Searching for clues, CRISPR-Cas system becomes a pathogen’s detective

CRISPR-Cas system has been developed since 2013 to become the most promising technique for gene editing. Dr. Feng Zhang’s laboratory has been, without any doubt, one of the CRISPR-Cas technology development pioneers. During 2013 and 2014 his group adapted the CRISPR-Cas9 technology to the deletion of regions and fragments of DNA for generating knock-out (KO) models, as well as for the insertion of foreign DNA fragments into specific genomic regions (knock-in models). In 2015 his group characterized the Cas9 nuclease crystallographic structure [1] and diversified the research in order to gain more insights about other Cas nucleases like the Cas12 (Cpf1) [2]. In 2017 his group identified the RNA nuclease Cas13 [3], proposing a method for RNA editing in living cells [4, 5]. The same year they developed a tool, based on the Cas13 nuclease specific properties, for the detection of pathogen’s nucleic acids [6] so-called SHERLOCK (High-Sensitivity Enzymatic Reporter UnLOCKing).

Recently, Zhang’s group published an article in Nature Protocols describing the use of SHERLOCK as a diagnostic platform which combines a DNA or RNA preamplification stage together with their detection with the nucleases Cas12 or Cas13, respectively. SHERLOCK allows multiplexed, portable and ultra-sensitive detection of DNA or RNA from clinically relevant samples. 


Figure 1: SHERLOCK components and workflow. Adapted from Kellner, M.J et al. Nat Protoc 14, 2986–3012 (2019)

How does SHERLOCK Works?

Cas13 is an RNA-guided RNase that produces multiple cleavage sites in single-stranded areas of an RNA target. Cas13 also exhibits a collateral activity which leads to the trans cleavage of bystander RNA molecules (Figure 1B). It has been recently discovered that the DNA nuclease Cas12 also display collateral activity. Researcher then combined the highly specific target recognition with the subsequent RNase activity to cleave a reporter molecule, developing a new approach for specific detection of foreign nucleic acids in clinical samples. The reporter molecule is a single-stranded nucleic acid molecule with a fluorophore in one side and a quencher in the opposite side. Once the nucleic acid is cleaved by the Cas13 collateral activity, both molecules are separated and the fluorescence can be detected. 

SHERLOCK includes a pre-amplification step (typically by recombinase polymerase amplification or RPA) that can amplify either RNA or DNA and introduce a T7 RNA polymerase promoter, allowing RNA transcription and subsequent detection by Cas13. Amplificated nucleic acids are incubated with Cas13 nucleases coupled with crRNA designed against pathogen’s sequences. Once the Cas13 binds to the target RNA, it will cleave the sequence and, with its collateral activity, will also cleave the single stranded RNA reporter molecule, releasing the fluorophore and allowing the detection of the signal (Figure 1C). The pre amplification reaction and the CRISPR collateral detection can either be run sequentially with a transfer step in between (two-steps detection procedure), or as single combined mixture (one-por detection procedure).

SHERLOCK is compatible with multiple readouts, either fluorescence detection or lateral flow detection, depending on the reporter molecule choice. Furthermore, either Cas12 or Cas13 enzymes can be used for detection of DNA or RNA, respectively, depending on the introduction of a T7 RNA polymerase promoter during pre-amplification and a T7 RNA polymerase during the detection reaction to generate RNA for Cas13 collateral activation. Fluorescence detection can be performed either as an endpoint readout or in real time with a plate reader or another compatible fluorometer. 

Advantages and limitations of SHERLOCK

SHERLOCK is highly sensitive and specific, being capable of detecting a single molecule in 1µl (2 aM) of both DNA and RNA targets. Cas13 does not catalytically activate when there are two or more mismatches in the crRNA: target duplex. Choosing the right primer and crRNA combination can markedly increase the speed of detection. As mentioned before, RPA can be combined with the Cas13 detection for a one-pot assay, although the viscosity of the mixture can reduce robustness of detection, leading to potentially higher false-negative rate and less sensitivity. One-pot assays have both benefits and disadvantages: it is possible to achieve detection in as little as 10-15 minutes for targets in the femtomolar concentration range and in less than an hour for targets in the attomolar concentration range. As well as being faster, there is also less risk of contamination and it is easier to obtain quantitative results due to the real-time detection properties. However, it is also less sensitive than two-steps SHERLOCK, and experimental optimization is often more challenging. One-pot SHERLOCK is more useful for time sensitive, high-throughput or quantitative application, as well as applications with increased contamination risk, whereas two-step SHERLOCK is more useful for applications with challenging sample inputs such as quick extractions from body fluids. Another advantage of SHERLOCK is the low cost of its components, as a typical single-plex reaction is estimated to cost as little as $0.60.

Applications of SHERLOCK

As SHERLOCK can be defined as a reprogrammable amplification system, it can be applied in any situation that requires sensitive detection of a DNA or RNA target. It has been used for the detection and genotyping of bacterial and viral infectious disease agents, including distinguishing single-nucleotide variants and finding antibiotic resistance genes [6]. The most powerful applications of this technique allowed for the discrimination of targets that differed only a single base pair, and successfully detected Zika and Dengue virus directly from patient urine and serum samples. Furthermore, SHERLOCK can distinguish between different virus or bacteria strains due to the specificity of the crRNA used. The single-nucleotide specificity of SHERLOCK has also been applied for genotyping patient samples and detection of cancer-associated mutations from circulating cell-free DNA, even in samples in which the target is present at an abundance of 0.1% compared to the background [6, 7]. 

The revised method, named SHERLOCKv2, can simultaneously detect one DNA target and three ssRNA targets in a single reaction. In SHERLOCKv2, the Cas13 together with Csm6 – and auxiliary CRISPR type III associated nuclease – can increase about 3- to 5-fold signal sensitivity [7]. Moreover, a technique which combines SHERLOCK together with HUDSON (Heating Unextracted Diagnostic Samples to Obliterate Nucleases) allows pathogen detection directly from body fluids of the patient without nucleic acid extraction [8].

Maybe we will see further applications of the method in the coming years.


Related articles

CRISPR-CasФ: A new all-in-one hypercompact genome editor

Beyond gene editing: the diversity of CRISPR-Cas system applications

What are the advances in CRISPR technology?

Recent advances in CRISPR technology-PART I: CRISPR-Cas9 Alternatives

Recent advances in CRISPR technology-PART II: Innovations

Nature Biotechnology: New base editors change C to A in bacteria and C to G in mammalian cells


References

1.  Nishimasu, H., et al., Crystal Structure of Staphylococcus aureus Cas9. Cell, 2015. 162(5): p. 1113-26.

2.  Zetsche, B., et al., Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 2015. 163(3): p. 759-71.

3.  Smargon, A.A., et al., Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28. Mol Cell, 2017. 65(4): p. 618-630 e7.

4.  Abudayyeh, O.O., et al., RNA targeting with CRISPR-Cas13. Nature, 2017. 550(7675): p. 280-284.

5.  Cox, D.B.T., et al., RNA editing with CRISPR-Cas13. Science, 2017. 358(6366): p. 1019-1027.

6.  Gootenberg, J.S., et al., Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, 2017. 356(6336): p. 438-442.

7.  Gootenberg, J.S., et al., Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science, 2018. 360(6387): p. 439-444.

8.  Myhrvold, C., et al., Field-deployable viral diagnostics using CRISPR-Cas13. Science, 2018. 360(6387): p. 444-448.


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