Building a Guide RNA for CRISPR as a 16-year-old
By: Ali Haider
Tags: Genetics, Science, Biology, Youth, Technology

One of the most challenging parts of succeeding in the STEM field for young aspirers is how we can prove ourselves. How can we get the more powerful people in the fields’ attention?

For the past few months, I have been trying to earn an internship or opportunity to work in a lab to develop my skills with gene editing. But because of COVID-19 that has been quite difficult.

I knew that I needed an alternative to these options and came across….

AT HOME KITS

which are quite expensive and take a long time to arrive.

I decided to keep researching and I came across Benchling.

Benchling main page

Benchling is a fantastic application that is a very popular program for people interested in the field of biology. They focus on allowing people to have the same access to simulation and understanding without access to a lab.

There are two reasons I decided to do my experiments on this website instead of spending money and waiting for a kit.

1. Ability to View and Edit Plasmids

If you’re wondering what plasmids are, they’re basically disk-like shapes of DNA.

The ability to view and edit plasmids is something that allows me to explore different types of plasmids and how I can change them using different things such as restriction enzymes.

2. Ability to View and Edit DNA sequences

The next big thing that really caught my eye was the ability to view and edit plasmids that can lead to producing CRISPR guide RNAs.

My First Project

This leads me to the first completed experiment I did with Benchling, editing a yeast plasmid to incorporate green fluorescent protein (GFP). This means that the bacteria these plasmids form would glow green.

I managed to complete this project using something very underrated in the field of biology called restriction enzymes.

Now, I didn’t have any special training or knowledge to complete this project.

Instead of looking to start these projects on my own, I looked for tutorials on YouTube. For this project, I based it on this tutorial by Standford Biome:

Stanford Biome video

Let’s go through the process of how I was able to do this.

Found a template of yeast plasmid to use. I chose the template pxP420 which can be found on addgene.org

found from addgene.org

2. Found a restriction site that is considered a one cut enzyme. This means that it can be removed with a single cut from the plasmid. The restriction site I chose is called SAC I.

*An enzyme is a catalyst for chemical reactions in biology (GFP is also an enzyme which is why we switch it out for another)

shows the restriction site I cut out

3. Next, I added in TEF1 promoter that starts the transcription process of DNA →mRNA which needs to happen since GFP is considered an mRNA.

shows the TEF1 promoter

4. After this, I added in the Kozak sequence that allows for the process of translation (changing DNA into protein) to occur. GFP is a protein so it is essential we need a Kozak sequence to allow the plasmid to understand the GFP.

Kozak Sequence

5. GFP is added into the Plasmid. It was important that this was a eukaryotic green fluorescent protein as yeast is made of eukaryotic cells.

GFP in the plasmid

6. After adding in the GFP, we need to stop the translation process. How can we do this? By adding in the stop codon. This is quite easy to add in as it only 3 nucleotides long: TGA along with its corresponding nucleotides to form DNA.

stop codon is seen here

7. We stopped the translation process, but now we need to stop the transcription process. We can do this by adding in a terminator sequence. A terminator sequence is already found in this plasmid, so it was easy to copy this into after the stop codon. It is called the CYC1 Terminator.

CYC1 Terminator

8. Finally, in order to allow for polymers to occur (which GFP is since it is a protein) it is essential to add in an auxotrophic selection marker. This part wasn’t provided in the tutorial in Stanford Biome, but I managed to find a common marker found in yeast plasmids. This is called URA 3.

where URA3 is found

This is the final result

My Second Project

For this second project, I spent time building a CRISPR guide RNA. Before diving deeper into gene editing these past few weeks, I never knew it was possible for a 16-year-old like myself to be able to do this.

This is the process I went through in order to be able to create the guide RNA.

Again, this project was based on another YouTube video.

Video this experiment is based off of

1. The first thing I did was I found a human gene I can edit. This human gene is called SCO1.

SCO1 and the enzymes found in it

2. The next thing I did was I chose an exon to edit. An exon is basically the part of a gene with coding information that allows that part to be transformed into protein. I ended up choosing exon 2. Let’s dive a little deeper into the process of how I chose this exon that I did.

The tutorial that I followed wanted to cut out the entire gene with the sequence they will use for CRISPR. This means the sequence has to be early on in the gene so that it can destroy it.

We cannot choose a sequence from the 5 UTR region (really early on in the gene)

To have the most effective approach we can’t focus on the first exon as it won’t edit the next exon because it comes before it.

Here you can see the second exon we focused on. If I chose the 4 transcript area before it, the next transcript (one on the top) I’m currently highlighting wouldn’t be affected.

3. The next thing I did was I asked Benchling to create a list of potential guide RNAs I can use in CRISPR.

the list of potential guide RNAs I can use

When looking at this chart you see a few things. What are these?

Position: Where the sequence are located in the gene

Strand: Positive means it can be transcribed into a protein, negative means it cannot

Sequence: Nucleotide sequence of the strand

PAM: PAM sequence is something very important in CRISPR. It indicates to the CRISPR mechanism the strand it is looking for as it will have a special three-nucleotide sequence after it. This also ensures the protein used by CRISPR doesn’t cut out the crRNA.

On-Target Score: Efficiency of the strand being used as guide RNA based on how it will perform through the on-target cut.

Off-Target Score: Efficiency of the strand being used as guide RNA based on how it will perform through off-target potential issues.

4. Finally, I looked for the best guides I can use based on the on-target score and off-target score and whether the sequence was found where transcripts are.

Visual Image on the sequence map of the sequence I chose to use as guide RNA

The sequence I chose is found in position 2 583 in the gene.

5. All in all, this would be the crRNA that would form the gRNA when combined with tracrRNA. I could use the crRNA I created and send it to a lab to get a copy of the CRISPR mechanism I would create with this.

For a better Visual Look of how I did these projects check out my two latest YouTube Videos:

First experiment video

Second Experiment Video

Thanks for reading!