CRISPR systems show promise for COVID-19 diagnosis. Current methods like PCR and antigen tests have limitations like long times, high costs, and need for trained operators. CRISPR uses Cas proteins and guide RNA to detect viral RNA or DNA. The Cas12 and Cas13 systems can detect single-stranded nucleic acids and generate a detectable signal through collateral cleavage of a reporter. Studies have used CRISPR/Cas12 to detect SARS-CoV-2 from clinical samples in under 2 hours using fluorescence or lateral flow strips. While CRISPR diagnostics are faster and cheaper than PCR, most platforms still require nucleic acid extraction and amplification before CRISPR detection.

1. CRISPR Systems for
COVID-19 Diagnosis
Presenter: Peng-Wen Liu
Advisor: I-Son Ng
National Cheng Kung University, Taiwan
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2. Introduction of SARS-CoV-2
Spike
protein
Envelope
protein
Membrane
protein
Nuclear capsid
protein and RNA
Ref: The New York Time. Coronavirus World Map: Tracking the Global Outbreak
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2):
A highly transmissible and pathogenic coronavirus that emerged
in late 2019 and has caused a pandemic of acute respiratory
disease, named ‘coronavirus disease 2019’ (COVID-19), which
threatens human health and public safety.
2
(Angiotensin converting enzyme-2)

3. Current clinical practices
qPCR Antigen rapid test
Methods
Clinical significance Gold standard method Screening test
Detection time 2~3 hours 15 min
Detection target RdRp* gene, N gene, E genea S protein, anti-SARS-CoV-2 antibody
Sensitivity 95% ~80%
Limitation 1. Professional operators
2. Time-consuming
3. High-cost (US: $100; TW: NT$3,500)
4. Hard to be realized in communities
Sensitivity varies within days of symptom onset
Ct value
RFU
SARS-CoV-2
Rapid Ag
C
T
SARS-CoV-2
Rapid Ag
C
T
*RdRp: RNA-dependent RNA polymerase
aThe target gene detection is according to the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel
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4. CRISPR system
• CRISPR is the abbreviation of
“Clustered Regularly Interspaced
Short Palindromic Repeat”.
• The system was first found as the
defense mechanism in bacteria
against virus attack.
• The system was then developed
as the gene editing tool which
can change DNA sequence or
modify gene function. 4
Horvath, P., & Barrangou, R. (2010). CRISPR/Cas, the immune system of bacteria and archaea. Science (New York, N.Y.), 327(5962), 167–170.

5. CRISPR mechanism
First, tracrRNA will recruit Cas
protein and binds with it. Next, the
Cas-sgRNA complex will recognize
the target sequence with crRNA.
Programmable
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6. CRISPR system for nucleic acid detection
dCas9 system Cas12 system Cas13 system
1. Can only react with dsDNA.
2. The nuclease activity of Cas9
protein was deactivated.
3. The recognition ability was
remained for gene labeling.
1. Can react with ssDNA/
dsDNA.
2. Having collateral cleavage
activity to cut ssDNA
reporter for detection.
1. Can only react with ssRNA.
2. Having collateral cleavage
activity to cut ssRNA
reporter for detection.
Ref: Wang M, Zhang R, Li J. CRISPR/cas systems redefine nucleic acid detection: Principles and methods. Biosens Bioelectron. 2020;165:112430.
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7. Moon J, Kwon HJ, Yong D, et al. Colorimetric Detection of SARS-CoV-2 and Drug-Resistant pH1N1 Using CRISPR/dCas9. ACS Sens.
2020;5(12):4017-4026.
Figure. (A) Schematic illustration of virus detection based on CRISPR/dCas9. Viral lysate and biotin-PAMmer are added into
a dCas9/gRNA complex-immobilized microplate. Next, streptavidin-HPR and TMB substrate solutions are added to the
microplate. Finally, yellow color is observed in the presence of the virus. (B) (i) Sequence of gRNA, target RNA, and biotin-
PAMmer. (ii) Electrophoretic mobility shift assay for binding test of RNA and biotin-PAMmer with dCas9/gRNA complex
(A) (B) (i)
(ii)
Viral sample pretreatment:
10 μL of TCEP/EDTA 50 °C 5 min + 64 °C
5 min
37oC
60 min
25oC
30 min
1. TMB substrate
2. 2.5 M sulfuric acid
This platform can directly detect target
RNAs without cDNA amplification.
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8. Moon J, Kwon HJ, Yong D, et al. Colorimetric Detection of SARS-CoV-2 and Drug-Resistant pH1N1 Using CRISPR/dCas9. ACS Sens.
2020;5(12):4017-4026.
Linear equation:
y = 0.145x +0.354
Figure. (A) Plot of OD450 nm versus the concentration of SARS-CoV-2 N1 RNA (0, 0.1, 1, 10, and 100 nM). Inset is a photograph of the
microplate after the detection of SARS-CoV-2 N1 RNA using CRISPR/dCas9. Right plot is the linearly fitted line of the left plot.(B)
Photograph of microplate and the corresponding heat map after the detection of SARS-CoV-2 in clinical samples using CRISPR/dCas9.
In the presence of COVID-19 patient samples, the color of all dCas9/gRNA (CoV-2 N1, N2, and N3)-immobilized wells turns yellow.
(A) (B)
COVID-19 positive
COVID-19
negative
No. 1, 2, and 5: Nasopharyngeal aspirates
No. 3 and 4: Sputum
No. 6-8: Control nasopharyngeal aspirate
1. The higher the concentration of RNA, the
more distinct the yellow color was observed.
2. However, it becomes difficult to differentiate
the low viral concentration samples and
negative control with naked eye.
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9. Chen, J. S., Ma, E., Harrington, L. B., Da Costa, M., Tian, X., Palefsky, J. M., & Doudna, J. A. (2018). CRISPR-Cas12a target binding
unleashes indiscriminate single-stranded DNase activity. Science (New York, N.Y.), 360(6387), 436–439.
Fig. (A) Model for PAM-dependent and PAM-independent activation of cis- and trans-cleavage by Cas12a.
(B) Representative Michaelis-Menten plot for LbCas12acatalyzed ssDNA trans cleavage using a dsDNA or ssDNA activator.
Measured kcat/Km values reported as mean ± SD, where n = 3 Michaelis-Menten fits. V0, rate of catalysis.
TS: target strand
NTS: non-target strand
PAM: protospacer adjacent motif
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(A) (B) Catalytic efficiency
The NTS of the dsDNA activator helps stabilize the Cas12a
complex in an optimal conformation for trans-ssDNA cutting.

10. Milenia HybriDetect kit@TwistDx
Fig. (A) Schematic of SARS-CoV-2 DETECTR workflow. Conventional RNA extraction can be used as an input to DETECTR (LAMP
preamplification and Cas12-based detection for E gene, N gene and RNase P), which is visualized by a fluorescent reader
or lateral flow strip. (B) Schematic of DETECTR coupled with lateral flow readout. The intact FAM-biotinylated reporter
molecule flows to the control capture line. Upon recognition of the matching target, the Cas–gRNA complex cleaves the
reporter molecule, which flows to the target capture line.
Broughton JP, Deng X, Yu G, et al. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020;38(7):870-
874.
Amplification:
62 °C for 20–30 min
Lateral flow assay within 5 min
Collateral cleavage
for 10 min
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11. Milenia HybriDetect kit@TwistDx
-Positive- -Negative-
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https://www.milenia-biotec.com/en/tips-lateral-flow-readouts-crispr-cas-strategies/

12. Fig. (A) Comparison of fluorescence to lateral flow.
(B) LoD for DETECTR assays. Fluorescence values using SARS-CoV-2 DETECTR assay (n = 6) using SARS-CoV-2 N2 gene IVT RNA.
(C) Performance characteristics of fluorescent SARS-CoV-2 DETECTR assay.
Broughton JP, Deng X, Yu G, et al. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020;38(7):870-
874.
(A)
(B)
(C)
Fluorescence signal of LbCas12a detection assay
on RT–LAMP amplicon for SARS-CoV-2 N gene
saturates within 10 min.
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PPV/NPV: positive/negative percent agreement

13. Kellner, M.J., Koob, J.G., Gootenberg, J.S. et al. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc 14, 2986–
3012 (2019).
Fig. (a) CRISPR−Cas13 RNA targeting complex components. (b) Reporter unlocking via CRISPR−Cas13 collateral RNase activity. (c)
SHERLOCK detection assay. RPA, Recombinase polymerase assay. T7, T7 RNA polymerase.
HEPN: higher eukaryotic and
prokaryotic nuclease domains Activated by binding
with target RNA
Single strand RNA
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14. Kellner, M. J., Koob, J. G., Gootenberg, J. S., Abudayyeh, O. O., & Zhang, F. (2019). SHERLOCK: nucleic acid detection with CRISPR
nucleases. Nature Protocols, 14(10), 2986–3012.
Fig. Detection can be performed or a single-plex colorimetric lateral flow reaction.
Timing: 40 min ~ 2 hr
Hybridetect
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1 hr incubation
Step 1. 25 min incubation – Isothermal amplification (RPA) of the extracted nucleic acid
Step 2. 30 min incubation – detection of pre-amplified viral RNA sequence using Cas13.
Step 3. 2 min incubation – visual read out of the detection result by eye using a
commercially-available paper dipstick.

15. Most of the CRISPR-based detection platforms still require nucleic acid extraction
and amplification prior to detection. 15

16. Conclusion
d
1. Compared with qPCR and rapid antigen test,
the CRISPR-base diagnostic platforms have the
advantages of speed, accuracy, low cost and
versatility. Current systems includes: dCas9,
Cas12 and Cas13, etc.
2. RPA and LAMP isothermal amplification
approaches are typically used in CRISPR-based
diagnostic works to amplify target genomic
sequences.
3. CRISPR-based diagnostic platforms are still only
capable of qualitative detection and cannot
quantify the desired nucleic acid content.
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17. THANK YOU !