SHERLOCK of Molecular Biology: New CRISPR-CAS System Developed

In the past decade, CRISPR-CAS system has been a topic of interest among molecular biologist all around the world. And because of this, the development of this system as a tool for scientists is also commendable. Here is another proof of the same but before we talk about SHERLOCK, let us brief you about what CRISPR is and how does it function, so as to appreciate the current scenario better.

In nature, Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes (CRISPR-Cas) adaptive immune systems is present in many Bacteria and almost all Archea. The function of this system is to protect the host from invading viruses. This system functions in 3 steps i.e. Adaptation then Transcription then Interference. Segments of foreign nucleic acids (spacers) were added in the CRISPR array in between pairs of direct repeats and upon transcription these forms the CRISPR RNAs (crRNA). These crRNAs guide the CAS proteins to target and cleave related sequences in the respective invader genomes. Generally, all CRISPR-CAS system involves these same steps although implementation of these steps and proteins involved in the system vary according to the species of the host organism.

Recently, a scientist from MIT and Harvard developed a system called Specific High Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK). This system leverages the programmable Endonucleases in CRISPR-based diagnostics of nucleic acids. This in vitro system has attomolar (10-18) sensitivity for detection. The heart of this system lies in the specific CAS protein involved, CAS13a. This protein is different than others in two aspects. First being the single effector (that is the organ or cell that acts in response to a stimulus) RNA-guided RNases nature of the protein and second is the fact that it cleaves not only the targeted RNA but also causes “collateral” degradation of the nearby Non-Targeted RNAs. This second property can be used as a tool for in vitro RNA detection by non-specific degradation of labeled reporter RNA allowing real-time detection or in vivo RNA detection by triggering programmed cell death.

Cas13a enzyme for SHERLOCK was selected after testing different orthologs for RNA-guided RNase activity. The currently used Cas13a is from Leptotrichia Wadei (LwCas13a). The detection sensitivity of approximately 50 fM was achieved with this enzyme which was not enough for many detection methodologies. Attomolar sensitivity was achieved by using a combination of Cas13a based detection with enzymes involved in isothermal amplification steps. Of the different combinations tried and tested, combination of Recombinase Polymerase Amplification (RPA), T7 RNA polymerase (for transcription of amplified DNA to RNA) and target detection by Cas13a Collateral cleavage action was found to achieve attomolar sensitivity. This sensitivity was tested for both DNA and RNA and was also verified by ddPCr and qPCR methods. Attomolar sensitivity was also maintained when all the components were combined in a single reaction. This proved that this platform can also be a useful tool in point of care diagnostics.

The advantage of SHERLOCK over ddPCr and qPCR:

1)      SHERLOCK shows less variation than ddPCR and qPCR and RPA. This was measured by Coefficient of Variation (the spread of variability on both the sides of a mean value) across replicates.

2)      All the components of SHERLOCK can be lyophilized and subsequently rehydrated without losing the attomolar sensitivity.

3)      SHERLOCk can work with different sample types such as Urine, Serum, Saliva etc. where titers are very low.

4)      No Nucleic acid purification is required for using SHERLOCK (verified through reaction having only 2% serum)

5)      SHERLOCK can also be used for Bacterial strain Genotyping and shows low cross-reactivity when different strains are multiplexed in a single reaction.

 

 Other Validation tests performed Using SHERLOCK:

1)      Effectiveness in Infectious Disease applications that require high sensitivity: Effective

2)      Effectiveness of components on lyophilization: Effective

3)      Effectiveness to detect target in different sample types: Successful

4)      Bacterial pathogen detection and Bacterial genotyping: Successful

5)      Low-frequency Cancer Mutation Detection in cell-free (cf) DNA: Successful

The sensitivity of SHERLOCK was increased even more by introducing single base miss matches in crRNA: target duplex enabling Cas13a to discriminate between targets differing by a single base. SHERLOCK was also proven to be an excellent choice of diagnostic tool in Human genotyping by Single Nucleotide Polymorphism (SNP) detection.

Applications of SHERLOCK in Diagnostics and forensics and Molecular genetics are endless. Development of SHERLOCK has also opened up a new school of thought that involves modification of CRISPR and its use in different ways.

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