Lab 2: CRISPR-Cas9 Gene-Editing in Yeast Flashcards
When was CRISPR-Cas9 first discovered?
In 1987 by a team of Japanese researchers led by Yoshizumi Ischino
However the function and purpose remained unknown
What allowed researchers to begin to understand the function of the CRISPR system?
Work with Streptococcus thermophilus allowed researchers to begin to understand the function of the CRISPR system as a bacterial immune defense system against invading viruses.
2005-2007
What did Jennifer Doudna and Emmanuelle Charpentier win the nobel prize for?
- In 2012-13, they characterized the Cas9 enzyme and demonstrated it can be programmed as a genome-editing tool
- They won the nobel prize in 2020 for this discovery.
Give examples of the CRISPR-Based genome editing use in food science.
- Nutrient-enrichment in crops (e.g., golden rice)
- Climate change resistant crops (e.g., drought resistant maize)
- Disease prevention in crops (e.g., upregulation of plant defense genes)
- Improved aroma/flavour (e.g., production of hop-associated flavours - without the hops)
What are challenges associated with CRISPR-based genome editing in food science?
-
Regulation of GMOs and consumer acceptance
- Poor public perception of GMOs
- Different nations have different regulations on the use of GMOs
-
Crop biodiversity
- Commercialization of CRISPR-edited crops (monocultures and low genetic diversity)
-
Bioethics
- Ethics of editing genomes in animals/humans
- Risk of unintended impacts
CRISPR-Cas9 technology allows for […] at […] in the genome.
CRISPR-Cas9 technology allows for precise edits at specific locations in the genome.
What are the 3 main components of CRISPR-Cas9 technology?
- Cas9
- Guide RNA (gRNA)
- Donor DNA
What is Cas9?
An RNA-guided nuclease that creates targeted double-strand breaks in DNA
An RNA-guided nuclease that creates targeted double-strand breaks in DNA
Cas9
What is guide RNA?
a programmable RNA sequence (typically 20 nucleotides long) that matches and binds to the target genomic region, directing Cas9 to induce a double-strand break at that site
Cas9 cuts the genomic DNA 3bp upstream of the PAM sequence.
a programmable RNA sequence (typically 20 nucleotides long) that matches and binds to the target genomic region, directing Cas9 to induce a double-strand break at that site
Guide RNA
What is Donor DNA?
a repair template designed to introduce the desired edit at the break site
a repair template designed to introduce the desired edit at the break site
Donor DNA
Once Cas9 induces a double-stranded break, the cell needs to repair the damaged DNA.
How is this done?
Homologous recombination
What is homologous recombination?
- An undamaged DNA sequence with similarity, or ‘homology’, to the broken DNA, is used as a template for accurate repair
- The sequence from the homologous strand is copied onto the damaged strand
Once Cas9 induces a double-strand break, the cell needs to repair the damaged DNA.
Researchers can leverage homologous recombination to repair Cas9 induced doublestrand breaks using […].
Researchers can leverage homologous recombination to repair Cas9 induced doublestrand breaks using designed repair templates (donor DNA).
This approach enables many kind of edits including insertion, deletion, and single nucleotide changes.
What are the kinds of edits enabled by designed repair templates (i.e., donor DNA)? [3]
- Gene insertion: Donor DNA contains a new gene flanked by sequences homologous to cut site.
- Gene deletion: Donor DNA includes only sequences homologous to upstream or downstream of the gene
- Single nucleotide changes: Donor DNA matches the target sequence except for a single nucleotide alteration. Often can be used to induce a specific amino acid change in the protein sequence.
Describe gene insertion.
A type of edit enabled by designed repair templates (i.e., donor DNA).
Donor DNA contains a new gene flanked by sequences homologous to cut site.
Describe gene deletion.
A type of edit enabled by designed repair templates (i.e., donor DNA).
Donor DNA includes only sequences homologous to upstream or downstream of the gene.
Describe single nucleotide changes.
A type of edit enabled by designed repair templates (i.e., donor DNA).
- Donor DNA matches the target sequence except for a single nucleotide alteration.
- Often can be used to induce a specific amino acid change in the protein sequence.
In order to edit a genome using CRISPR-Cas9, what is required?
- The three CRISPR components need to be present within the cell
How are Cas9 and gRNA expressed?
Typically from a plasmid that has been transformed into the cell
How is donor DNA expressed?
Typically transformed as a 100 nucleotide DNA fragment into the cell
What is the PAM sequence?
Protospacer Adjacent Motif
Essential DNA motif for CRISPR-Cas9 Targeting
For Cas9, a guide RNA sequence can’t be designed to target just any region in the genome, it must be […]
Adjacent to a PAM sequence; Cas9 must first recognize a PAM sequence adjacent to the gRNA sequence, before inducing a double-strand break.
Protospacer Adjacent Motif
What is the PAM sequence for Cas9?
5’-NGG-3’; meaning the gRNA must be adjacent to either AGG, CGG, TGG, or GGG.
Why is the PAM sequence essential?
- The CRISPR system evolved in bacteria as an adaptive immune mechanism against invading viral DNA
- During an infection, bacteria cut-and-paste a snippet of viral DNA, and store it in a CRISPR array in its genome
- If the same virus reinfects, the stored sequence is transcribed into a guide RNA, which binds to the viral DNA and directs Cas9 to create a double-strand break, effectively neutralizing the virus
How do bacteria prevent self-targeting of the Cas9 enzyme?
- To prevent self-targeting, bacteria only incorporate viral DNA segments adjacent to 5’-NGG-3’ (PAM) motifs but do not store the PAM motif in its own genome.
- Requiring Cas9 to cut only at regions adjacent to a PAM sequence ensures it targets only viral DNA and not the sequences stored in its CRISPR array
What is the objective of lab 2?
To use CRISPR-Cas9 technology to delete the ADE2 gene in Saccharomyces cerevisiae, and produce a red-coloured yeast strain.
What is ADE2?
- ADE2 specifically converts P-ribosylaminoimidazole (AIR) to Pribosylaminoimidazolecarboxylate (CAIR)
- Deletion of ADE2 results in an accumulation of AIR in the vacuole of the cell, and
- AIR has a red pigment, and thus accumulation effectively results in a colour change of the yeast cell from beige to red
- The yeast must be grown in media containing purine, as it can import it into the cell
Describe the use of CRISPR-Cas9 to selectively disrupt ADE2.
Step 1: Design ADE2 gRNA and insert it into plasmid construct with CRISPR-Cas9, and a selection marker (G418 Resistance)
Step 2: Design donor DNA that creates deletion of ADE2 gene
Step 3: Transform CRISPR-Cas9 plasmid and donor DNA into yeast. Cas9 and gRNA expressed and causes double-stranded break in ADE2; donor DNA is template for repair of ADE2.
Step 4: Select for S. cerevisiae cells that have the CRISPR-Cas9 based on growth on G418 antibiotic, and for ADE2 deletion based on red-pigment
Step 5: Amplify edited ADE2 gene via Polymerase Chain Reaction (PCR)
Step 6: Confirm deletion of ADE2 via gel
electrophoresis
We are responsible to perform steps 3 to 5 in the lab. Other steps will be prepared for us.
What will occur on day 1 and day 2 of the lab?
Day 1: CRISPR-Cas9 Gene Editing and Yeast Transformation
Day 2: Polymerase Chain Reaction
How can guide RNA be designed to target ADE2?
- Identify 20 base-pair region that contains a 5’-NGG-3’ PAM sequence
- The guide sequence can be ordered and
synthesized from biotech companies as
complementary top and bottom strands- To clone the gRNA into the CRISPR-Cas9 plasmid, overhangs homologous to plasmid regions are also needed
What are the components of the pWS173 plasmid? [6]
Designed for replication and expression in both E. coli
and S. cerevisiae.
-
Cas9 enzyme from Streptococcus
pyogenes under a Saccharomyces
cerevisiae PGK1 promoter -
Insertion site for gRNA separated by two BsmBI restriction enzyme sites. sgRNA under a Saccharomyces
cerevisiae tRNA promoter - G418R resistance gene: Provides resistance to Geneticin (G418) (antifungal)
- KanR resistance gene: Provides resistance to Kanamycin (antibacterial)
-
Two sites that allow replication of the plasmid in bacteria/yeast
-
ORI: Origin of replication for
bacteria -
2u ori: Origin of replication for
yeast
-
ORI: Origin of replication for
- Designed repair templates (i.e., donor DNA), the ADE2 sgRNA, under a Saccharomyces
cerevisiae tRNA promoter inserted using the restriction enzyme sites.
How is designed sgRNA inserted into CRISPR-Cas9 plasmid?
- BsmBI restriction enzyme cleaves CRISPR-Cas9 plasmid at specific DNA sequence, generating an overhang sequence
- Overhang sequence of restriction enzyme cut is complimentary to the overhang designed in sgRNA (gact)
After insertion of ADE2 deletion sgRNA into CRISPR-Cas9 Plasmid, what does it additionally contain? [3]
Lab 2
-
Cas9 enzyme under a Saccharomyces
cerevisiae PGK1 promoter -
ADE2 sgRNA under a Saccharomyces
cerevisiae tRNA promoter - G418R resistance gene: Provides resistance to Geneticin (G418) (antifungal)
How is donor DNA designed to edit ADE2 gene?
Order 100 base-pair sequence consisting of 50 homologous nucleotides to upstream and 50 homologous nucleotides to downstream ADE2 gene
What are the major components of a yeast transformation?
- Yeast cells
- Lithium acetate
- Polyethlene glycol (PEG)
- Carrier salmon sperm DNA
- DNA to be transformed
- Heat-shock at 42 degrees
Used to introduce CRISPR-Cas9 machinery and donor DNA into yeast cell.
Describe yeast cells used in transformation. [2]
- Best when they are in ‘mid-log’ phase (undergoing DNA replication)
- Homologous recombination efficiency is higher during DNA replication
What is the purpose of lithium acetate in yeast transformation? [2]
- Neutralize negative charges on the cell membrane and DNA
- Disrupts cell membrane
What is the purpose of polyethlene glycol (PEG) in yeast transformation?
- Helps bring plasmid and donor DNA close to cell membrane to facilitate uptake
What is the purpose of carrier salmon sperm DNA in yeast transformation?
- Protects plasmid and donor DNA from cell endounucleases and the cell wall
The cell wall has been suggested to bind to DNA during the transformation process. The addition of the salmon sperm DNA can thus reduce the possibility of the desired DNA (in this case CRISPR plasmid and donor DNA) from binding to the cell wall, as the cell wall may be overloaded with the salmon DNA.
What is the purpose of DNA to be transformed in yeast transformation?
- CRISPR-Cas9 plasmid and donor DNA
- Include negative control (no plasmid added)
What is the purpose of heat shock at 42 degrees in yeast transformation?
- Induces pore formation within cell membrane
How do we select for yeast cells that contain (1) the recombinant plasmid and (2) the ADE2 deletion?
-
G418 Agar plates
- G418 is an antifungal that disrupts protein synthesis in the yeast cell, causing it to die
- G418R is a gene that provides resistance to Geneticin by inactivating geneticin
- Cells that contain CRISPR plasmid can grow on G418 agar, cells that do not contain plasmid will die
-
Media containing purine
- Ade2 specifically converts P-ribosylaminoimidazole (AIR) to Pribosylaminoimidazolecarboxylate (CAIR)
- Deletion of ADE2 results in an accumulation of AIR in the vacuole of the cell, and
- AIR has a red pigment, and thus accumulation effectively results in a colour change of the yeast cell from beige to red
- The yeast must be grown in media containing purine, as it can import it into the cell
How can we confirm gene editing was successful?
Amplify edited ADE2 gene with
Polymerase Chain Reaction (PCR) and confirm edited ADE2 via gel electrophoresis and sequencing.
What do we need to combine to amplify edited ADE2 gene with Polymerase Chain Reaction (PCR)? [5]
- Extracted DNA from yeast
- Free dNTPs (A/G/C/Ts)
- Short 20 nucleotide primers that are
homologous to upstream/downstream
regions of ADE2 - High fidelity DNA (Taq) polymerase with proofreading ability
- Buffer that contains enzyme co-factors like Mg2+
How can we confirm edited ADE2 following amplification with PCR?
- Add amplified DNA product to wells of an agarose gel
- As DNA is negatively charged, DNA will migrate from the anode (-ve) to cathode (+ve)
- Agarose gel contains pores: small DNA strands move faster through gel than larger DNA strands
- Wildtype ADE2 (no gene editing) is 1716 bp in length
- As primers that amplify ADE2 are
upstream/downstream of the gene, they add 236 bp to the amplified DNA.- Therefore the DNA band on the gel will be 1952 bp
- Deletion of ADE2 will result in a PCR product of 236 bp
Delection of ADE2 will result in a PCR product of […].
Deletion of ADE2 will result in a PCR product of 236 bp
What is zymolase?
An enzyme that has glucanase activity and degrades the yeast cell wall
Describe DNA extraction with zymolase enzyme.
- Incubate at 37 degrees for 30 minutes - zymolase is active
- Incubate at 95 degrees for 10 minutes - break open the cell
Where does Cas9 exactly cut the DNA?
Cas9 cuts the genomic DNA 3bp upstream of the PAM sequence.
Does Cas9 only cut the DNA once, or does it cut on both sides of the gRNA sequence?
- Cas9 does not cut the guide RNA sequence.
- Cas9 introduces one double-stranded break at a specific location dictated by the gRNA and PAM sequence.
- This means that both DNA strands are cut at the same site, leading to a single DSB rather than multiple cuts.
The purpose of the carrier salmon sperm DNA is to protect the plasmid and donor DNA from cell endonucleases and cell wall.
Please clarify the ‘and cell wall’ component.
The cell wall has been suggested to bind to DNA during the transformation process. The addition of the salmon sperm DNA can thus reduce the possibility of the desired DNA (in this case CRISPR plasmid and donor DNA) from binding to the cell wall, as the cell wall may be overloaded with the salmon DNA.
Compare the purpose of the Lithium Acetate and the salmon sperm DNA in the yeast transformation process.
The CRISPR DNA must pass through both the cell wall and the cell membrane to get into the yeast cell.
- The purpose of LiAc is to neutralize negative charge repulsion between the cell membrane (composed of phospholipids with phosphate groups that are negatively charged), and DNA (phosphate groups negatively charged).
- The cell wall (different cellular and molecular structure than cell membrane) is composed of protein and polysaccharide molecules. The purpose of the salmon sperm DNA is to increase efficiency of uptake of CRISPR DNA through various means, like limiting endonuclease degradation of the CRISPR DNA, as well as limiting non-specific interactions for example between the CRISPR DNA and cell wall.
