADE2)
What is Adenine biosynthetic gene 2 (ADE2)?
=4
- ENCODES THE ENZYME
phosphoribosylaminoimidazole carboxylase (or, simply the Ade2 protein). - This ENZYME CATALYSES a step in the ‘de novo’ PURINE NUCLEOTIDE BIOSYNTHETIC PATHWAY
- In ade2 MUTANTS, theCELLS ARE DEPRIVED OF ADENINE AND RED PIGMENT ACCUMULATES, RESULTING IN A PINK PHENOTYPE.
- A MUTATION IN THIS GENE AFFECTS ‘CELL METABOLISM’, making this an EXAMPLE of a METABOLIC MUTATION.
Adenine2 (Ade2) gene editing: ‘mutating Ade2 gene stops the…’
Mutating Ade2 gene stops the ADENINE BIOSYNTHESIS PATHWAY AT A SPECIFIC STEP.
Adenine2 (Ade2) gene editing:
PATHWAY PROCESS
+
SIMPLIFIED PROCESS DIAGARMS X 2
SLIDE 3
*Ade2 gene encodes Ade2 protein that processes the intermediate
P-ribosylamino inidazole (AIR).
*If Ade2 is functional, the next compound, CAIR, is made, and there is no buildup of:
AIR
*If Ade2 edited to a nonfunctional gene, the intermediate compound….BUILDS UP
AIR
Buildup of ….., which has a …..colour, causes a buildup in the cells, making the cells pink
AIR
PINK
PINK
How do we inactivate the ADE2 protein? =4
- Carry out GENOME EDITING on the ADE2 gene
2 * Target Cas9 to the start of the gene and allow it to CREATE a ds break
3 * Provide aREPAIR TEMPLATE (containing premature STOP codon
sequences) to be used WHEN THE CELL FIXES THE CUT BY HOMOLOGOUS RECOMBINATION(NHEJ does not work well in yeast)
4* The PROTEIN will NO LONGER be TRANSLATED FROM THE mRNA made.
CRISPR gene editing combines two main components, and two steps
WHAT ARE THE 2 COMPONENTS?
- CAS9 plasmid, to make CAS9 protein + guide RNA complex
- Homology-directed DNA HDR fragment to repair and edit targeted DNA sequence using the cells homology-directed DNA repair mechanism
CRISPR gene editing combines two main components, and two steps
WHAT ARE THE 2 STEPS?
- Recognition and DS cut at targeted DNA sequence, recognized by the cell as DNA damage
- Homology-directed repair DNA (HDR) fragment to be integrated into, and replace, the DNA region damaged by the CAS9-guide RNA cut
pCAS9/guide RNA plasmid.
WHAT DOES IT DO?
- This is a cloning/expression plasmid vector that contains two genes relevant to CRISPR function.
- First, it encodes the ‘CAS9 protein.’
- Second, it encodes the ‘20nt guide RNA’ that targets the pCAS9 protein to the targeted gene.
- When transformed into the host cells (in our case, the transformed yeast cells), the expressed CAS9 protein combines with the 20 nt guide RNA to form a targeted nuclease that will make a double stranded (ds) cut within the targeted gene
WHAT HAPPENS AFTER ….’The pCAS9/ guide RNA plasmid is transformed into the yeast cells.’ 3
1.The cells then use the plasmid DNA as a template to transcribe the guide RNA,
as well as transcribing the RNA encoding CAS9.
- This CAS9 RNA is then used to produce CAS9 protein by translation.
- These interact to form
the pCAS9-guide RNA
ribonucleoprotein
(RNP = RNA+protein) complex
pCAS9/guide RNA plasmid. DIAGRAM
SLIDE 6
pCAS9/guide RNA plasmid.
‘GUIDE RNA …EXPRESSION MODULE’
This is where the 20 nt guideRNA sequence is inserted.
It is expressed from a yeast promoter within the module, so that the guide RNA is transcribed and makes the guide RNA in the yeast cells.
pCAS9/guide RNA plasmid.
‘Ori for Yeast’
- This is the origin of replication for yeast
cells. - Since we are doing the gene editing in yeast, the genetically modified pCAS9
plasmid with the guide RNA sequence must be able to be replicated and maintained as
a permanent genetic element (also called a
“replicon”) in the eukaryotic yeast cells
pCAS9/guide RNA plasmid.
‘Ori for bacteria’
- As with all cloning vectors, pCAS9 has an origin of replication for bacteria cells.
- This allows the plasmid to be maintained and used
for basic cloning applications (such as inserting the guide RNA sequence) in the prokaryotic bacteria cells