Question: Present a question. What is the question that you want to answer? (for
example: is it possible to introduce wt CFTR sequence into the mutant CFTR allele and
have it function as CFTR is predicted to?).
2. Introduction: Describe background of gene/condition, etc. What does your audience
need to know about this? What causes it? What is the specific sequence change . How many people are affected? What are the effects? Why is this an
important question to address? [If you are proposing a novel creation, such as GFPkitties, please instead describe why this would be a useful or profitable venture, and
how it is similar and different from previous items on the market, like “GloFish”]. Please
be sure to cite sources to describe the background information, including info about the
genes, the role of the genes to be targeted, overview of disease or condition resulting
from mutant alleles (if applicable), or an overview of the function of the genes to be
altered/fused (if applicable).
3. Proposed Strategy: How will you address this? Please be specific. What will the
constructs look like? Please describe your strategy for creating the sgRNA construct and
the donor DNA construct, and please provide a diagram that includes the relevant
sequence. In your diagram, please show: target sequence, PAM, sgRNA sequence,
where mutation occurs (if applicable), where cut site/s is/are. If creating a fusion
protein, please show in your diagram where your inserted gene will go. Also, please
show what the sequence of your donor DNA is (no need to include entire gene sequence
for GFP or other inserted protein for fusion protein projects, just indicate the gene with
a labeled rectangle (as in the lab protocols), with the relevant homologous sequence.
4. Delivery: Please comment on how you would deliver the components needed for
CRISPR to happen, and to which cell population within the organism. Our Moodle site
has a review paper that describes different ways of delivering CRISPR components that
you may find helpful.
5. Results: Please describe how you would determine if CRISPR was successful. Please
describe what the phenotypic effects of the CRISPR would be on the patient or
organism, and also, describe how you would confirm with PCR that the gene edit was
successful.
