CRISPR and CRISPR gene editing - Part 1: What is CRISPR?

What is CRISPR?

CRISPR. Credit: ExploreBiotech

In recent years, CRISPR gene editing has become the salient solution in eliminating congenital diseases such as sickle cell anemia. This is especially so as the 2020 Nobel Prize was given to scientists, Emmanuelle Charpentier and Jennifer Doudna, for developing the CRISPR gene editing methodology. I have read about this in many web articles that explain CRISPR in layman's terms. However, close to none delve deeper into the nitty gritty of the CRISPR gene and CRISPR gene editing. Hence, I took a deep dive to explore what is underneath the surface of this colossal iceberg. Join me on part 1 of this 2-part series on CRISPR and CRISPR gene editing, where we first explore CRISPR in depth!

What is CRISPR?

CRISPR, which stands for clustered regularly interspaced short palindromic repeats, is an immune mechanism, of prokaryotes such as bacteria, which is used to identify the genetic material of pathogens. The system uses CRISPR-associated (Cas) proteins to cut the foreign genetic material, shielding the prokaryote from the invading organism.

Take note that CRISPR is a genetic sequence found in the DNA of prokaryotes and its length can vary from a few hundred to several thousands of bp (base pairs) long. Base pairs refer to two nitrogenous bases in a double-stranded DNA helix which are bound together by hydrogen bonds ( A-T, C-G). The bases are Adenine (A), Thymine (T), Cytosine (C), and Guanine (G). A pairs with T while C pairs with G, in a double-stranded DNA helix. The below shows what a base pair comprises in a double-stranded DNA. 

What constitutes a base pair in a DNA double helix. 

Credit: Okinawa Institute of Science and Technology (OIST) Graduate University

What does a CRISPR locus consist of?

A CRISPR locus basically refers to an entire continuous genetic sequence of CRISPR. CRISPR loci refers to multiple CRISPR genetic sequences in a strand of DNA, for instance. 

A CRISPR locus consists of repeats, spacers, and Cas genes. 

Repeats

Repeats are short and palindromic DNA sequences, which are usually 20 to 50 bp long. They occur at regular intervals, meaning that, for example, in a particular CRISPR locus, there is consistently 30 bp between successive repeats.  

Palindromic sequence means that in a double-stranded DNA, the reading of bases from a sequence on one strand in one direction (5' to 3' for example) is identical to the reading of bases on the other strand in the same direction (5' to 3'). Meaning, that sequences read the same forward and backward on opposite strands. Remember that the strands in a double-stranded genetic material are antiparallel so the same direction (5' to 3') refers to the opposite direction for each strand. Hence, do not get confused by the mirror image of the bases in the palindromic sequence, from the image below.

A palindromic genetic sequence in a double-stranded genetic material. 

Credit: Dr Tushar Chauhan/ Genetic Education Inc.

Spacers

Spacers are unique DNA sequences retrieved from destroyed pathogens. These sequences act as a record of past infections and, thus, aid in faster recognition of pathogens that have attacked the prokaryote previously. 

Cas genes

Cas genes are gene sequences that give rise to Cas proteins when translated by ribosomes. The Cas proteins are responsible for the CRISPR functionality, may it be transcription or cleaving infectious organisms.

How does CRISPR work?

The CRISPR mechanism operates in 3 important stages - Adaptation, expression, and interference. 

Adaptation (Spacer Acquisition)

As aforementioned, spacers are originally part of the invasive pathogens that attack the prokaryote. For pathogens that the prokaryote has never encountered before, a Cas protein (individual) or complex of Cas proteins (multiple proteins attached to each other and arranged in a chain-like structure) tries to identify a specific genetic sequence on the invader called protospacers (get it? they are the precursor to spacers). The presence of a protospacer adjacent motif (PAM), a very short sequence of DNA beside the protospacer, is crucial for recognition. 

The Cas protein or complex identifies and removes the protospacer in the pathogen's  and incorporates it into the bacterial genome as a new spacer within the CRISPR array. The new spacer is typically added nearest to the leader sequence (a region near the CRISPR promoter, which initiates transcription). 

This arrangement ensures that if the same invader appears again, the CRISPR array can be rapidly transcribed (Produce RNA from DNA), producing a crRNA (CRISPR RNA) that includes the spacer sequence. This crRNA guides Cas proteins to recognize and target the invader DNA efficiently, as the crRNA's spacer is similar to the protospacer region of the invader's genome.

What adaptation is, in a nutshell. Credit: crispreducation

Expression

The CRISPR sequence, including repeats and spacers, is transcribed into long precursor RNA, called pre-crRNA. Thereafter, CAS proteins or RNAses (another type of protein) cut the pre-crRNA into individual crRNAs, each containing a spacer and part of a repeat. The spacer region of each crRNA is complementary to the protospacer region of a specific pathogen's DNA or RNA, allowing each crRNA to guide Cas proteins to recognize and target that specific invader.

Interference

The crRNA forms a complex with specific Cas proteins. Then, the crRNA brings the complex to the invader DNA. The PAM-binding domain of one of the CAS proteins binds with the PAM of the invader DNA to help the CAS proteins distinguish between the invader DNA and the prokaryote's own DNA. Then the Cas proteins unwind the invader DNA, thereby exposing the protospacer sequence for the cRNA to bind. Thereafter, the cRNA binds with the protospacer (with its complementary spacer sequence), which confirms that the DNA is indeed the invader's. This results in the Cas protein or the complex of Cas cleaving the invader DNA.

It is important to note that Adaptation will not take place for invaders that have been encountered before. When meeting such invaders, expression would occur first, followed by interference.

Another key point to account for is the fact that adaptation is about acquiring the spacer whilst interference is for destroying the pathogen. Although both involve cleavage at the protospacer region of the invader DNA, adaptation is meant for CRISPR to possess the ability to encode for the specific cRNA whilst interference is meant to compromise the integrity of the invader DNA. The adaptation stage has no crRNA while the interference stage does. 

What are the types of CRISPR systems?

As earlier mentioned, CRISPR systems' operation could depend on an individual Cas protein or a complex. Whether a CRISPR system uses a single Cas protein or a complex, is contingent on which class the system is classified under.

Class 1 (more than 1 Cas protein)

This class involves the use of multi-protein complexes. Each Cas protein would have a role in the immune surveillance system. An example of such a complex would be an array of Cas protein that require Cas 3 to perform the role of DNA degradation. The array of proteins could conduct multiple cuts to progressively destroy the invader DNA.

Class 2 (single Cas protein)

This class involves the use of a single Cas protein which would essentially be the jack of trades; it performs all the tasks necessary of Cas proteins in the CRISPR system. An example of such a protein would be Cas 9. The protein just conducts a single cut of the invader DNA, which is adequate enough to disrupt the integrity of the invader DNA, thereby destroying it.

How does a CRISPR system distinguish between self and invaders?

There are 2 features that are key in ensuring that the prokaryote's CRISPR system does not confuse itself and invade pathogens.

PAM sequences in invader DNA

These sequences are present in the invader DNA but absent within the repeats of the prokaryote's CRISPR sequence. Hence, it allows the CRISPR system to easily target the 

Absence of PAM sequences in CRISPR sequence

The absence of PAM sequence is the repeats of the CRISPR array makes sure that the Cas proteins do not accidentally bind to the host CRISPR sequence.

Could a CRISPR system fail?

Yes a CRISPR system can fail in detecting and neutralising threats. There are mainly 2 ways in which pathogens could develop mechanisms to evade the CRISPR immune surveillance system

Mutation of PAM sites

The base pairs of the PAM sequence in invading DNA could be mutated to give a different genetic code that could not be recognized by the CRISPR system, thereby evading it. 

Anti-CRISPR proteins

These proteins from the invading organisms could bind to the Cas proteins and inhibit them from performing their role, thereby preventing either adaptation or interference. or both of them. 

Next up in this series, we will explore what CRISPR gene editing is, in-depth, and the benefits it provides for humanity. If you'd like to hear about more topics pertaining to genetics and share your opinions on today's article, please list them down in the comments. Thanks a lot for reading!

References

1. Derry, W. B. (2020). CRISPR: Development of a technology and its applications. The FEBS Journal, 287(23), 5989–6003. https://doi.org/10.1111/febs.15621

2. Barrangou, R., & Marraffini, L. A. (2014). CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity. Molecular Cell, 54(2), 234-244. https://doi.org/10.1016/j.molcel.2014.03.011

3. Brouns, S. J., Jore, M. M., Lundgren, M., Westra, E. R., Slijkhuis, R. J., Snijders, A. P., ... & van der Oost, J. (2008). Small CRISPR RNAs guide antiviral defense in prokaryotes. Science, 321(5891), 960-964. https://doi.org/10.1126/science.1159689

4. Makarova, K. S., Wolf, Y. I., Alkhnbashi, O. S., Costa, F., Shah, S. A., Saunders, S. J., ... & Koonin, E. V. (2015). An updated evolutionary classification of CRISPR-Cas systems. Nature Reviews Microbiology, 13(11), 722-736. https://doi.org/10.1038/nrmicro3569

5. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096), 816-821. https://doi.org/10.1126/science.1225829

6. van der Oost, J., Jore, M. M., Westra, E. R., Lundgren, M., & Brouns, S. J. (2009). CRISPR-based adaptive and heritable immunity in prokaryotes. Trends in Biochemical Sciences, 34(8), 401-407. https://doi.org/10.1016/j.tibs.2009.05.002