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CRISPR > CRISPR LAB - INTRODUCTION > Flashcards

CRISPR LAB - INTRODUCTION Flashcards

1
Q

In lab we are working with:…..

because…

A

‘Saccharomyces cerevisiae’

  1. easy to GROW
  2. easy to TRANSFORM with DNA
  3. easy to SELECT FOR GENETIC VARIANTS
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2
Q

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?

A

Adenine biosynthetic gene 2 (ADE2)

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3
Q

What is Adenine biosynthetic gene 2 (ADE2)?
=4

A
  1. ENCODES THE ENZYME
    phosphoribosylaminoimidazole carboxylase (or, simply the Ade2 protein).
  2. This ENZYME CATALYSES a step in the ‘de novo’ PURINE NUCLEOTIDE BIOSYNTHETIC PATHWAY
  3. In ade2 MUTANTS, theCELLS ARE DEPRIVED OF ADENINE AND RED PIGMENT ACCUMULATES, RESULTING IN A PINK PHENOTYPE.
  4. A MUTATION IN THIS GENE AFFECTS ‘CELL METABOLISM’, making this an EXAMPLE of a METABOLIC MUTATION.
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4
Q

Adenine2 (Ade2) gene editing: ‘mutating Ade2 gene stops the…’

A

Mutating Ade2 gene stops the ADENINE BIOSYNTHESIS PATHWAY AT A SPECIFIC STEP.

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5
Q

Adenine2 (Ade2) gene editing:

A

PATHWAY PROCESS

+

SIMPLIFIED PROCESS DIAGARMS X 2

SLIDE 3

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6
Q

*Ade2 gene encodes Ade2 protein that processes the intermediate

A

P-ribosylamino inidazole (AIR).

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7
Q

*If Ade2 is functional, the next compound, CAIR, is made, and there is no buildup of:

A

AIR

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8
Q

*If Ade2 edited to a nonfunctional gene, the intermediate compound….BUILDS UP

A

AIR

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9
Q

Buildup of ….., which has a …..colour, causes a buildup in the cells, making the cells pink

A

AIR

PINK

PINK

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10
Q

How do we inactivate the ADE2 protein? =4

A
    • 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.

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11
Q

CRISPR gene editing combines two main components, and two steps

WHAT ARE THE 2 COMPONENTS?

A
  1. CAS9 plasmid, to make CAS9 protein + guide RNA complex
  2. Homology-directed DNA HDR fragment to repair and edit targeted DNA sequence using the cells homology-directed DNA repair mechanism
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12
Q

CRISPR gene editing combines two main components, and two steps

WHAT ARE THE 2 STEPS?

A
  1. Recognition and DS cut at targeted DNA sequence, recognized by the cell as DNA damage
  2. Homology-directed repair DNA (HDR) fragment to be integrated into, and replace, the DNA region damaged by the CAS9-guide RNA cut
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13
Q

pCAS9/guide RNA plasmid.

WHAT DOES IT DO?

A
  1. This is a cloning/expression plasmid vector that contains two genes relevant to CRISPR function.
  2. First, it encodes the ‘CAS9 protein.’
  3. Second, it encodes the ‘20nt guide RNA’ that targets the pCAS9 protein to the targeted gene.
  4. 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
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14
Q

WHAT HAPPENS AFTER ….’The pCAS9/ guide RNA plasmid is transformed into the yeast cells.’ 3

A

1.The cells then use the plasmid DNA as a template to transcribe the guide RNA,
as well as transcribing the RNA encoding CAS9.

  1. This CAS9 RNA is then used to produce CAS9 protein by translation.
  2. These interact to form
    the pCAS9-guide RNA
    ribonucleoprotein
    (RNP = RNA+protein) complex
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15
Q

pCAS9/guide RNA plasmid. DIAGRAM

A

SLIDE 6

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16
Q

pCAS9/guide RNA plasmid.

‘GUIDE RNA …EXPRESSION MODULE’

A

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.

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17
Q

pCAS9/guide RNA plasmid.

‘Ori for Yeast’

A
  1. This is the origin of replication for yeast
    cells.
  2. 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
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18
Q

pCAS9/guide RNA plasmid.

‘Ori for bacteria’

A
  1. As with all cloning vectors, pCAS9 has an origin of replication for bacteria cells.
  2. 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
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19
Q

pCAS9/guide RNA plasmid.

‘CAS9 ORF and RNR2 PROMOTER’

A
  1. This is the CAS9 gene that produces the CAS9 protein.
  2. It is transcribed from the RNR2 yeast promoter, so that the protein is produced in yeast cells.
20
Q

pCAS9/guide RNA plasmid.

‘Kan RESISTANCE YEAST CELLS…./ KAN RESISTANCE BACTERIA’

A
  1. Resistance to the antibiotic Kanamycin.
  2. These are the same coding region, expressed from different promoters, one that is active in bacteria, and one that is active in yeast cells.
  3. This allows the same Kan
    resistance protein to be
    expressed both in E. coli
    and yeast, providing the
    same selectable marker for
    both types of cells.
21
Q

pCAS9/guide RNA plasmid

how many bp?

A

pCAS 8761 bp

22
Q

pCAS9/guide RNA plasmid. diagram

A

slide 7

23
Q

Designing the guide RNA….What sequence does the CAS9/guide RNA recognize?’ = 4

A
  1. *It recognizes 20 nucleotide sequence on the genomic DNA that you select
    — The guide RNA recognizes this specific 20 nt sequence
  2. *It is very important to note the last three bases in the selected target sequence (underlined).
  3. *This is called the Protospacer Adjacent Motif, (PAM).
  4. *Any target sequence that is not followed by this three base sequence (NGG, where N = any base, and G = guanine bases) will not be recognized.
24
Q

The guide RNA sequence itself cannot contain the PAM sequence. explain….

A

The guide RNA sequence itself cannot contain the PAM sequence.

—*** Thus, the guide RNA for this sequence, identified by CRISPR direct, would be three bases shorter than the target sequence, with the PAM removed from the 3’ end

25
Q

Designing the guide RNA (continued..)….’The 3’ end of the DNA target sequence must have…’

= 4

A
  1. The 3’ end of the DNA target sequence must have a proto-spacer adjacent motif (PAM) sequence (5’-NGG-3’).
  2. The 20 nucleotides upstream of the PAM sequence will be your targeting sequence (crRNA), and Cas9 nuclease will cleave approximately three bases upstream of the PAM.
  3. The PAM sequence itself is absolutely required for cleavage, but it is NOT part of the guide RNA sequence and therefore should not be included in the guide RNA itself.
  4. The target sequence can be on either DNA strand
26
Q

How do you select where you want to edit? = 5

A
  1. Within any strand of DNA, a number of unique 20 nt sequences that are followed by a PAM will be identified.
  2. ‘CRISPRdirect’, as well as other available programs, can help identify the best sequences to use, including
    such factors as melting temp, GC content, presence of restriction sites, palindromes, etc.
  3. Also use other information, such as where you want to make or correct a mutation (restore to wild type).
  4. If the gene is a non-functional mutant, you would want to select a region near the mutant site.
  5. May want to edit within a promoter, within a specific exon, maybe within a region that codes for a substrate
    binding site, a protein-protein interaction domain, or a nucleic acid binding domain.
27
Q

Designing the guide RNA… in the experiment

A

1 *For these experiments, we selected sequences near the 5’ region of the coding sequence (ORF).

2 *This because we wanted to inactivate the Ade2 gene by editing in three stop codons.

3 *A stop codon at the 5’ end of the gene has a better chance of making a non-functional protein than one at the 3’ end.

28
Q

Designing the guide RNA (continued..).. simplified 3

A
  1. Identify a PAM (INGG) sequences in the DNA sequence you would like to target
  2. Determine the 5’ start of the actual sgRNA targeting sequence by counting 20 nucleotides upstream of the PAM sequence
  3. Determine the actual sgRNA targeting sequence
29
Q

Designing the guide RNA (continued..)

‘Ade2 is about 1700 nt long. We selected a region, identified on CRISPR direct, that was near the 5’ end of the gene’ explain…6

A
  1. Ade2 is about 1700 nt long.
  2. We selected a region, identified on CRISPR direct, that was near the 5’ end of the gene
  3. The ATG (AUG) start codon and and the encoded amino acids for this region of the gene are shown highlighted in bold.
    – Note that some 5’ non-coding sequence is also shown.
  4. Several sequences were identified using CRISPR direct, the one we selected to use was:
    5’ ATTGGGACGTATGATTGTTGAGG 3’
  5. Although several additional sequences were identified, we selected this sequence because it was
    the 5’ most sequence that could be used for CRISPR.
  6. This gives us the opportunity of introducing one or more stop codons near the start of the RNA,
    resulting in early termination of translation and ensuring that a non-functional protein is created.
30
Q

Designing the guide RNA….diagrams

A

slide 8

slide 9

slide 10

slide 11

31
Q

Designing the guide RNA (continued..)

‘All of this is done by us before you come
in to the lab:…’ = 3

A

1 * Once we have chosen our guide RNA sequence, we order two complimentary oligonucleotides that correspond to this sequence

2 * When the oligos arrive, we anneal them together to form a short ds-DNA fragment

3 * That fragment is then cloned into the gRNA site in the pCas plasmid

32
Q

Double Strand (DSB), or DS cut: WHAT IS IT?

WHAT DOES IT DO?

WHAT DOES THE PROCESS DO?

A
  1. A DSB made in the targeted gene by the CAS9/guide RNA nuclease.
  2. A break in both strands of the DNA double helix, caused by the gRNA guided Cas9 protein
  3. The DS cut is recognized by the cell as DNA damage, and elicits a DNA repair mechanism called Homology
    Directed Repair (HDR)
33
Q

HDR

A

= Homology Directed Repair

34
Q

Designing the HDR Template…6

A
  1. A DNA repair mechanism that uses DNA homology to repair a DSB.
  2. Used to insert novel DNA sequences into a genome by flanking the desired sequence with DNA regions homologous to the sequences flanking an induced DSB.
  3. Broken DNA region, DS Break
  4. HD Repair fragment, use as a template to repair damaged DNA region
  5. HDR Fragment inserted into damaged region by cells DNA repair mechanism.
  6. New TAG Stop codon introduced into HDR fragment
35
Q

Designing the HDR Template…

‘The HDR Fragment is inserted into the gene at the site of the original DNA break.’ EXPLAIN

A
  1. The HDR Fragment is inserted into the gene at the site of the original DNA break.
  2. This occurs through the
    cell’s innate DNA repair machinery, which uses the HDR sequence as the model sequence to repair the broken DNA.
  3. The entire DNA region, ‘including and surrounding both sides of the DS break,’ are replaced with the DNA
    fragment from the HDR Fragment.
36
Q

Designing the HDR Template DIAGRAM

A

SLIDE 14

37
Q

Designing the HDR Template FEATURES… =

A
  1. This is the HDR fragment for Ade2.
  2. It is 126 BP in length
  3. It had three stop codons edited into it
  4. Note also, the HDR fragment contains no PAM sequence following the 20 NT recognition site
38
Q

5’ AAC AGG CTC AAC ATT AAG ACG GTA ATA CTA GAT GCT GAA AAT TCT CC ATG GAT TCT AGA ACA GTT GGT ATA TTG GGA GGG GGA ‘TAA’ TTG GGA CGT ATG ATT GTT ‘TAG TAA’ GCT AAC AGG CTC AAC ATT AAG ACG GTA ATA CTA GAT GCT GAA AAT TCT CC 3’

WHY THREE STOP CODONS…

A

Prevents any chance of read through, ensures that we get an Ade2 mutant, that does not produce a functional Ade2 protein.

39
Q

Designing the HDR Template…

‘Note that the PAM sequence has been edited out of the HDR fragment as well. Why?’

A

Prevents any further recognition by CAS9/guide RNA once the HDR has been integrated

40
Q

Note that the PAM sequence has been edited out of the HDR fragment as well…

Designing the HDR Template

A

SLIDE 16

41
Q

DIAGRAM OF THE CRISPR PROCESS…SLIDE 18

A

IMPORTANT NEED TO LEARN

42
Q

The yeast get transformed with two pieces of DNA: 2

A
  1. The pcas plasmid that encodes the pcas9 protein and the guide RNA
  2. The PCR-amplified HDR DNA
43
Q

Cotransformation process….

A

1 *The CAS9 protein/guide RNA is from the transformed plasmid.

2 *The HDR repair fragment is directly transformed into the cells

3.The DS cut and HDR repair
processes occur together
within the yeast cells, this
results in editing of the
selected region of the
targeted gene

44
Q

Cotransformation process …diagram

A

slide 20 important

45
Q

How can we tell if the ADE2 protein is inactivated? = 5

A

1* We will plate out our transformed yeast onto YPD plates that contain G418 (geneticin)

2 * The G418 is similar to Kanamycin and will mean that only yeast that have taken up the pCAS plasmid will be able to grow

3 * (The YPD just has all the nutrients yeast need to grow)

4 * If the colonies are pink it means they contain ade2 (mutated ADE2)

  1. Remember that the ADE2 inactivation will lead to pink yeast
46
Q

CRISPR labs: PROCESS…

A
  • Practical session 1 – PCR amplification to create the HDR template
  • Practical session 2 – Bioinformatics presentation on designing gRNA for CRISPR; PCR product cleanup and analysis
  • Practical session 3 – Co-transformation of 1) pCAS/gRNA plasmid and HDR template, 2) pCAS/gRNA plasmid only, and 3) negative control (without plasmid and template) into yeast cells
  • Practical session 4 – Short presentation on more CRISPR research and brainstorming activities; Examination of colonies in three initial plates and colony transfer
  • Practical session 5 – Examination of colonies in re-streaking plate Results Sheet – At the end of CRISPR lab manual Online turnitin submission by 11:59 pm on 26 May 2023 (10 marks)

CRISPR (3 decks)

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