Using Gene KO to Engineer Stem Cell Lines — What’s the big deal?

Stem cells have huge potential.

I’m 99.87% sure you probably already knew that.

But, did you know that we can also engineer stem cells?

I really hope you didn’t, but even if you do, keep reading. 😉

Before we dive into the juicy stuff, let’s make sure we’re all on the same page.

Yes, I made that abbreviation up. 😉

Q: I know what stem cells are, but what are stem cell lines?

A: Stem cell lines are a group of stem cells that are cultured in vitro. They can be used for several different purposes and in different fields.

Q: Why are they so important? What can they be used for?

They are used widely in regenerative medicine, and can be used to study early human development. They can also be used in different therapies to replace damaged or diseased cells and tissues.

Q: What does ‘Gene KO’ mean?

A: KO stands for ‘knockout’. So gene knockout, is essentially making a gene in an organism inactive and lose its function through gene manipulation.

Q: Why is this important?

A: Being able to perform gene knockout allows for scientists to learn about the function of different genes and their purpose.

Q: How do we perform gene KO?

A: There are many different methods, and an overall process of how we can go about doing this. Some of these methods include conditional knockout, homologous recombination, gene editing and more. I’ll go into more-depth of the general process of how this all works a little later in this article.

Q: What is sgRNA?

A: This stands for single-guide RNA molecule. Guide RNAs guide the insertion or deletion of a target region target DNA region of interest and directs the Cas nuclease there for editing.

Q: What is recombinase?

A: An enzyme that promotes genetic recombination. Recombination in genetics is the rearrangement of genetic material, especially by crossing over in chromosomes or by the artificial joining of segments of DNA from different organisms.

Some abbreviations to note:

hPSC = Human pluripotent stem cell

iKO = induced gene knockout

Let’s dive in :)

Today in science, we are able to express and delete certain genes. This is a huge tool as it allows us to figure out the function of each of these genes in biology. This is essentially what gene knockout does. By being able to make certain genes inactive, it allows scientists to figure out how a system will work without it, thus allowing them to distinguish its function.

Engineering Human stem cell lines with inducible gene knockout using CRISPR/Cas9

Woah…I know, that was a lot of words.

Making hPSC lines with inducible gene knockout is challenging, but a few scientists teamed together, and as always, something magical happened 🦄

They tried to achieve this by combining CRISPR/Cas9 software, and both the Cre/LoxP and Flp/FRT system.

The Flp protein (like Cre), is a tyrosine family site-specific recombinase. This specific family of recombinases performs its function through a type IB topoisomerase mechanism causing the recombination of two separate strands of DNA. Recombination is carried out by a repeated two-step process.

Flp protein

FRT sites are mostly used for inducing deletions or “flipping out” of markers in transgenic constructs. When there are two FRT sequences behind one another, the DNA sequence that is between these two sites will be deleted after inducing flippase.

Cre recombinase is a tyrosine recombinase enzyme derived from the P1 bacteriophage. The enzyme is a member of the integrase family of site specific recombinase. It is known to catalyze the site specific recombination event between two DNA recognition sites, which are known as LoxP sites.

Cre recombinase

Through this combination of both softwares and CRISPR/Cas9 they found that dual-sgRNA targeting is needed for biallelic knock-in of FRT sequences to surround the exon.

The exon is a segment of a DNA or RNA molecule containing information coding for a protein or peptide sequence.

They then developed a strategy to simultaneously insert an activity-controllable recombinase-expressing cassette and remove the drug-resistance gene, which would speed up the creation of iKO hPSC lines.

Gene cassettes are small, discrete mobile elements. A gene cassette generally comprises a single gene and a downstream 59-base element, which is a recombination site.

Applications of this experiment

This was used to establish hESC (human embryonic stem cell) lines and iPSC (induced pluripotent stem cell) lines with induced gene knockout of SOX2, PAX6, OTX2 and AGO2.

These 4 genes exhibit diverse structural layout and temporal expression patterns. The availability of iKO hPSC lines will substantially transform the way we examine gene function in human cells.

If you enjoyed this article (or would like to chat more about induced gene knockout 😉) please connect with me on LinkedIn! For more information and for any questions you may have, send an email to christina.a.vadivelu@gmail.com!

I’d also love it if you followed me on Medium 😁!

On a path to impact billions. Yeah, billions.

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