Reporting Mechanism
@Adrian Villanueva @Yujeong Oh @Marko Susic
Each reporting mechanism is dependent on the CRIPSR system used for the project i.e. Toehold switches are generally more geared towards CRISPR/Cas9 Systems.
Toehold Switch (Cas9)
Toehold Switches Activating An Reporter Protein
(From Team ODYSSEE - Winner of best diagnostics project 2019)
Figure 1. Illustration of toehold switch
The toehold switch has this hairpin loop such that it prevents a ribosome from binding to the start site of the RNA sequence. We would then need a complimentary trigger that would bind to the RNA sequence right before the loop, causing the hairpin to unwind and allow for translation to occur. In our case, we can code for a reporter gene.
Common reporter genes:
- GFP (Green fluorescent protein)
- Luciferase
Potential:
- Chromoprotein; colorimetric detection
This Toehold Switch is normally coupled with CRISPR Cas9 systems. A study by K. Pardee (2016) shows detection of Zika virus using CRISPR-Cas9 system and toehold switch:
- Theory: Toehold switch sensors are programmable synthetic riboregulators that control the translation of a gene via the binding of a trans-acting trigger RNA. The switches contain a hairpin structure that blocks gene translation in cis by sequestration of the ribosome binding site (RBS) and start codon. Upon a switch binding to a complementary trigger RNA, sequestration of the RBS and start codon is relieved, activating gene translation (Figures 2A and 2B ) (Green et al., 2014). To allow for colorimetric detection of trigger RNA sequences, the sensors can be designed to regulate translation of the enzyme LacZ, which mediates a color change by converting a yellow substrate (chlorophenol red-β-D-galactopyranoside) to a purple product (chlorophenol red).
References for Toehold Switch
Quenched ssDNA Reporter (Cas12)
Figure 1. An example of a quenched ssDNA reporter coupled with Cas12 System
Due to the ability of the Cas12a enzyme to trigger collateral ssDNA cleavage after detection, the Cas12a system can be coupled with a fluorescent quenched ssDNA reporter to easily relay a visible signal to the user.
attomolar sensitivity with DETECTR protocol (Chen et al., 2018)
References for Quenched ssDNA Reporter
Lateral Flow Readout using FAM-Biotin Reporter (Cas12)
0.24 fM sensitivity
- Detection using a FAM-biotin labelled ssDNA reporter molecule
- Biotin-labeling is commonly used for non-radioactive labeling and purification of proteins and other target molecules.
- Uncleaved reporter molecules are captured at the first line.
- The cleaved reporter molecules after indiscriminate cleavage activity of Cas12 enzyme are captured at the second line
- Fluorescent signal was detectable <1 min
- Visual signal by lateral flow was detectable within 5 min
- Universal LFA kit by HybriDetect; applicable to genomic product detection
Case study:
https://www.nature.com/articles/s42003-020-0796-5#Sec8
- Detection of African swine fever virus DNA using RAA+CRISPR-Cas12a and lateral flow
- 3 minutes to be detected with the lateral flow chip
- Detection of SARS-CoV-2 RNA using RT-LAMP+CRISPR-Cas12a and lateral flow
- 2 minutes to be detected with the lateral flow chip (Milenia HybriDetect 1, TwistDx.)
- RT-LAMP: 20-30 minutes, Cas12: 10 minutes
References for Lateral flow readout
- Detection using a FAM-biotin labelled ssDNA reporter molecule
Turbidity-based Readout using Liquid-liquid phase separation (Cas12)
- Long nucleic acid polymers and positively charged polyelectrolytes can undergo liquid-liquid phase separation into a polymer-rich and a polymer-depleted phase
- LLPS increases solution turbidity, which is visible to the naked eye.
- Solution with target becomes transparent; whereas, the solution without target is still turbid
- Adding charged single stranded poly(dT) DNA as a reporter
- Detection sensitivity at a micromolar level; less sensitive compared to FQ reporter
- Only one research paper related to this mechanism
References for Turbidity-based readout
(Author, year) Link Column (Spoelstra, 2019) https://www.biorxiv.org/content/10.1101/471482v2.full.pdf Untitled Untitled
Electrochemical sensing using hpDNA reporter (Cas12)
"Under the optimal conditions, as low as 30 pM target DNA was detected in about 60 min with 3.5 orders of magnitude dynamic range from 50 pM to 100 nM."
- An electrochemical technique has received much attention due to easy construction, cost-effective, miniaturization, rapid response, high selectivity, and sensitivity.
- a universal CRISPR/Cas12a-based electrochemical biosensor, termed E-CRISPR, has been established for DNA and protein detection.
- conventional linear ssDNA reporter is still employed to assemble on the electrode as a sensing interface, which may compromise the analytical performance of the biosensor.
In the study by Zhang (2020),
- A hairpin DNA (hpDNA) linked with methylene blue (MB) tag is employed to investigate the interfacial cleavage activity of Cas12a for developing an electrochemical DNA sensor.
- The electrochemical differential pulse voltammetry (DPV) measurements were performed.
- Principle:
- crRNA bind to Cas12a for the formation of a CRISPR complex
- CRISPR complex recognizes and cuts target DNA based on crRNA sequence and PAM sequence
- nonspecific ssDNA cutting by CRISPR complex
- hpDNA consisting of a thiol group at the 5′ terminus and a methylene blue (MB) tag at the 3′ terminus is covalently modified to a gold electrode (GE) through S–Au bonding.
- In the absence of the target, the Cas12a cannot cleave the hpDNA reporter.
- So, the formation of a stem-loop structure brings the MB tag near the GE surface, resulting in a high redox response being detected. In the presence of the target, the ssDNase activity of Cas12a is activated, cleaving the loop region of hpDNA into short fragments and leading to the dissociation of the stem part of hpDNA.
- The melting temperature (Tm) of the duplex stem decreases from about 48 °C to lower than 10 °C (estimated with the IDT Oligo Analyzer, under the conditions of 10 mM Mg2+ and 100 mM Na+), therefore releasing the MB from GE and decreasing the peak current. Therefore, the established electrochemical biosensor could convert per target recognition event into numerous disintegration of the hpDNA reporter on the interface for highly sensitive electrochemical DNA biosensing.
Reagents:
- NEBuffer - 1 M NaCl - 0.5 M Tris - HCl - 0.1 M MgCl2 - 0.01 M DTT - 7.9 pH
- Cas12a
- crRNA
- U RNase inhibitor
- Target DNA.
- Prepared hpDNA biosensor
Reagents used are stable.
References for Electrochemical sensing
(Author, year) Link Column (D. Zhang, 2020) https://pubs.acs.org/doi/10.1021/acssensors.9b02461 Untitled Untitled
Quenched ssRNA Reporter (Cas13)
Figure 1. An illustration of a quenched ssRNA reporter coupled with Cas13 System
Similar mechanism as the quenched ssDNA Reporter, this reporter exploits the ability of Cas13 to cleave ssRNA after detection. After detection, the Cas13 enzyme cleaves nearby fluorescent quenched ssRNA reporters to relay a visible signal to the user.
- By scaling up the pre-amplification RPA step, we found that LwaCas13a could give detection signal for 200, 80, and 8zM input samples and allow for single-molecule volume inputs of 250μL and 540μL (fig. S21A–B), and PsmCas13b could detect 200zM input samples in 250μL reactions (fig. 21C). (Jonathan S. Gootenberg et al.)
Low-cost fluorescence reader
- Cas13a paper-based nucleic acid assay
- All necessary reagents can be lyophilized (freeze-dried) and stored on filter paper; detector could be used to apply SHERLOCK in low resource settings
- 6.8 nM Fluorescein sensitivity
- Pocket-sized and under 15$ cost
- Sample distinguishable from background activity within 20 minutes
Figure 3. Portable low-cost detector
References for Quenched ssRNA Reporter
Lateral Flow Readout by destruction FAM-biotin reporter (Cas13)
- Lateral-flow readout based on the destruction of a FAM-biotin reporter
- Allows for detection on commercial lateral flow strips
- Abundant reporter accumulates anti-FAM antibody-gold nanoparticle conjugates at the first line on the strip, preventing binding of the antibody-gold conjugates to protein A on the second line
- Cleavage of reporter would reduce accumulation at the first line and result in signal on the second line
- Rapid closed tube assay format in which the entire SHERLOCK reaction is performed in a one-pot assay without any sample purification (demonstration in the paper)
- We tested this design for instrument-free detection of ZIKV or DENV ssRNA, and found that detection was possible in under 90 minutes with sensitivities down to the 2 aM condition (Jonathan S. Gootenberg et al.)
Electrochemical Microfluidic Biosensor (Cas13)
10 pM Sensitivity
- Used on miRNA in original paper (likely could be adapted)
- Low-cost electrochemical biosensor developed using dry film photoresist (DFR) technology
- Detection of enzymatically produced hydrogen peroxide takes place in the electrochemical cell
- After RNA targeting (off-chip), cleaved and non-cleaved reporter RNAs bind to the immobilized streptavidin on the microfluidic chip
- Fluorescein antibodies coupled to glucose oxidase (GOx), which are only capable of binding to uncleaved reporter RNAs, are introduced to enable enzymatic reading of the assay
- By pumping glucose through the microfluidic biosensor, GOx catalyzes its substrate, producing hydrogen peroxide which is amperometrically detected in the electrochemical cell
- The resulting amperometric signal is directly proportional to the amount of immobilized GOx (bound to uncleaved reRNA), and therefore inversely proportional to the concentration of target sample RNA
Readout Time Comparison
Sensitivity Comparison
| Reporting mechanism | CRISPR-Cas12 (DETECTR) | CRISPR-Cas13 (SHERLOCK) |
|---|---|---|
| Fluorescence | 1aM | 80zM |
| FAM-Biotin (Lateral Flow) | 0.24fM | 2aM |
| Electrochemical (E-CRISPR) | 30pM | 10pM |
Figure. For Reference
References
| Author | Link |
|---|---|
| Richard Bruch et al. | https://pubmed.ncbi.nlm.nih.gov/31663165/ |
Others:
Gold nanoparticles
Shen Q, Nie Z, Guo M, et al. Simple and rapid colorimetric sensing of enzymatic cleavage and oxidative damage of single-stranded DNA with unmodified gold nanoparticles as indicator, Chem Commun, 2009(pg. 929-31)
- there is no paper that combine this technique with CRISPR technique
- length-dependent adsorption of ssDNA to gold nanoparticles (AuNPs) via electrostatic interactions: with longer ssDNA sequences adsorbing more slowly to AuNPs compared with shorter sequences.
- In the absence of nuclease S1, the intact ssDNA oligonucleotides adsorb slowly to the AuNPs (Figure 5).
- The addition of salts triggers the aggregation of AuNPs resulting in a red-to-blue colorimetric signal.
- However, the addition of nuclease S1 results in the enzymatic cleavage of DNA into shorter fragments, which assembly rapidly upon the AuNP surface. The subsequent addition of salt fails to promote aggregation due to electrostatic repulsion between the AuNPs, and thus no color change is observed.