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CRISPR Project Explained
CRISPR Project Explained
CRISPR Project
The end goal of this project is to procure and grow a cell line of Calu-3 (lung epithelial cells). Once grown to 70-90% confluency, the cells will be transfected (introduce DNA into eukaryotic cells) with both a plasmid containing sequences that encode proteins and RNA used for "prime editing" (CRISPR 3.0). The basics of prime editing and what it can accomplish in regards to editing DNA can be found here. The plasmid will also contain an eGFP sequence adjacent to the sequence for the CRISPR proteins, allowing the CRISPR proteins to have an eGFP tag when expressed in the cell. Once the transfection process is complete, the cell lines will be viewed under a fluorescent microscope in room 515. The presence or absence of fluorescence will suggest whether or not the CRISPR proteins were expressed in the cell. DNA isolation and sequencing techniques before and after transfection will be used to study the incorporatino of an edit in the cell's genome.
The importance of the Cas9 gene in gene editing can be found online, but the overall idea is as follows. Cas9 is a DNA sequence found in many species of bacteria. This sequence codes for the synthesis of a protein that can identify and cleave foreign DNA. With this power, the possibility of using the proteins produced from the Cas9 gene to cut a mutated CFTR gene away and replace it with the correct CFTR gene becomes relevant.
Because the Calu-3 cells will be obtained from a lab in Columbia, Missouri that Dr. Norimatsu has connections with, we are first exploring this transfection process with cells that are available locally. An ATSU student of Dr. Baer’s lab currently cultures/grows melanoma cell lines. He has been generous enough to donate some of these cells to us. Thus, we will be attempting our first transfection(s) using these cells. The following tasks are what have been completed thus far in preparation for the transfection.
On 1/16/19, a plasmid containing a sequence of Cas9 and eGFP was ordered by Tina. This plasmid arrived on 1/28/19, and came with the plasmid DNA incorporated into E. Coli cells. These cells were contained in an agar stab.
On 1/29/19, Gabe transferred the cells contained in the agar stab onto a plate via quadrant streaking technique. The streak was successful and individual colonies formed on the plate. The plate has since been placed in Fridge B.
To prepare the control of this experiment, Macey transformed JM109 cells with the eGFP Plasmid obtained from Dr. Cox’s lab on 1/30/19. The transformation was successful and individual colonies formed on the plate. The plate has since been placed in Fridge B.
On 2/4/19, Tina and Gabe completed two separate single colony inoculations, one from the Cas9 + eGFP Plate and the other from the eGFP Plate. These inoculations have been placed in the shaker incubator and will incubate overnight at 37C and 200 RPM. Macey and Julia will perform a Maxiprep on 2/5/19 after the incubation period is complete.
On 2/12/19, Noah and Gabe ran a gel with both the eGFP Plasmid and the Cas9+eGFP Plasmid that had recently undergone Maxiprep. The gel results suggested a good presence of the plasmid DNA, confirmed by Dr. Norimastu on 2/18/19.
A list of transfection reagents as well as media needed for the growth and culturing of the melanoma cells has been made and is being modified based on advice provided by Aaron. This list can be found in the CRISPR folder on the google drive.
This is the transfection protocol that is used by Aaron. We will use it as well:
https://assets.fishersci.com/TFS-Assets/LSG/manuals/lipofectamine3000_protocol.pdf
To-do:
Perform a Nanodrop on both the eGFP and Cas9 + eGFP DNA
Order all cell growth media and transfection reagents needed
Grow and passage melanoma cells to correct confluency
Transfect melanoma cells
View transfected melanoma cells under fluorescent microscope
Continue to put all updates on slack and in the notebook on the Google Drive. This document is simply here to provide context to everyone.
Prime Editing
Prime Editing
Seeing as how there are >75,000 genetic variants that result in diseases like Cystic Fibrosis, the experimentation of genome editing technology has drastically increased in recent years. Techniques like the original CRISPR-Cas9 function by inducing a double strand break in the DNA, which allows for errors since indels (inserts and deletions) are prone to be added at the sites of editing. Base editing can avoid making double stand breaks in DNA, but is severely limited in the edits that can be made (can only carry out C→T, G→A, A→G, and T→C). Prime Editing is new technology synthesized by Liu et. al and described in their 2019 paper that is able to carry out multiple types of mutation and editing without creating double strand breaks; it is described as a "'search-and-rescue' genome editing technology that mediates target insertions, deletions, all 12 possible base-to-base conversions, and combinations thereof in human cells without requiring DSBs or donor DNA templates."
The original prime editing technology, called PE1, is made of a prime editing complex. This complex consists of a Cas9 nickase domain coupled with a reverse transcriptase domain, and a guide RNA specifically referred to as pegRNA. Addgene's Blog provides a simplified explanation of the mechanism of prime editing:
First, an engineered prime editing guide RNA (pegRNA) that both specifies the target site and contains the desired edit(s) engages the prime editor protein. This primer editor protein consists of a Cas9 nickase fused to a reverse transcriptase. The Cas9 nickase part of the protein is guided to the DNA target site by the pegRNA. After nicking by Cas9, the reverse transcriptase domain uses the pegRNA to template reverse transcription of the desired edit, directly polymerizing DNA onto the nicked target DNA strand. The edited DNA strand replaces the original DNA strand, creating a heteroduplex containing one edited strand and one unedited strand. Lastly, the editor guides resolution of the heteroduplex to favor copying the edit onto the unedited strand, completing the process.
A great video that helps explain how prime editing works can be found here.
Design CRIPSR 3.0/Prime Editing Plasmids
Calu-3 Cells
Calu-3 Cells
What is Calu - 3?
Calu - 3 is a human lung epithelial cancer cell.
What is Cas - 9 Protein?
Cas - 9 Protein is an enzyme that naturally occurs in some bacteria of the streptococcus species. In these bacteria, it acts in immune defense by recognizing foreign DNA sequences and splicing them to remove them from the cell’s genetic makeup. Aside from immunity, Cas - 9 can be used to remove or inactivate unwanted genes.
What is Guide RNA?
Guide RNA is a sequence that directs Cas-9 to a corresponding sequence to be deleted once it is transfected into the cell
What is Transfection?
Transfection entails introducing nucleic acids or an entire protein to a eukaryotic cell.
What is a Lipofectamine?
Lipofectamine is a transfection reagent that facilitates the process of inserting a desired protein into a cell.
Calu - 3 Spotlight & Relevance
Calu - 3 Cells have large amounts of CFTR membrane proteins and chloride channels within their cells, and thus contain the CFTR gene that codes for these relevant Cystic Fibrosis Proteins. The overall goal of the CRSPR project is to procure Calu - 3 Cells to freeze and grow. After growing the cells, they will be transfected with Cas - 9 and Green Fluorescent Protein (GFP). Transfection will be carried out using lipofectamine and Opti-MEM medium for optimal efficiency. Once transfected, we will look, through a microscope, for GFP expression (denoted by glowing green residue). The amount of GFP expression will suggest directly how much Cas - 9 is also expressed in that cell. With enough of a percentage of Cas - 9 expression, the cell may have the ability to remove or inactivate unwanted/defective genes, such as a mutated CFTR gene. We cure Cystic Fibrosis. Boom.
Growing Calu - 3 Cells
Because Calu - 3 Cells are cancerous in nature, they have the ability to divide indefinitely, and their abundance is therefore useful when cultured. In our case, the CFTR genes are of interest.
Culturing Calu - 3 Cells
Using in vitro methods, Calu - 3 Cells have been cultured under the following conditions and medium:
Temperature - 37C
5% CO2 & 95% O2
Media: Eagle’s Minimal Essential Medium (EMEM) OR Dulbecco’s Modified Eagle’s Medium (DMEM) OR Opti-MEM
EMEM - Culture containing amino acids, salts, glucose, and vitamins that is used to maintain cells in tissue culture
DMEM - Contains about 4x as much of vitamins, amino acids, and glucose. It contains iron and phenol red
Opti-MEM - Contains insulin, transferrin, hypoxanthine, thymidine, and trace elements. This medium contains lower serum levels. Serum can lower the efficacy of transfection, so this medium is great for transfection.
Transepithelial Resistance (TER) - The measure of electrical resistance across the monolayer of epithelial cells. The electrical resistance measured is directly proportional to the membrane’s integrity.
The general trend of Calu - 3 Cell culturing technique seems to be as follows:
1. Eagle's Minimum Essential Medium (EMEM) and/or Dulbecco's Modified Eagle's Medium (DMEM)
*Most cultures that used DMEM also used Ham's F-12
2. 7-15% Fetal Calf Serum (FCS)
3. Varying amounts of Penicillin and Streptomycin
4. 5% CO2 and 95% O2
5. Media contained in either transwell plates or tissue culture flasks
6. 37C
7. Split when cells are somewhere between 80-95% confluent
Sources
https://www.hindawi.com/journals/ccrp/2010/394578/
http://www.pnas.org/content/pnas/suppl/2011/08/31/1110144108.DCSupplemental/sapp.pdf
https://www.atcc.org/products/all/HTB-55.aspx#culturemethod
http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.875.888&rep=rep1&type=pdf
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3515819/?tool=pmcentrez
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4025061/?tool=pmcentrez
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2013912/?tool=pmcentrez
This article has extra information about transfection of the cells using Fugene 6 as the transfectant
Shelf Lives:
Shelf life of EMEM by itself stored at 2-8C is 12 months. Shelf life of EMEM/Fetal Bovine Serum Soln. is 3-4 weeks stored at 2-8C. Trypsin-EDTA and any other media such as Fetal Bovine Serum by itself stored at -20C or lower is good for 5 years.
Cryovials:
TPP and NUNC brands
GuideRNA and Cas - 9
CRISPR Project (Slideshow)
CRISPR Project (Slideshow)
https://docs.google.com/presentation/d/1vsTNHxZwGmSpG78nobxZj6yWaumHFj6aSYR7_dEE2Gk/edit#slide=id.p
Transfection (Slideshow)
Transfection (Slideshow)
https://docs.google.com/presentation/d/1SSegAk58qRUBvIUHPTFYpCrVuZ8wULBl98upVpg16OI/edit#slide=id.p
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