Changing the World of Detection: Cas 13

I don’t even think a 5-yr old can write this bad. But our body does…

Cells contain DNA, comprised of four letters: A, C, T and G. Those letters code for proteins which allow your body to function. Change a letter or multiple, and that will change the protein, to something your body may not want.

In the example above, the code that makes the world great does not truly describe the world on 5/30/2020. The “top” in the second sentence makes no sense. The duplication of sentences, deletion of letters and mis-spelling are all errors.

But, we can see them. With our eyes or genetic sequencing. And when you can see a problem, it’s a lot easier to fix it.

In genetic sequencing, finding that problem may require more expertise, equipment, time, and money. And fixing the problem is not as simple as erasing a letter…not yet.

Genetic Engineering

Your body duplicates DNA a lot every single day. And eventually, your body will copy a letter wrong during the process of transcription. Or, there may be an insertion of a letter, a deletion, a duplication, etc. Most of these mutations are harmless.

The different types of mutations.

Over time, we have found certain mutations that cause a certain disease, by looking at a patients genetic code. Now that we know the specific problem, it’s hard to leave it unsolved. And that’s why we started the field of genetic engineering.

If curing disease was as simple as erasing a letter and re-writing the desired code, OH BOY! That vision was a long ways away with the current genetic engineering tools, ZFN and TALEN.

Now, with the development of a cheaper, quicker and more precise editing tool in CRISPR, that “OH BOY” is just on the tip of my tongue.

CRISPR Family

Many people are familiar with CRISPR Cas 9. An enzyme that can target your problem and make it disappear. But, Cas 9 isn’t alone. It’s got a family with a variety of specialities.

One member of that family is Cas 13. Not only can it make specific types of problems disappear, but it can also recognize those problems with little equipment, expertise, and time needed!

Cas 13: The Key Parts

Most CRISPRs are great at locating genetic code and cutting it. But, what type of genetic code it locates and cuts can differ. Oh, and some of them have a secret weapon!

HEPN (aka the scissors)

Cas 13 has 2 HEPN domains (a part of a protein that can function by itself). These domains exhibit Ribonuclease (RNase) activity, which commonly catalyzes the degradation of RNA.

That’s the main difference between Cas 9 and Cas 13, the latter cuts RNA and the prior cuts DNA.

We can edit these HEPN domains to make one or both of them inactive. These possibilities bring forth more applications for Cas 13.

ssRNA (aka The Guide)

The single-stranded RNA (ssRNA) is the specific sequence of genetic code that you want to locate in the body. When attached to the Cas 13 complex, it will bring the scissors to the target site with a matching RNA to the ssRNA.

Unlike Cas 9 which requires a trcRNA along with cRNA, Cas 13 only needs cRNA to locate its target.

We will identify the gene we want to erase and choose the best sequence for our ssRNA. The ssRNA is designed based on:

  • The number of potential off-target matches — what is the chance the ssRNA binds to the undesired RNA. Possibly bringing the eraser to a perfectly fine word, instead of the problem. This is often determined by how closely related the sequences are
  • Highest potential cleavage efficiency — if this ssRNA the best at locating and allowing the cut of the target
The ssRNA (bottom) is complementary (the letters are pairs, A pairs with T and G pairs with C) to the target RNA.

PFS (aka PAM)

The protospacer flanking site works just like the PAM. In order for the ssRNA to bind to the target sequence and allow the cut, a small letter sequence needs to appear before the target.

PAM for CRISPR Cas 9 functions like the PFS for Cas 13

Like Cas 9, the PAM site is determined by the orthogonal of Cas 13. For example, the SpCas 9 is a version of Cas 9 that comes from the bacteria streptococcus pyogones, carrying the NGG PAM Sequence.

There are a variety of orthogonals for Cas 13, but quite a few of them don’t even require a PFS.

This means we are not restricted to what we can target unlike Cas 9. +1 for Cas13

Trans / Collateral Cleavage (aka the Secret Weapon)

Most of the Cas family, including Cas 13, perform site-specific cleavage/cuts.

Cas 13 starts by locating and cleaving a specific target, but after that target is cut…it goes into Tasmanian devil mode. From scissors to paper shredder that will now cut any RNA which bumps into it.

After cleaving the target, the HEPN domains move closer together and form a catalytic-active site. The site is exposed, unlike in Cas 9, allowing it to trans cleave RNA due to it’s RNase activity.

You may think: “Cutting up your own RNA, that’s stupid.”

I would say: “Reporters”

When the Cas 13 complex is inserted into the body or a test sample, RNA reporters are also inserted. These reporters have a probe and a quencher. The fluorescent probe wants to light up, but when attached to the quencher, there is no light present.

So, if Cas 13 has found its target, it will start trans cleavage. Also cleaving the reporter, detaching the probe from the quencher, allowing the probe to light up!

If sequence = present: light up. Else: Dark

Applications

  1. RNA Knockdown — Is there a piece of RNA causing a problem. Yes. Okay, Cas 13 will cut it out. When the RNA tries to repair itself with no template, it will fail and become inactive. This allows us to understand the RNA’s function.

2. Agriculture genotyping — By using Cas 13’s trans cleavage property, we can check for off-site genetic changes caused by GMOs or pathogens in crops. Allowing us to stay safe, and help farmers produce higher yields.

3. Cancer Detection — With Cas 13’s secret weapon, a liquid biopsy can be tested for the presence of cancer. It has already been tested on melanoma cancer, which has a single mutation.

4. Single Point Mutation Diseases — Cystic Fibrosis and Sickle Cell Anemia are both diseases caused by the mutation of one letter. ONE LETTER. Using dCas 13 (dead Cas 13: both HEPN domains are inactive) and ADAR, we have been able to turn a base pair A to G.

dCas 13 just brings ADAR to the target site and ADAR solves the problem.

Viral Detection with SHERLOCK

Incentive

As previously mentioned, identifying problems can take time, equipment and expertise.

Prime example: Right NOW! Testing for SARS-COV-2 (the virus) is crucial to slowing down the spread. Yet, it takes days for results. Current methods include:

  1. Nucleic Acid (A, C, T, G) Detection via RNA Sample — After extracting RNA through a swab, it is amplified using PCR, increasing the number of SARS-COV-2 particles. Some type of fluorescent instrument will then tag a sequence if the virus is present.
  • Limitations: Requires Equipment and Expertise

2. Antibody Paper Strips — This paper strip contains antibodies that will lock to SARS-COV-2 antigens. Results return in 1 hour!

  • Limitations: Less sensitive because there is no amplification

3. Serology — A strip containing antibodies that will detect antibodies formed in a SARS-COV-2 response.

  • Limitation: It will only detect if you had SARS-COV-2, likely unnoticed since if you were asymptomatic.

Now, imagine you live in Africa during the Ebola outbreak, way more deadly than COVID-19 (the disease caused by SARS-COV-2). Where there was little to no equipment and expertise, so your best option was to send tests across the world to receive results!!!

Adding more of the current materials will only increase labour. It’s not that “we need more tests,” we need more rapid tests.

Point is: There is a huge incentive to create a sensitive, specific and flexible way to test for viral diseases.

SHERLOCK does exactly that.

Sherlock is identifying the crime in your body. A trespasser or forger?

The Process

Using Cas 13’s ability to trans cleave, HUDSON to extract nucleic acids, Isothermic Amplification to increase the copies of SARS-COV-2 and a visual readout, detecting SARS-COV-2 and other viral infections became fast, adaptable, accurate and as readable as a pregnancy test!

Check out how the process works below

1. Cas 13 Preparation

It’s clear you have to identify the virus you want to target and a specific sequence within the RNA.

The Cas 13 and ssRNA doesn’t change from patient to patient because that would require genome sequencing, already helping you identify the problem.

2. DNA or RNA Extraction

After extracting a sample through the nostrils or saliva, we have to isolate the DNA/RNA from the cell. Originally, this would require skill and equipment, but not with HUDSON.

With the use of lyse viral particles, we can break down the cell while preserving its integrity. We also want to remove high levels of Ribonuclease which affect the Cas 13 reaction.

This can all be achieved by simply heating and cooling DNA/RNA and a buffer.

3. Amplification

If an extracted sample only contains a few SARS-COV-2 sequences, Cas 13 may not recognize the virus’ presence. But, we don’t want to use any equipment…

Solution: Isothermal Amplification — Using heating and cooling tactics along with enzymes to duplicate strands of DNA.

Above is the process of RPA, a type of isothermal amplification.

  1. Recombinase enzyme creates matching primers for the 3' and 5' ends of DNA
  2. A Single Stranded Break Occurs, allowing the overhang of complete DNA over the matching primers
  3. Additionally added primers kickstart the DNA Polymerase reaction, where complementary letters will be added to the matching primers.

Voila! Now you got 2 more target strands. This process is crucial not only for its field use, but also for its ability to amplify DNA to RNA by using the T7 RNA Polymerase.

4. Cas 13 Detection

Finally, we can add the Cas 13 complex, including the ssRNA and the HEPN domains into the amplified DNA. Oh, don’t forget those reporters either.

If SARS-COV-2 is present, the reporters will be cut and give off their fluorescent light.

Results can then be read on a paper strip that is dipped into the test sample. If the reporter is intact, it will come to a halt at a control point on the strip. On the other hand, residue for the separated reporters will not come to a complete halt.

Results

  • Cost of Materials: $6
  • Sensitivity: 97%
  • Specificity: 100%
  • Time: < 1 hour
  • Expertise Needed: None

Researches are coming close to performing the complete reaction at a constant temperature and no-opening of tubes. Currently, they are testing different families and orthogonals of Cas 13, but nothing has been approved… yet.

SHERLOCK Advancements

You have already heard about visual readouts, but SHERLOCK is only getting cooler and cooler…as expected.

  • Multiplexing — Previously, Cas 13 has been used to diagnose the Zika, Dengue, West Nile and Yellow Fever viruses! All viruses with very similar symptoms and genetic codes. But, why run 4 different tests?

That’s why researchers designed a way to test for multiple viruses at once. Different families of Cas 13 will target certain RNA during trans cleavage. So by introducing multiple variants of Cas 13, we can observe the cleavage of specific reporters.

Research Article: https://pubmed.ncbi.nlm.nih.gov/29449508/

Who knows, maybe SHERLOCK will be able to detect if you have any form of cancer!

  • Quantitation — Detecting the amount of a virus in a sample and the strain of the virus by using different amount of primers in the RPA process

Key Takeaways

Time is of the essence when detecting diseases. Cas 13 gives us a specific and flexible way to detect a variety of genetic identifiers, and even genetically engineer RNA.

  • Genetic Engineering helps fix the errors in our genetic code
  • Cas 13 is apart of the CRISPR family, each member with their unique applications
  • Cas 13 uses 2 HEPN domains two cut RNA, compared to DNA
  • Cas 13 will start cutting any RNA that binds to it after cutting its target, this is called trans cleavage
  • Cas 13 has many applications, one huge one is viral detection by taking advantage of trans cleavage

Before You Go

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