03/​04/​2021

Covid Mutation Test with CRISPR and HybriDetect

At the end of 2019 the first infec­tions of the coron­avirus (SARS-​​CoV-​​2) in humans have been identified in Wuhan, China. Since then the covid 19 outbreak has been distributed over the entire world with millions of people infected and a lot of reported deaths.

Now, more than 1 year that the pandemic started and the many people infected, it is no surprise that covid mutants could be identified, given that this is in the nature of such viruses. This new coron­avirus variants are at the suspicion to infect humans more effec­tively than the wild type and possibly may lead to more severe courses of the disease. Three major variants of SARS-​​CoV-​​2 are currently under the focus of attention, all belonging to the so-​​called N501Y mutation. These are the UK coron­avirus variant N501Y.V1 (B 1.1.7 variant), the South African coron­avirus N501Y.V2 and the Brazilian coron­avirus N501Y.V3.

In order to keep infection rates low an accurate identi­fi­cation of the virus mutations in SARS-​​CoV-​​2 positive people is strongly needed. Point mutations are difficult to diagnose using qPCR tests. So far mutations are identified by genome­se­quencing of the virus. However, due to the sophis­ti­cated equipment and the long turn-​​around-​​time this is only done in very specialized labora­tories from laboratory staff.

At present there is a high demand of simple and reliable identi­fi­cation of the N501Y type coron­avirus mutations which can be done in any lab.

New Publi­cation brings hope for fast and easy detection of Covid mutations

Just recently Kumar et al. published a paper on their devel­opment of a CRISPR based FnCas9 (Francisella novicida Cas9) method combined with the lateral flow dipstick Milenia HybriDetect called RAY (Rapid Variant AssaY). This test is designed for the simple and accurate detection of the British, South African and Brazilian coron­avirus. Some months ago, in 2020, this group has also developed a test for the detection of the wild type of SARS-​​CoV-​​2 which is called FELUDA (FnCas9 Editor Linked Uniform Detection Assay). FELUDA is also a CRISPR based FnCas9 method and has been described in our blog earlier. The test devel­opers already published in September 2020 that FnCas9 shows a very high speci­ficity and could therefore be an ideal tool for the detection of point mutations. In their new paper they benefit from this feature of FnCas9 and were able to success­fully detect all N501Y-​​type mutations with a simple paper strip test.

 

Devel­opment of RAY to detect coron­avirus variant

Due to the lack of fast antigen based tests to identify SARS-​​CoV-​​2 mutant variants there is a high need of fast and cheaper mutant detection than the gold standard sequencing. The authors showed, that RAY can detect the covid infection and the presence of common N501Y mutation in one test with a high test sensi­tivity.

The researchers partic­u­larly concen­trated on the SARS-​​CoV2 mutation N501Y, because it is present in all three new virus variants. A major step was to design a guide RNA (sgRNA) for the N501Y mutant, resulting in binding of FnCas9 to the mutant, but not to the wildtype.

First the group searched for the Cas9 PAM (Proto­spacer adjacent motif) sequence (NGG) in the surrounding area of the known mutations. The Cas9 protein requires the short specific PAM sequence to bind to the target sequence. The test devel­opers recog­nized 10 sites targetable for FnCas9. One was the N501Y mutation which is present in all three new covid mutants.

Second they designed a FnCas9 sgRNA for the N501Y mutation. To design RAY, they took advantage of the fact, that FnCas9 is unable to bind targets with 2 mismatches at the PAM proximal 2nd and 6th position (which was previ­ously reported). The wildtype of the virus is not addressed with this sgRNA and gives no signal.

The S-​​gene region addressed by the sgRNA for the detection of SARS-​​CoV2 used in the FELUDA test, which is not affected by the mutations, is used as an internal positive control.

Kumar et al. validated RAY on another SARS-​​CoV-​​2 mutation (T716I) and could show, that the principle of the test can be applied to other genetic variants of the virus.

SARS-​​CoV-​​2 Mutation Test on HybriDetect: RAY

To improve the sensi­tivity and the test perfor­mance compared to electrophoresis based identi­fi­cation of the virus mutations, the test devel­opers adapted RAY on Milenia HybriDetect lateral flow dipsticks (like FELUDA). Therefore, the reverse transcription and ampli­fi­cation of the S-​​gene region of SARS-​​CoV-​​2 containing N501Y mutation was done using biotiny­lated primers. The guideRNA was FAM-​​labeled (see Fig. x) and a catalytical inactive FnCas9 used. In a next step, the biotiny­lated amplicons and the RNP (gRNA +FnCas9) were incubated for 10 minutes at 37°C. To get a visible result on HybriDetect dipsticks, the sample was mixed with HybriDetect buffer and a dipstick placed for 2 to 5 minutes in the reaction.

 

For the covid mutation test, the FELUDA sgRNA is used in one reaction.

Fig. 1.: Lateral Flow Assay using FnCas9 for Covid19 detection. (1)

For the SARS-​​CoV-​​2 mutation test, the detecion by the S-​​gene sgRNA means, that the sample is SARS-​​CoV-​​2 positive. The detection by both sgRNAs – S-​​gene and N501Y – impli­cates, that a mutated variant of the coron­avirus was detected in the sample.

The authors tested RAY on samples which were tested positive for SARS-​​CoV-​​2 by qPCR and sequenced for the detection of N501Y muation. The results showed, that RAY was able to identify all samples with genetic variants of the virus, even the samples with very high Ct values.

Fig. 2.: Covid Mutation and Wildtype Detection on Patient Samples (2)

Advan­tages of RAY compared to sequencing tests

The test can be done with a regular thermo­cycler within 1 hour. The detection of the amplicons is done via our lateral flow strips and results can be reported within 5 minutes after starting the run of the test strips just by visual inspection. Therefore, the entire procedure from the time of taking the sample until the result can be reported takes only around 75 minutes. This test could be used for rapid diagnosis of mutated covid19 virus variants and therefore has the ability to increase test capacity which is highly needed. Still sequencing needs to be done to get infor­mation about new virus mutations occurring.

Do you want to know more about our products and news About Milenia Biotec? Please follow us on LinkedIn
https://​www​.linkedin​.com/​c​o​m​p​a​n​y​/​m​i​l​e​n​i​a​-​b​iotec

Refer­ences
  1. Azhar, M., Phutela R., Kumar M., Ansari A.H., Rauthan R., Gulati S., Sharma N., Sinha D., Saumya S., Singh S., Acharya S., Paul D., Kathpalia P., Aich M., Sehgal P., Ranjan G., Bhoyar R., Singhal K., Lad H., Patra P.K., Makharia G., Chandak G.R., Pesala B., Chakraborty D., Maiti S: Rapid, accurate, nucle­obase detection using FnCas9 https://​doi​.org/​10​.1101 (2020)
  2. Kumar M., Gulati S., Ansari A.H., Phutela R., Acharya S., Kathpalia P., Kanakan A., Maurya R., Vasudevan J.S., Murali A., Pandey R., Maiti S., Chakraborty D: RAY:CRISPR diagnostic for rapid and accurate detection pf SARS-​​CoV2 variants on a paper strip https://​doi​.org/​10​.​1101​/​2021​.​02​.​01​.​21250900 (2021)