CRISPR 101

It’s about time you learned the full capabilities of CRISPR with a focus on Cas 9

All the tech we make, everything, will eventually lead us to a world of biological enhancement. Where coding for genetic traits is as simple as coding a website.

AI and Quantum computing for drug discovery, simulations, and rapid detection of cures. IoT devices to extract biological information.

And most recently, the discovery of a fast and low-cost gene-editing tool in CRISPR. You likely know it as a complex that can cut a targeted site in our DNA. And that’s what somebody did…

He Juinki edited 2 copies of the CCR5 gene in one girl and only one copy in another girl. Hoping, these girls have a low chance of developing HIV.

Aside from clear consent, forged medical records and a global consensus on the actions taken, there’s a bigger problem…

We don’t know the full effects of editing a gene. Are you more at risk for a certain disease now? If HIV is a complex disease, caused by multiple genes, will it even solve the problem?😔

CCR5 is also related to brain function, meaning He may have made especially intelligent human beings🧠! At the same time, the girls may be at larger risk for the West Nile virus and severe flu.

We have constantly been moving towards human enhancement, so will this be accepted one day? Still, this is nowhere near the full capabilities of CRISPR. 🤯 That makes me more scared.

CRISPR was not Developed it was Discovered

Bacteria have been able to defend against viruses for over centries. Why? CRISPR. And only in 1987 did we discover these clustered DNA repeats.

1. After the viral DNA is inserted into the bacteria, Cas 1 and Cas 2 will identify the new DNA and cut out a specific sequence from that DNA (protospacer).
2. That is inserted into the CRISPR sequence/array, where a variety of viral DNA is collected and separated by spacers (think of papers with writing as the viral DNA, and in between each of those papers is a blank paper).
3. If that viral DNA appears again a cRNA (CRISPR RNA) is transcribed from the sequence. It will then pair with tracrRNA. This complex will locate the viral DNA and Cas 9 will cut it out.

• cRNA + tracrRNA = Guide RNA which contains a complementary sequence (the DNA pairs match up) to the targeted DNA
• Cas 9 Endonucelase= Protein that cuts DNA

Protospacer Adjacent Motif (PAM)

What’s stopping CRISPR from just cutting the target DNA in the CRISPR array? PAM.

When Cas 1 and Cas 2 store the viral DNA in a CRISPR array, they actually start looking for a PAM, a DNA sequence of 2–6 base pairs downstream (right-left or 3' — 5') the target DNA.

Here, the PAM sequence is NGG, where N can be any base.

CRISPR will only make the cut if it recognizes the PAM array (usually 3–4 bases). Lucky for us, the PAM sequence in a CRISPR sequence is “GTT”.

Sometimes the target sequence may not have this “NGG” PAM. This is why there are different Cas endonucleases. The common Cas 9, Streptococcus Pyogenes, reads the PAM “NGG”, which forms spCas 9. SaCas and NmeCas also recognize different PAMs.

We don’t want to be limited to what genes we can edit due to a PAM sequence. Although there are many Cas endonucleases, there is not one for every PAM sequence that is also short like NGG. That's why researchers have looked into xCas9, which can recognize a broad range of PAM sequences.

Types of CRISPR Cas 9

Cas 9 contains two molecular scissors, RuvC and HNH enzymes, which perform the cut on the desired sequence. We can edit and disable these enzymes to form different types of Cas 9 with different functions. ✂️

Cas 9 Wild Type: The one You’re Familiar With

We design a guide RNA (gRNA) with a cRNA and tracrRNA complex, rather than the organism. The guide sequence, with the help of PAM, brings us directly to the gene of interest.

Both molecular scissors are active, and therefore cleave both strands of the DNA.

After the double-stranded break (DSB), two things can happen:

1. Non-Homologous end joining (NHEJ) — The DNA tries to repair itself even though there are no DNA bases to make a pair. This results in random mutations that can get quite… well, wild.
2. Homologous Directed Repair (HDR) — We can control the repair process by including a repair template, allowing for more control.

Applications

Gene Knockout is the largest benefit of Wild Type Cas 9. When NHEJ occurs, the cell will have a hard time repairing the DNA often leading to the inactivation of that gene.

Imagine there was a gene that did no good (fairly unlikely), you could just knock it out.🤯🤯

Destroying a gene may not sound the best, but it helps us understand that genes function. How may it be related to certain traits or diseases? A gene will remain a variable until we can isolate it, just like in math.

Cas 9 Nickase

Here, we can either disable the HNH or RuvC enzyme so that only one strand of DNA is cut. The rest of the process involving recognition stays the same.

Disabling the HNH enzyme cleaves the complementary DNA to the RNA. Whereas RuvC cleaves the non-complementary strand.

The paired Cas 9 Nickases performs a PAM-in cut where the PAMs are side by side. A PAM-out cut would have the targeted sequences side by side and PAMs on opposite ends.

Described in the picture, you can use a single complex to cleave one strand or multiple Cas 9s can be used to cleave both strands.

Applications

For controlled gene editing, where you wish to fix the mutations in a gene but not disable it, Cas 9 nickase is your go-to guy. Especially in PAM-out sequences because it causes target sequences to overlap. To do this, you would need a pair of Cas 9 Nickase. ✂️✂️

We can much more accurately pair DNA if one side is filled out due to pairing rules.

dCas 9 (dead Cas 9) or Null Mutant

Where both HNH and RovC are inactive and therefore no cuts can be made. Though, the complex can still bind to DNA. So, that sounds pretty useless…right?

Wrong.

Applications

We can literally control a gene and its ability to make proteins without cutting it out! Meaning the edits are reversible. When genes can’t make proteins, they’re not expressed and essentially can’t do much. 🤫

We can do this by activating and repressing genes. One approach looks at fusing dCas to epigenetic modifiers like methylation and histone modifiers. This controls how tightly packed DNA is. The closer packed, the harder for proteins to come and transcribe the DNA.

dCas-Sam — fusing transcriptional activators which can activate genes 10–1000 fold! This is currently being used for HIV eradication.

dCas-Krab — A transitional repressor to decrease gene expression by 5–10 fold!

Body: Oh, look it’s a cancer gene

dCas 9: Let me just turn it off 🤯🤯🤯

dCas9: Yeah, I can also label specific parts of DNA for analysis.

Key Takeaways

• CRISPR is a key part of our future that's already existed for centuries
• There are multiple variants of CRISPR Cas 9, some with two molecular scissors, others with one or none
• Each variant has specific applications thanks to their strengths and weaknesses

As we continue to discover and develop types of CRISPR, we must consider their overall applications. What if He had used dCas 9 to just silence a gene important for bodily function, like the production of haemoglobin?

Understanding the full capabilities will allow for more discussions around what should be accessible in shaping our biological future. For who? What types of CRISPR technology and what use cases, like agriculture and genetic enhancement?

Especially CRISPR use today given the key limitation, we don’t know the full effects of editing a gene.

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