Figure 1. Schematic diagram of Cas protein recognition and cleavage of target sequences. Image taken from Mammoth Bio's official website. The Cas protein specifically recognizes and binds to the target sequence through the guide RNA (for dsDNA targets, a PAM sequence is required), forming a ternary complex, which excites trans-cleavage activity while cleaving the target sequence in cis, and converts the surrounding nucleic acid sequence Make indiscriminate cuts. At present, the identified Cas proteins with cis- and trans-cleavage activities are mainly concentrated in Cas12 and Cas13 proteins, which recognize (/cut) DNA sequences and RNA sequences, respectively.

2. CRISPR signal reporting system

Since the Cas protein cuts the surrounding nucleic acid sequence only after specifically recognizing and binding the target sequence, it can be determined whether the target sequence exists in the system to be tested by detecting whether the reporter sequence is cut.

Almost all CRISPR diagnostic methods complete the diagnostic process by adding some detectable exogenous probes to the system.

Lateral Flow Test Strips

 

Zhang Feng's team launched SHERLOCKv2, introducing nucleic acid test strips as a signal reporting system, which is very convenient for POCT use.

 

They introduced two small molecular markers FAM and biotin at both ends of the probe. The commercial nucleic acid test strips they used marked the anti-FAM antibody with colloidal gold, and the detection line on the NC membrane was coated with streptavidin that could bind biotin. This method detects nucleic acid amplification products containing both FAM and biotin.

 

Zhang Feng's team pioneered the interpretation of the test strip itself, using the quality control line of the sandwich test strip as a detection line to detect whether the probe is cut.

 

They adjusted the concentration of the probe so that when the probe was not cut, all the colloidal gold was concentrated in the lower line, and once the probe was cut, the colloidal gold would cross the lower line to the upper line to develop color.

Figure 2. How lateral flow chromatography test strips work. The picture is taken from the official website of Milenia Company. The initial use of the test strip was to detect products containing both FAM and Biotin (as shown in the picture above). After Zhang Feng developed its CRISPR use, Milenia also demonstrated its CRISPR diagnostic use on its official website.

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Figure 3. How the CRISPR diagnostic lateral flow method works. The picture is taken from the SHERLOCKv2 system

The design of interpreting the test strips of the sandwich method through the competition method is very ingenious. The advantage is that you can get rid of the dependence on the instrument and move towards POCT better.

 

Some time ago, the James Collins team developed the popular mask system that can detect the new crown, which also uses this method.

 

However, this test strip has disadvantages, and the concentration of the probe must be adjusted to adapt to the test strip. In addition, it is difficult to do multi-index detection in this way, and it is difficult to add internal quality control while testing, but there have been some new developments in this area.

Figure 4. Interpretation of CRISPR diagnostic lateral flow assays. The picture is taken from the SHERLOCKv2 system. As shown in the rightmost test strip in the figure above, when testing negative samples, there will also be a faint T line, which will cause trouble for the interpretation of the test results.