Gene Editing is Now… Easy?

Nikhita Srinivas
9 min readFeb 1, 2021

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Human bodies are incredible. They are made of an average of 37.2 trillion cells. Each one of those cells has an estimated 30,000 to 120,000 genes and that is what makes us who we are. The craziest thing is that we can edit those genes. We can literally modify the fundamental block of life.

Now this might sound like sci-fi or something out of a movie but it is surprisingly simple. It is actually simple enough that a high schooler can design their own experiment and that’s exactly what I did!

The Basics

Genes are essentially segments of DNA. DNA contains the recipe or code that is used to create proteins that make our bodies functions. Each DNA molecule is in the shape of a double helix (think of a spiral staircase that has hundreds of millions of steps). Each step of the staircase has 1 of 4 different molecules called nucleotide bases. Those 4 bases are Adenine (A), Thymine (T), Guanine (G), and Cytosine (C). The bases are arranged in groups of 3. Each group codes for different instructions. For example, GTT (Guanine, Thymine, Thymine) codes for something different from GCT (Guanine, Cytosine, Thymine).

DNA can also be complementary to each other. Thymine and Adenine are complementary pairs, and Guanine and Cytosine are complementary to each other. For example, the complementary DNA sequence of AGTGGC would be TCACCG. ← without this ability, gene editing wouldn’t be possible

Our genotype (or genome) is our special combination of genes. Our special combination of A, T, G, and C. That combination of genes is unique to you and it is what makes you, you! Our phenotype is the way those genes physical show themselves. For example your genotype might be not having the gene for brown eyes and your phenotype would be your eyes appearing blue. Basically, a change in the genome can alter the phenotype and result in physical changes.

There are different types of cells in a person’s body. There are heart cells, liver cells, muscle cells, and many more. These cells all look different, and they do different things so you would assume that they had a different genotype since they are not physically the same. However, each cell in our body contains pretty much the same DNA. The reason why they look different and do different things is because different genes are expressed in different cells.

In fact, overall, only 1% of our genes are expressed, which is crazy to think about!

It’s like having a huge encyclopedia but there’s only one coherent sentence every 100 pages. In between those individual sentences, there’s random, unreadable, and seemingly unusable letters and shapes. You would probably be confused and wonder why those random characters were there in the first place. That encyclopedia is similar to the human genome.

So why is so much of the human genome not being used?

In order for a gene to be expressed, different sequences of DNA have to be present and in a specific order. Those other sequences are:

  1. Coding sequences- This is the part of the gene that actually codes for proteins. It contains introns and exons. Introns are not expressed, and exons are expressed. Exons are the 1%. They are that one sentence in the encyclopedia.
  2. Promoters- These are highly specific sequences of DNA and these happen before the coding gene. Without the promoter sequence, the gene cannot be expressed.
  3. Transcription Initiation Sites- The site is also called the 5’ UTR (5 prime UTR) for “Un- Translated Region” which means that none of that DNA is part of the coding section. This comes right before the coding DNA sequence.
  4. Transcription Stop Sites- This region of the gene happens after the coding sequence and is also called the 3’UTR. The actual coding sequence lies between the 5’UTR and the 3’UTR.

This all comes together with gene editing. Now, we can insert and delete genes. We can knock in genes which expresses them, and we can knockout genes which stops their expression completely. We can meddle with DNA, and edit humans at the most basic level by changing the genotype and the subsequent phenotype! 🤯

The most popular way to do this is by using CRISPR Cas9.

Overview of CRISPR Cas9

CRISPR is an acronym. It stands for Clustered Regularly Interspaced Short Palindromic Repeats. It is the most popular form of gene editing because it is relatively simple. It only has 2 main components: a guide RNA (gRNA) and a CRISPR associated protein (Cas).

**ok what does any of that mean?

  1. RNA is a molecule that is extremely similar to DNA. There are some differences, however. Unlike DNA, RNA is single stranded. They are both made of 4 nucleotide bases but even here there are differences. Instead of Thymine, RNA contains Uracil (U). There are different types of RNA in the cell. They include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and guide RNA (gRNA). An important thing to note is that an RNA strand can also be complementary to a DNA strand.
  2. Another thing to note is what an enzyme it. An enzyme is a biological catalyst which means it speeds up reactions and processes in cells. In this case, the main enzyme used is a nuclease. This enzyme speeds up the process of cutting DNA.

Now… back to CRISPR!

The Guide RNA (gRNA) is a specific RNA sequence that finds the target DNA region in the genome and leads the Cas nuclease there for editing. The Guide RNA itself it made of 2 parts: crispr RNA and tracr RNA

  • CRISPR RNA (crRNA) is a 17–20 nucleotide sequence that is complementary to the target DNA. This is how the gRNA finds the target DNA region… it just searches for its match!
  • TRACR RNA is what binds the Cas nuclease to the DNA.

The CRISPR associated nuclease (Cas) is a non- specific endonuclease (a fancy word for a type of enzyme that cuts DNA). There are many different versions of the Cas nuclease but Cas9 is the most widely known. After it is led to the specific DNA locus (location) by the gRNA, it makes a double stranded break. That means it cuts through both strands of DNA.

However, the Cas nuclease will only bind to the target sequence if there is a specific sequence present. That sequence is called the Protospacer Adjacent Motif or PAM. Therefore, scientists can only target locations that contain PAM sequences which limits a lot of options for gene editing.

In the cell, the double stranded break is then fixed by non-homologous end joining (NHEJ). NHEJ is one of the cell’s major repair pathways which kicks in when a break in DNA occurs. However, this process is error prone and often results in insertions or deletions of extra nucleotides at the break. This leads to mutations that can completely knockout the gene’s function (stop gene expression).

There’s no one size fits all for CRISPR Cas9. Each genome and specific gene we want to edit requires its own specially designed CRISPR Cas9 complex.

How do we create one?

This really interested me. Knowing how CRISPR works is one thing but being able to design a Cas9 molecule that could actual edit DNA makes everything suddenly become real.

When designing a CRISPR experiment, the crRNA part of the gRNA is the component that changes and allows customization but when actually designing a CRISPR complex, sgRNAs are extremely useful.

sgRNA stands for single guide RNA and it is a single RNA molecule that contains both the custom designed crRNA sequence and the tracrRNA sequence. Scientists can either use a crRNA and a tracrRNA separately OR they can put them together and just use a sgRNA. They are much easier, and are used a lot more.

The easiest way to design an sgRNA is by using software tools.. The most popular tools are:

· Synthego design tool

· Desktop genetics

· Benchling

I decided to use Benchling.

In Benchling, the first thing I had to decide was which gene I wanted to edit. I knew I wanted to edit a human gene and I decided on the human SCO1 gene.

Benchling is really great because when you import the gene, it automatically annotates it for all editable exons, and you can also import different versions of the gene on top of each other, so you have a better understanding of the best places to cut.

The next step for me was deciding which exon to edit.

On Benchling’s linear map, it shows the gene and the different exons I can edit. I decided on the 2nd exon. I wanted to knockout the entire gene so I knew I had to cut towards the beginning so it would completely stop gene expression. I didn’t want to pick the 1st exon because it wasn’t included in all forms of the gene and I wanted to maximize the efficiency of the sgRNA.

After I picked the exon, Benchling automatically created a list of potential guide RNA sequences I could use.

Before 2016, I would have to analyze all of the experimental conditions and even then, there was a huge chance that I would create a CRISPR guide that targeted the wrong exon. BUT luckily, in 2016, the Doench Study analyzed thousands of guide RNAs, and created design and scoring rules for picking the right exon. They even compiled all the information in human and mouse genome libraries, so they are easy to access! The rules predict the target accuracy of different gRNAs so you can pick the best one!

The study also came up with off- target and on- target activity scores. The off- target scores tell the inverse probability of the Cas9 complex not binding to the right location (so the higher the better). The on- target score is the cleavage efficiency of Cas9. This basically means the score represents how efficient the complex is at actually cutting the DNA.

Along with the list of potential guide RNA sequences, Benchling also provided the on and off target scores for each sequence. I sorted the exons from highest to lowest on- target activity scores and picked the ones with the highest. In general, it is recommended to select at least two different sequences, so you minimize the chance of completely missing the gene. I chose the strands at positions 2583 and 2598.

Now I had designed the sgRNA that I needed to create a CRISPR experiment. The next step would be to send to it a lab and let them actually create it. There are two main ways to do this: synthetically generating the sgRNA or making the guide using a DNA template. Even with DNA templates, there are multiple ways to create the guides.

1. Plasmid- Expressed sgRNA

In this method, the sgRNA is copied in a plasmid vector which is put into the cells. Plasmid vectors are small, circular, DNA molecules that hold DNA fragments or genes. The cells use their RNA polymerase enzyme to generate the sgRNA. RNA polymerase is an enzyme that creates the complementary strand of RNA from a DNA template.

Cloning the guide RNA plasmid normally requires around 1–2 weeks of lab time before the actual experiment.

2. In Vitro- Transcribed sgRNA

Another method for making sgRNA involves creating the sgRNA outside of the cell. A DNA template is designed that contains the guide sequence and an RNA polymerase. Once inside the body, the RNA polymerase creates the sgRNA.

This method only requires 1–3 days but is prone to errors and normally needs purification before it can be used in experiments.

CRISPR Cas9 in its simplicity and efficiency has revolutionized gene editing. Newer softwares like Synthego and Benchling allow not just researchers, but high schoolers like me to design experiments fast and with ease. It is a simple way to visualize and learn more about the gene editing process and it continues to push the industry faster into the future!Sources

Hi! I hope you learned something and enjoyed reading this article! If you would like to connect, please reach out to me on Linkedin!

Talk to you soon :)

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Nikhita Srinivas

17 Years Old- Passionate about FemTech and Gene Editing- TKS