Team:Warwick/Design

Theory by TEMPLATED

What is CRISPR/Cas9

The CRISPR-Cas9 system

Advantages and Disadvantages

Use of CRISPR/Cas9 in our system

What is CRISPR/Cas9

The Idea

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) are prokaryotic DNA segments containing short repeated base sequences. The CRISPR-Cas9 system has two constituent parts:

  • The Cas9 enzyme

    This is the ‘active part’ of the system. It essentially acts as a pair of scissors that cut the double-stranded DNA at the target location, enabling editing at a specific point within the genome

  • The guide RNA (gRNA)

    The single stranded gRNA ensures that the Cas9 enzyme cuts the genome in the correct location. In nature, the gRNA is found as two single stranded RNA that bind have complimentary sites. It is a small (~ 20 base pairs) modifiable RNA sequence composed of two parts: target-specific CRISPR RNA (crRNA) and auxiliary trans-activating crRNA (tracrRNA).
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The CRISPR-Cas9 system: Step by step DNA cutting in nature

dCas9 Binding

  1. A bacteriophage infects a bacteria, inflicting damage that the cell survives.

  2. The bacteria digests the bacteriophage DNA, producing a copy of a 20nt section of the bacteriophage genome and incorporating it into a CRIPSR array within the bacteria's genome as a protospacer.

  3. A recurrence of infection from the same bacteriophage triggers a response from the bacteria.

  4. The bacteria compares the DNA from the bacteriophage against the CRISPR array, expressing the CRISPR RNA (crRNA) that matches.

  5. The crRNA binds with trans activating CRISPR RNA (tracrRNA), forming the full guide RNA (gRNA).

  6. Cas9 enzyme binds to the Cas9 handle on the tracrRNA section of the gRNA.

  7. The Cas9 enzyme compares the 20nt section of the crRNA against multiple places on the bacteriophage genome, binding when it finds a complimentary section next to a NGG-3' section called the protospacer adjacent motif (PAM).

  8. The Cas9 enzymes expresses dual nuclease functionality, cutting both strands of the DNA it identifies.

  9. The bacteriophage genome is cleaved by Cas9 preventing successful infection of the host cell.

This system can be modified by synthetic biologist to allow targeted DNA cutting. The 20nt crRNA section can be edited to allow the Cas9 enzyme to target almost any position on a genome, provided it contains a PAM. By combining the crRNA and tracrRNA into a single guide RNA (sgRNA), editing is simplified and further secondary structures can be added to the sgRNA further altering the function of the system. The Cas9 enzyme can also be edited to reduce its nuclease action so that it only cuts a single strand of the target DNA, or eliminate it entirely so that it acts more as a targetable DNA binding enzyme.

Genome editing by CRISPR/Cas9 can range from single base changes, to insertion or deletion of entire genes.

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Genome Editing: A step by step CRISPR guide

  1. Target DNA sequence is identified and a complementary strand of gRNA is designed.

  2. Cas9 enzyme binds to the gRNA, targeting it towards a specific DNA sequence.

  3. Cas9 binds to the target PAM site.

  4. Cas9 enzyme cuts both strands of the double-stranded DNA.

  5. When the cell detects the damage inflicted upon the DNA by the dCas9, it attempts to repair the two strands.

  6. Genome mutations induced by flaws in cell DNA repair mechanisms results in a base changes due to non-homologous end joining.

  7. Introducing sections of DNA with similar ends to both sides of the cut allows homology-directed repair to occur, allowing the insertion of the introduced DNA into the cut site.
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Advantages and Disadvantages of CRISPR technologies in gene editing

CRISPR technology is one of the most recently developed gene editing tools. Its predecessors include ZNFs (Zinc Finger Proteins) and TALENs (Transcription Activator-like Effector Nucleases). The advantages of CRISPR technology compared to these previous methods include:

  • Simplified design targeting – targeting does not rely on unpredictable DNA-protein interactions that are difficult to modify. As the desired binding site can be changed, sgRNAs can be designed easily and cheaply to allow complementary binding to the majority of sequences within the genome.

  • Simultaneous transcription – as the targeting of Cas9 is programmable, we can design for targeting of multiple sites within the same system.

However, as with many existing methods, there are some drawbacks to this form of gene editing. If the sgRNA is not fully complementary to the DNA sequence, it may be unable to bind, preventing the required transcription of the gene. There is also an issue with off-target binding, however as we are using the modified nuclease-deficient Cas9 the potential harm caused by off-target binding is greatly reduced. In systems where highly specific binding is an absolute necessity, as it currently exists, CRIPSR/Cas9 may have too frequent off-target binding for it to be reliable enough in these situations. However there are multiple developments in CRISPR technology to reduce the level of off-target binding: reducing the length of the crRNA can lower off-target binding by reducing overall binding efficiency; or using two single-strand cutting Cas9 targeting opposite strands at the same site to create the double strand cut so that two systems need to fail before an off-target cut occurs.

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Use of CRISPR/Cas9 in our system

We elected to use CRISPR/Cas9 as the DNA targeting system in our device over using more conventional DNA binding proteins due to the inherently modular nature of CRISPR. By creating an activation system that relies on CRISPR we create a system whereby modifying 20 nucleotides can target the activation system to completely different genes. Or by including two gRNAs in one device, activate multiple genes simultaneously without the need for introducing further large enzymes. Through this we future-proof our idea by allowing it to be used for a multitude of targets, allowing the other parts of our system to be interchangeable without needing to rework the entire system.

Furthermore, as CRISPR relies on folding RNA, it opens up RNA detection as a possibility for methods of detecting stimuli. With the introduction of RNA aptamers that can potentially bind a multitude of compounds, it becomes possible to create a detection system for each of those compounds by only modifying the gRNA while maintaining the rest of the system.

As the nature of our detection system requires activation and regulation of transcription, not strand cutting, we use a deactivated version of the Cas9 enzyme – dCas9. This prevents the cleavage of the double stranded DNA, but still allows binding to the specific target site. In the majority of previous research, the role of dCas9 as a repressor has been analysed. In our project, Spirosensr, we aim to investigate and utilise dCas9 as an activator. One of the key features of our detection system is it’s easily altered modular nature. CRISPR/Cas9 technology is well suited to our project, as the cutting and binding of DNA is easily controlled to change specificity.

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Why CRISPR/Cas9

The Warwick iGEM 2016 team dedicated a lot of time to researching novel technologies that were potential project focal points. We elected to use CRISPR technology as it possesses a myriad of potential uses, whilst being a very precise method of genetic manipulation. Given that this field of research is relatively new, we were excited by the concept of investigating potential novel applications of the CRISPR/Cas9 system.

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