Diagnostic CRISPR Tools and Techniques

Lateral Flow Assays using Genome Editing Tools

What is CRISPR/Cas?

The CRISPR / Cas system acts as an immune system equivalent that is anchored in the genome of bacteria and archaea. The underlying mechanism protects the organism from phage infections by sequence specific destruction of “unknown” nucleic acids. The CRISPR/Cas-system has the ability to learn, remember and adapt. (1, 2)

This defense system is based on regions of repeating DNA-sequences, called “Clustered Regularly Interspaced Short Palindromic Repeats, also referred to as CRISPR. In addition, CRISPR-associated proteins (Cas proteins) are required for successful defense. Transcribed CRISPR-RNAs are able to guide Cas

protein(s) to the viral genome. A characteristic feature of Cas proteins is the endonuclease activity, which causes the specific degradation of viral nucleic acids. These combined features of sequence specific recognition and cutting have been used for the development of genome editing tools. (3, 4)

Figure 1. The steps of CRISPR-mediated immunity. adapted from Molecular Cell 54, April 24, 2014. (5)
Importance of Diagnostic Alternatives in the Point-of-Care Field

There is a great need for diagnostic alternatives that are suitable for simple, fast, specific, sensitive, and inexpensive early detection of pathogens. Simple handling and the avoidance of expensive and complex devices are considered to be particularly important, especially for third world countries or regions with limited lab capacities. (6, 7)

This deficiency became very clear in the years 2014 to 2016 during the Ebola outbreak (8). Today we are experiencing an even more extreme situation. The Sars-CoV-2 pandemic affects the whole world and safe, scalable diagnostics is one of the most important issues today. As part of WHO’s response to the outbreak, the R&D Blueprint has been activated to accelerate diagnostics, vaccines and therapeutics for this novel coronavirus (9).
What Makes CRISPR Associated Enzymes Attractive for Diagnostic Purposes?

In recent years, CRISPR/Cas-based detection systems have increasingly come into focus as serious diagnostic alternatives. But why are some Cas proteins particularly suitable for molecular biosensing?
Within the Cas protein classification model, all diagnostically relevant Cas proteins originate from class II. These are multidomain proteins that are guided to the target nucleic acid by associated single stranded RNAs. Another essential feature of diagnostically relevant Cas proteins is a so-called collateral activity, which occurs after a target sequence has been successfully recognized. Single-stranded RNA-/ DNA-fragments are efficiently degraded in a short period of time due to this collateral activity. And this function makes these proteins such interesting tools in diagnostics. By using molecular reporters, the collateral activity can be used for signal generation in a diagnostic approach. Reporters are short DNA- or RNA-fragments that have defined labels at their ends. Reporter cleavage leads to the generation of a detectable signal.

CRISPR/Cas-dependent Reporter Cleavage via Collateral Activity

Different Cas proteins have special characteristics and differ in some relevant criteria such as: size, recognition of nucleic acid type, collateral degradation of ssDNA or ssRNA (10). Table 1 gives a brief overview of the three diagnostically relevant Cas proteins and their specific features.

Table 1. Characteristics of commonly used CRISPR-associated Proteins for diagnostic purpose

CRISPR/Cas-Systems and Lateral Flow Readout with HybriDetect

CRISPR/Cas-based detection methods can be combined with a simple Lateral Flow Readout. Therefore the universal lateral flow platform HybriDetect is the perfect tool for a sensitive, rapid, equipment-free and simple visualization of test results (11). The general mechanism is explained in the following Figure 3.

Figure 3. General mechanism of CRISPR/Cas-mediated detection of nucleic acids via HybriDetect Lateral Flow. (A) The presence of the genetic target leads to a positive test result. (B) The absence of the specific genetic target leads to a negative test result.

The interpretation of the HybriDetect dipstick is extremely simple. If the intensity of the T-line exceeds the T-line intensity of the negative control, the test is interpreted as positive. At the same time, the C-line intensity decreases in clear positive results. The simple and intuitive interpretation of the test strips is illustrated in the following figure. About 200 copies of artificial virus RNA can be clearly detected using the CRISPR/Cas-based detection method in combination with HybriDetect lateral flow assay.

Figure 4. Detection of synthetic ZIKA virus ssRNA using SHERLOCK with 1 hour of LwaCas13a reaction, followed by Lateral Flow with Milenia HybriDetect. (12)

CRISPR/Cas-detection methods are mostly combined with pre-amplification steps. Isothermal amplification such as LAMP or RPA are the most frequently used techniques. Combinations of these methods are named SHERLOCK, DETECTR or HOLMES. Recently, it has been proved that these CRISPR/Cas-methods are able to detect pathogenic viral genomes at attomolar levels in a simple, rapid and low-equip Point-of-Care-approach using the Milenia HybriDetect.

# Detection of Zika Virus , SHERLOCK_CRISPR/Cas13-System (12,13)
# Detection of Dengue Virus, SHERLOCK_CRISPR/Cas13-System (12)
# Detection of Human Papillomavirus (HPV) -16 and -18, CRISPR/Cas12a-System (14)
# Detection of SARS-CoV-2, CRISPR/Cas12a-System, CRISPR/Cas13-System (15,16)


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André Breitbach
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