This week we looked at the process of designing a genetic modification in any gene of a mammalian or plant. Coming from a design background I found the week particularly difficult and had to quickly catch up on a lot of terminology including nucleotides, DNA polymerase, and plasmids among others. I attached some of the definitions I came across and helpful resources at the bottom of the page to help other designers/non-bio backgrounds understand the content as well!
To start the design process I first needed to pick a gene to modify. As a beginner, I chose LRP5 from Prof. Church's list of potential human genome modifications. Mutations in LRP5 can cause considerable changes in bone mass since it helps regulate bone mineral density. A loss of function mutation of this gene would cause osteoporosis while a gain-of-function mutation would increase bone mass. One potential negative to note is that extra strong bones could result in low buoyancy.
After selecting LRP5, I chose the genome editor CRISPR/Cas9. This editor requires a guide RNA and a Cas9 protein. To design the guide RNA and PAM site I used Benchling as recommended in class. I left the guide length at 20 and set the Cas9 protein to SpCas9 with a NGG PAM site to start. The results turned out quite promising!
LAB
For this week's lab we needed to determine our blood type from our DNA. To do this we first isolated DNA from saliva at home.
The materials needed are:
TA DROP-OFF!
Because of Covid 19 we don't have access to the lab. But thankfully we have awesome TA's to help us along! After extraction, I stopped by the MIT media lab to drop off my sample.
DNA QUANTIFICATION
DNA quantification was used to check the DNA concentration of the saliva samples. DNA absorbs UV light, and different nucleotides absorb light of different wavelengths but the overall peak is 260nm. We can check the purity by loading the sample to the Spectrophotometer machine and looking at the peak which should coincide with 260nm. Other peaks may signal impurities.
PCR
PCR stands for polymerase chain reaction and is a method used to rapidly make millions to billions of copies of a specific DNA sample. The process involves denaturation which breaks the strands in two followed by primer annealing where primers are incorporated to the solution to define the regions to copy and then taq polymerase kicks in! The taq polymerase extends the primers, synthesizing new strands of DNA.
GEL ELECTROPHORESIS
The image above shows the results of the PCR visualized through gel electrophoresis. Gel electrophoresis is a technique in which fragments of DNA are pulled through a gel matrix by an electric current and it separates DNA fragments according to size. The gel shows that the exon 6 amplicon is slightly shorter than the exon 7 amplicon which was expected. There are also no visible byproducts meaning the primers worked well and were specific to the regions of interest.
SANGER SEQUENCING
The PCR samples were then cleaned using NEB PCR and DNA clean-up kit and submitted for sanger sequencing. In Sanger Sequencing, the target DNA is copied many times, making fragments of different lengths. Fluorescent "chain terminator" nucleotides mark the ends of the fragments and allow the sequence to be determined. After the reaction is done, the fragments are run through a long thin tube containing a gel matrix in a process call capillary gel electrophoresis. As each fragment crosses the finish line at the end of the tube it is illuminated by a laser, allowing the attached dye to be detected.
RESULTS
Nucleosides -> molecules put together by cells before they are turned into Nucleotides. They include Adenosine, Cytidine, Guanosine, and Thymidine Nucleotides -> when a cell adds a phosphate to a nucleoside. Nucleotide molecules commonly abbreviated as A,C,G,T are the building blocks of DNA
DNA Polymerase -> a protein enzyme used by the cell to construct DNA by assembling nucleotides.
Plasmids -> genetic structure in a cell that can replicate independently of chromosomes.
Vector -> a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell where it can be replicated or expressed
Recombinant DNA (rDNA) -> DNA molecules formed by laboratory methods of genetic recombination that bring together genetic material from multiple sources.
Codon -> a group of three ribonucleotides in an RNA transcript. Anti-Codon -> the complement to the three ribonucleotides in the RNA transcript.
PAM -> a specific sequence of nucleotides, about 2-6 base pairs, that follows the DNA region targeted for cleavage by the CRISPR system.
gRNA -> a specific RNA sequence that recognizes the target DNA region of interest and directs the Cas nuclease there for editing.
NHEJ Repair -> the most common pathway to fix a double strand break, however it is not very precise
PCR -> stands for polymerase chain reaction and is a method used to rapidly make millions to billions of copies of a specific DNA sample. It relies on using the ability of taq polymerase to synthesize new strand of DNA complementary to the offered template strand.
Pahara, Justin, and Julie Legault. Zero to Genetic Engineering Hero: the Beginner's Guide to Programming Bacteria at Home, School & in the Makerspace. 2019.
To extract the DNA, you rub your tongue along your gums and spit into a marked glass container until you get about 1.5 teaspoons of Saliva. This is surprisingly hard! Afterwards you add one drop of dish washing soap which breaks open the cells that are in the saliva and releases DNA. Next, add a tiny pinch of salt. This encourages the neg charged DNA molecules to bond into sodium DNA clumps.
To collect the DNA, carefully add cold alcohol and then a cloudy thin layer forms between the saliva and alcohol. The cloud is the DNA! I didn't quite get a cloud, but I saw the formation of long string like material. I collected this thread with a toothpick and packed it to drop off later for analysis.
For detailed instructions to try this at home check HERE
TERMS AND REFERENCES
After sanger sequencing we received the results and used Benchling's sequence alignment tool to locate the nucleotides at specific locations which would determine our blood type.
The diagram on the left shows the exons of the ABO gene and in order to determine the blood type you look at two of them: exon 6 and exon 7. At exon 6, some people have a small deletion. This was not present in my sample. Then in exon 7 you look at the nucleotides at three positions nt 467, nt 703, and nt 1096 to determine the Blood type.
By using Benchling's sequence alignments I was able to determine that the nucleotides present at those locations for my sequence are C,A, and A meaning that I am blood type B!
DNA CLUMP!
BENCHLING RESULTS
After inputting the parameters, I examined the first Exon and sorted the gRNA sequences by Off-Target Score and then looked at the corresponding On-Target Score. Immediately I was surprised by the Off-Target scores. Many of them were above 50. The sequence I ended up selecting did not have the highest off target score, but was 3rd highest and also corresponded with a promising On-Target Score over 50.