CRISPR Cas-9 Questions
Write three different ideas you have about why CRISPR-Cas9 technology could be more useful for gene therapy and/or gene-cutting tools.
1. It is relatively simple to complete. 2. It can also be modified to add a new sequence in where one was previously cut. 3. It is much more specific than restriction enzymes because it has a longer nucleotide sequence and uses PAMs for identification
Describe two possible advantages of using NHEJ over using HDR in a gene editing experiment
1. NHEJ is much easier to perform. 2. As it usually induces a premature stop codon, it is very useful in silencing genes.
Describe two potential advantages and two potential disadvantages of administering such a therapy to liver cells only.
1. Only the liver cells may experience off-target effects. 2. There may be other cells types that need fewer LDLR for cell function
If you were to continue evaluating the candidate target sites for use in a therapy, what are two additional pieces of information or experiments that would help you?
1. the genome of the person who would be getting this therapy 2. Their specific mutation that is causing the disease state
What are two potential advantages and two potential disadvantages of administering a cystic fibrosis gene editing therapy by inhalation instead of orally, by injection, or another method?
Advantages: 1. Lung tissue, which is the most affected is treated 2. Off-target effects throughout the body are minimized Disadvantages: 1. Only lung tissue is treated, so other tissues affected by the lack of ion channels remained filled with mucus. 2. The inhalation may not be as effective as injection because the gases may not be able to penetrate the mucus and find the cells as easily.
Does the presence of alignment matches indicate higher or lower risk of off-target effects
Alignment matches indicate a higher risk of off-target effects
Do you think there should be differences between how off-target risk is evaluated for CRISPR-based therapies and for laboratory CRISPR experiments?
CRISPR-based therapies are affecting real people lives whereas laboratory CRISPR experiments just affect cell lines and laboratory animals. While caution should be used in both, extreme caution and extensive research should be used of CRISPR-based therapies that are actually going to be given to people.
Explain how CRISPR-Cas9 together with HDR could be used to change a single nucleotide, for instance changing a T to an A
Cas9 with HDR could be used to change a single nucleotide if researchers designed a donor template DNA with a desired sequence that was exactly the same except for that one base pair.
How would you decide whether the risk of off-target activity for a CRISPR-Cas9 therapy is low enough to be considered safe?
I would look at the specific off target effects for each person based on their genome. It would also depend if the Cas9 was going to effect all the cells in the body or just in a specific region. Because if only specialized cells were being used some off target effects would not matter because you would be able to cut genes that are not being used in that cell type.
Is this gene editing strategy (for CAD) an example of replacing, inserting, or deleting a sequence?
It would probably defined as deleting; however, we are not using HDR which typically has these three outcomes. Instead we are using NHEJ to induce mutations that will silence the gene instead.
Of the plates that show evidence of the lacZ gene having been cut, which also show evidence of the DNA cut having been repaired? Note that repairing DNA does not mean repairing the function of a gene.
Plate D will show evidence of being repaired because these colonies though unable to process lactose, will be alive unlike those that were cut in plate B
Which plates show evidence of the lacZ gene having been cut by Cas9?
Plates B and D will have the sgRNA to guide the Cas9 where to cut; however, plate B will most likely die because it does not have a DNA repair system which means the cells can't grow since they cannot repair the ds break. Though plates A and D contain the template to "fix" the double strand, Cas9 will not cut these plates since no sgRNA is present to direct them.
If the bacteria on the starter plates did NOt have a functional lacZ gene, what color would you expect the colonies to be
The colonies would be white because they would not be able to use/ break down the X-gal.
Using evidence from Table 2, explain in complete sentences why the bacterial colonies on the starter plate are blue.
They are blue because both starter plates have the lacZ turned on and X-gal has been added to the plate.
Should off-target effects be considered for nontherapeutric CRISPR in the laboratory. Why or why not?
Yes, because if the off target effects are not considered whatsoever then that is a variable not being considered. Additionally, those off target effects may indirectly affect the experiment.
Does the sgRNA bind to the PAM?
Yes, there is a spacer on the sgRNA that is within the Cas9 protein that binds to the protospacer sequence, opening up the double helix
What other cells could be edited for sickle cell anemia to achieve similar results? List two potential advantages an two potential disadvantages of editing these cells instead of hematopoietic cells
You could also edit the myeloid stem cell that then differentiates into the red blood cells. Advantages 1. Limit off target effects in white blood cells and lymphocytes 2. Fewer cell types are affected. Disadvantages: 1. White blood cells and lymphocytes may have unknown affects from sickle cell type that would be helped by gene editing. 2. By not editing the white blood cells and lymphocytes, they may recognize the new myeloid stem cell when it is introduced and attack it.
What is the probability that any base in the sequence is an adenine? How many times do you expect to find adenine in the human genome?
(0.25)(3,234,830,00)= 808,707,500
How many times would you expect to find a specific 20 base pair sequence in the human genome?
(0.25)^20= 9.09x10^-13 = 0.0029 times
How many times would you except to find an EcoRI cut site in a fragment of DNA 1,000,000 base pairs long?
(0.25)^6= 2.44x10^-4 = 89,753 times
What is the probability of finding a particular two-base sequence? How many times do you expect to find that sequence in the human genome?
Probability (1/4)^2 = 0.0625 = 202,176,875 times
In addition to inserting or exchanging sequences, it is possible to remove short sequences near a cut sit using HDR. Think of and describe an idea for how the donor template DNA sequence could be designed to cause such a removal. Use external resources about HDR as needed.
The gene could be cut using CRISPR-Cas9 and then the donor template DNA sequence could be for another gene or simply a noncoding repeat region of DNA or an intron.
Describe at least two other experiments that could be done to verify that chromosomal gene editing occurred in the bacteria.
1. A gel with dideoxyribose nucleotides to sequence the DNA 2. Growing the cultures on spectinomycin; only plate D should grow have significant growth.
In actuality, the DNA sequence of the human genome is not random. Some sequences including some very large sequences and are repeated many times throughout the human genome. Write two ideas you have for how this fact complicates the use of CRISPR gene-editing technology in humans.
1. CRISPR-Cas9 may cut something off target if the desired sequences has many repeats elsewhere in the genome. 2. The number of occurrences we calculated above will probably be way off depending on how frequently this sequence may happen or not happen to exist.
Describe two possible advantages of using HDR over using NHEJ in a gene editing experiment
1. HDR creates a more specific outcome. 2. HDR can modify or edit the gene.
How many nucleotides long is the guiding region of the sgRNA?
20 nucleotides
Describe in complete sentences how the requirement of a PAM sequence affects the flexibility of CRISPR-Cas9 gene editing
A PAM sequence makes the gene editing of CRISPR-Cas9 less flexible because it requires more specificity for the enzymes. The PAM sequence and the DNA sequence must match for Cas9 to bind and cut.
Explain how colony color can be used as evidence of the state of the lacZ gene in the bacteria.
A blue colony color indicates that the lacZ is still functional while a white colony color indicates that the lacZ gene is not functional.
Using mathematical evidence, explain why CRISPR-Cas9 gene-cutting technology, which uses a target sequence of 20 base pairs, is more specific than classic restriction enzymes.
A sequence from a restriction enzyme is said to occur 89,753 times while a 20 nucleotide sequence cut by CRISPR will probably only occur once (0.0029)
Describe in complete sentences how you would identify a target DNA cleavage site for CRISPR-Cas9 and design an sgRNA
A target DNA cleavage site would have PAM sites -5' NGG flanking both sides of the target. To design an sgRNA, just choose a 20 nucleotide sequence that is complementary to the sequence you want to cut and then add the approximately 80 scaffold region that stays constant for most sgRNAs.
One of your plates may have few if any colonies on it. Write a claim supported by evidence from your results about why colonies did not grow. Include reasoning for why your evidence supports your claim.
Again, this is probably plate B because with these unrepaired double stranded breaks, it is unable to grow.
Describe how disrupting PCSK9 would impact gene expression.
By disrupting PCKS9, you would have more LDL receptors to clear out more LDL from the bloodstream thus reducing the risk of CAD
Describe how editing CFTR would impact gene expression.
By editing the CFTR gene you could, the F508del mutation would be fixed and the ion channels would function normally.
How might an exact alignment match be an off-target cut site?
CRISPR-Cas9 cuts all sequences that are an exact match even if it is off target.
For this gene-editing strategy(sickle cell), why is it useful to edit the DNA of only hematopoietic stem cells?
Editing just the hematopoietic stem cells guarantees that all future cells will have the desired gene edit for the sickle cells. Additionally, mature red blood cells do not contain DNA so you can exactly edit their genomes.
Where does Cas9 cut the target DNA relative to the protospacer sequence?
It cuts the DNA 3 nucleotides upstream of the PAM
Is this gene editing strategy for sickle anemia, an example of replacing, inserting, or deleting a sequence?
It is an example of replacing because you are taking the thymine and replacing it with adenine
Is this gene editing strategy (for CF) an example of replacing, inserting, or deleting a sequence?
It is an example of replacing, because the old sequence is cut and then repaired with inserted template DNA via homologous recombination.
Write out a complete equation to calculate the predicted occurrence of a sequence of n length within a DNA fragment of X length.
Occurrence: (1/4)^n * X
What health problems could arise from off-target CRISPR-Cas9 activity?
Off-target CRISPR-Cas9 could cause many unintended health problems, many of which we may not even understand. This is much about the noncoding regions of DNA that tend to be highly repetitive that we do not understand and could be off-target cuts. Additionally, each person has a unique code, and each person may have a different susceptibility to CRISPR off target effects.
Why might there be multiple results from a single accession number?
The BLAST database searches the entire database, so although a 23 nucleotide sequence is very specific, it may happen to have more repeats than other sequences. Additionally, the database finds matches that may not be a 100% match but that are related and still may be targeted.
Explain how the differences between the IX and IX/ARA starter plates may influence gene editing in the laboratory activity.
The IX plate does not have arabinose so it is unable to use its HDR in its plasmid. IX/ARA will be able to use its HDR because it has arabinose in the media. This will allow it use the DNA template in the pLZDonor or pLZDonorGuide.
What happens to a bacterium if a double-strand DNA break is not repaired?
The cells will die. See plate B.
