Designer babies? The end of all diseases? Genetically modified humans that never age? When scientists first discovered the CRISPR genome in 1987, little did they know it would revolutionize the world of genetic engineering. Now, CRISPR/Cas systems are widely used to edit genes. Scientists, have even been able to remove malignancy of certain cancer cells using this technology. But what exactly is the CRISPR/Cas system and how does it work?
The CRISPR/Cas system is a form of an adaptive immune system that bacteria have evolved in order to protect themselves from any foreign genetic material -a.k.a DNA or RNA- that enters the cell. Just like our immune system protects us from getting the chickenpox twice, the CRISPR/Cas system protects bacteria from catching the same viral infection twice.
Before we talk about the details of the CRISPR/Cas system, let's try to understand an important characteristic of nucleic acids-a.k.a DNA or RNA- that allows them to interact with each other. All nucleic acids are polymers that are made from a combination of 4 different monomers known as nucleotides. DNA is made from nucleotides adenine, guanine, cytosine and thymine. RNA uses almost the same set of nucleotides; the only discrepancy is that it uses uracil instead of thymine. Adenine "base-pairs", or interacts, with thymine or uracil and guanine base-pairs with cytosine. Base-pairing allows nucleotides from one nucleic acid chain to interact with nucleotides from another (or the same) nucleic acid chain. DNA is a double helix because of this property: one strand of DNA base-pairs with its complementary strand to form a stable helical structure! Now that we know how base-pairing works, let's move back to the CRISPR/Cas system.
All CRISPR/Cas based immunity occurs in three steps: 1) acquisition, 2) transcription and 3) interference. During acquisition, the bacteria incorporates part of the viral DNA into its own genome; this part of the genome is called the CRISPR locus. There are "repeat" sequences that show up again and again in the CRISPR locus (figure 2). Viral DNA gets incorporated between these repeat sequences, introducing variable regions in the locus that are also known as "spacers". Acquisition is followed by transcription of the locus which produces a long RNA known as the CRISPR RNA (crRNA). This crRNA is further processed, to form multiple mature crRNAs that can recruit specific proteins in the cell and build an RNA-protein complex called "Cascade". Interference can finally happen; the Cascade complex facilitates base pairing of the crRNA with its target- the target here is the same viral DNA the crRNA originally coded for! Once base-pairing takes place, the Cascade complex recruits enzymes that can cleave the viral DNA and deactivate it. For a more detailed explanation of this mechanism, look at the video below:
Since 1987, a variety of CRISPR/Cas systems have been identified across bacteria. However, this post will discuss the very first CRISPR/Cas system that was originally identified: the "type I E" system in Escherichia coli. This is one of the most well studied CRISPR/Cas systems.
Although acquisition is the first step of the CRISPR/Cas based immunity, we will come back to the details of this mechanism later. Let’s start by looking at the transcription and processing of the crRNA. When the CRISPR locus gets transcribed, a long crRNA is made. This crRNA encodes for all the repeats as well as all the spacer regions of the CRISPR locus (shown in figure 3). This seems useless - the cell wants crRNAs that is specific for the DNA of just one type of virus. Thus, the crRNA needs to be processed and cleaved. This is done by a specific protein called CasE. CasE recognizes and binds to the repeat sequences in the crRNA and acts as “molecular scissors”, by cutting the crRNA at regions where it is bound. Cleaving results in the formation of many mature crRNAs, each of which is specific to just one type of virus. CasE stays bound to the crRNA and acts as a nucleation point for the next step - formation of the Cascade complex. For a more detailed explanation of transcription and processing, look at the video linked below.
The Cascade complex, in the type I E system, comprises of 11 protein subunits that assembles all around the crRNA- take a look at figure 4, doesn’t it look like a seahorse? During assembly, the crRNA-CasE complex first recruits the protein CasD. This is followed by six copies of CasC assembling all around the crRNA backbone. This assembly changes the conformation of the crRNA so that it can favorably base-pair with its target DNA. Once, CasC assembles, two copies of CasB is recruited to the complex. Finally, CasA, a large protein that plays an important role in the recognition of the viral DNA, joins the complex. Once, the Cascade complex assembles, it looks for its target DNA in the bacterial cell. The complex facilitates base pairing of the crRNA to its target DNA. For a more detailed explanation of Cascade formation look at the video below.
Once the Cascade complex binds to its target, one of two immune responses can take place: "interference" or "priming". Interference directly deactivates the viral DNA by recruiting an enzyme, Cas3. Priming, on the other hand, is another word for spacer acquisition; remember, this is the first step that takes place in the CRISPR/Cas based immunity. Priming incorporates the viral DNA into the CRISPR locus and creates memory of the infection in the bacteria.
There are two models that have been proposed for priming so far. The interference dependent model and the interference independent model. Cas3 along with another protein complex, Cas1-Cas2, plays an important role in both models. The Cas1-Cas2 protein complex is responsible for integrating the viral DNA into the CRISPR locus. Let’s talk more about the interference independent model.
In the interference independent model, once the Cascade complex assembles and binds to its target, the Cas1-Cas2 complex is recruited. The Cas1-Cas2 complex, recruits Cas3 and forms a Cas1-Cas2-Cas3 protein complex. This complex moves along the viral DNA looking for sequences it can use for spacer acquisition.
How does the Cascade complex decide whether interference or priming takes place upon target binding? It all comes down to the interaction of the viral DNA with the CasA protein in the cascade complex. A paper by Xue , et al. in 2016 has shown that CasA can have two different conformations depending on how it interacts with the viral DNA target. So how does the viral DNA interact with CasA in the first place?
CasA recognizes specific DNA sequences in the viral DNA, also known as "PAM sequences". This is also how Cascade distinguishes between self vs non-self DNA. You can imagine that if the Cascade complex had no way of discriminating between self vs non-self, it would probably incorporate its own bacterial DNA into the CRISPR locus, or even cleave its own DNA! This is analogous to autoimmune diseases, where an organism's immune system attacks itself. This definitely wouldn’t work out very well for the cell...
The region of CasA that binds to the the PAM sequence is known as the NTD (N-terminal domain). Proteins are often read from the N-terminal to the C-terminal end. If the interaction between the NTD region of CasA and the PAM sequence is tight, another region of CasA, also known as the CTD (C-terminal domain) gets locked. CasA, then adopts a closed conformation. This recruits Cas3, the enzyme that cleaves the DNA, and induces interference. If the interaction between CasA and the PAM sequence is weak, the CTD opens up. CasA then adopts an open conformation and recruits the Cas1-Cas2 complex. This complex does not cleave the DNA but induces priming. This mechanism is shown if figure 5. For more details about priming, look at the video below.
Scientists have modified the CRISPR/Cas system to make it an EXTREMELY powerful gene editing tool. The biggest change is in the type of Cascade complex they use to cleave their target DNA. Maybe, some of us have even heard about CRISPR/Cas9 gene editing. Cas9 is a type II CRISPR/Cas system and is easier to work with because Cascade assembly involves the recruitment of just one protein, Cas9, rather than 11 different ones. Cas9 can also cleave the target DNA on its own; thus, there is no need for the recruitment of any other enzyme (like Cas3) during interference.
Scientists have designed guide RNAs, analogous to crRNAs in the Type I E system, that can form complementary base pairing with the DNA sequence they want to edit. The guide RNA forms a targeting complex with Cas9, similar to cascade. When the complex finds its target, it base pairs with it and Cas9 cleaves the DNA. Isn’t this cool? In addition, scientists sometimes place another DNA sequence along with the guide RNA in the system; this sequence gets incorporated to the target DNA once it's cleaved. This means we can potentially delete incorrect genes and replace them with correct ones! Goodbye, cancer ! Or any other disease for that matter.
It's pretty obvious that the CRISPR/Cas system can have a huge impact on health and diseases. In fact, it's already being used in thousands of research institutes all over the world. It can also be used in other sectors including: the agricultural industry to modify crops and make them more resistant to certain pests, and the biotech industry to produce drugs and biofuels more efficiently. Of course, there will be ethical issues that have to be considered at every step of using this technology but I can’t wait to see what it’s going to do next!
References:
1) Xue, C., Whitis, N. R., & Sashital, D. G. (2016). Conformational control of Cascade interference and priming activities in CRISPR immunity. Molecular Cell, 64(4), 826–834.
2) André Plagens, Hagen Richter, Emmanuelle Charpentier, Lennart Randau; DNA and RNA interference mechanisms by CRISPR-Cas surveillance complexes, FEMS Microbiology Reviews, Volume 39, Issue 3, 1 May 2015, Pages 442–463.
Meet the Author
Smriti is a junior at Mount Holyoke who is graduating in 2019 with a bachelor’s degree in Biochemistry and a minor in Mathematics. She currently works in Professor Katherine Berry’s lab at Mount Holyoke College which studies RNA-protein interactions. Smriti plans to continue with research and will be applying to graduate schools next semester.
Figure 1: Gene editing using the CRISPR/Cas system. The CRISPR/Cas system acts as “molecular scissors”and cuts the target DNA to make modifications. Figure retreived from https://phys.org/news/2017-06-technique-enables-safer-gene-editing-therapy.html.
Before we talk about the details of the CRISPR/Cas system, let's try to understand an important characteristic of nucleic acids-a.k.a DNA or RNA- that allows them to interact with each other. All nucleic acids are polymers that are made from a combination of 4 different monomers known as nucleotides. DNA is made from nucleotides adenine, guanine, cytosine and thymine. RNA uses almost the same set of nucleotides; the only discrepancy is that it uses uracil instead of thymine. Adenine "base-pairs", or interacts, with thymine or uracil and guanine base-pairs with cytosine. Base-pairing allows nucleotides from one nucleic acid chain to interact with nucleotides from another (or the same) nucleic acid chain. DNA is a double helix because of this property: one strand of DNA base-pairs with its complementary strand to form a stable helical structure! Now that we know how base-pairing works, let's move back to the CRISPR/Cas system.
Figure 2: Three steps of the CRISPR/Cas based immunity in bacteria: acquisition, transcription and interference. Figure retrieved from, http://rna.berkeley.edu/crispr.html.
Figure 3: Processing of the crRNA to form mature crRNAs. The unprocessed crRNA in (a) has constant regions that are highlighted by blue and red as well as variable regions that have their own unique color. CasE recognizes the constant region highlighted in blue ,as shown in (b), and acts as molecular scissors, as shown in (c), to cleave the RNA and form mature crRNAs, as shown in (d).
Figure 4: Cascade complex bound to its target viral DNA. The 11 subunit cascade complex allows the crRNA to successfully base pair with its target. CasA recruits an enzyme, Cas3 that degrades the viral DNA.
Once the Cascade complex binds to its target, one of two immune responses can take place: "interference" or "priming". Interference directly deactivates the viral DNA by recruiting an enzyme, Cas3. Priming, on the other hand, is another word for spacer acquisition; remember, this is the first step that takes place in the CRISPR/Cas based immunity. Priming incorporates the viral DNA into the CRISPR locus and creates memory of the infection in the bacteria.
There are two models that have been proposed for priming so far. The interference dependent model and the interference independent model. Cas3 along with another protein complex, Cas1-Cas2, plays an important role in both models. The Cas1-Cas2 protein complex is responsible for integrating the viral DNA into the CRISPR locus. Let’s talk more about the interference independent model.
In the interference independent model, once the Cascade complex assembles and binds to its target, the Cas1-Cas2 complex is recruited. The Cas1-Cas2 complex, recruits Cas3 and forms a Cas1-Cas2-Cas3 protein complex. This complex moves along the viral DNA looking for sequences it can use for spacer acquisition.
How does the Cascade complex decide whether interference or priming takes place upon target binding? It all comes down to the interaction of the viral DNA with the CasA protein in the cascade complex. A paper by Xue , et al. in 2016 has shown that CasA can have two different conformations depending on how it interacts with the viral DNA target. So how does the viral DNA interact with CasA in the first place?
Figure 5: Mechanism showing how the cascade complex decides between interference and priming. Figure adapted from Xue et al. 2016.
The region of CasA that binds to the the PAM sequence is known as the NTD (N-terminal domain). Proteins are often read from the N-terminal to the C-terminal end. If the interaction between the NTD region of CasA and the PAM sequence is tight, another region of CasA, also known as the CTD (C-terminal domain) gets locked. CasA, then adopts a closed conformation. This recruits Cas3, the enzyme that cleaves the DNA, and induces interference. If the interaction between CasA and the PAM sequence is weak, the CTD opens up. CasA then adopts an open conformation and recruits the Cas1-Cas2 complex. This complex does not cleave the DNA but induces priming. This mechanism is shown if figure 5. For more details about priming, look at the video below.
Scientists have modified the CRISPR/Cas system to make it an EXTREMELY powerful gene editing tool. The biggest change is in the type of Cascade complex they use to cleave their target DNA. Maybe, some of us have even heard about CRISPR/Cas9 gene editing. Cas9 is a type II CRISPR/Cas system and is easier to work with because Cascade assembly involves the recruitment of just one protein, Cas9, rather than 11 different ones. Cas9 can also cleave the target DNA on its own; thus, there is no need for the recruitment of any other enzyme (like Cas3) during interference.
Scientists have designed guide RNAs, analogous to crRNAs in the Type I E system, that can form complementary base pairing with the DNA sequence they want to edit. The guide RNA forms a targeting complex with Cas9, similar to cascade. When the complex finds its target, it base pairs with it and Cas9 cleaves the DNA. Isn’t this cool? In addition, scientists sometimes place another DNA sequence along with the guide RNA in the system; this sequence gets incorporated to the target DNA once it's cleaved. This means we can potentially delete incorrect genes and replace them with correct ones! Goodbye, cancer ! Or any other disease for that matter.
It's pretty obvious that the CRISPR/Cas system can have a huge impact on health and diseases. In fact, it's already being used in thousands of research institutes all over the world. It can also be used in other sectors including: the agricultural industry to modify crops and make them more resistant to certain pests, and the biotech industry to produce drugs and biofuels more efficiently. Of course, there will be ethical issues that have to be considered at every step of using this technology but I can’t wait to see what it’s going to do next!
References:
1) Xue, C., Whitis, N. R., & Sashital, D. G. (2016). Conformational control of Cascade interference and priming activities in CRISPR immunity. Molecular Cell, 64(4), 826–834.
2) André Plagens, Hagen Richter, Emmanuelle Charpentier, Lennart Randau; DNA and RNA interference mechanisms by CRISPR-Cas surveillance complexes, FEMS Microbiology Reviews, Volume 39, Issue 3, 1 May 2015, Pages 442–463.
Meet the Author
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