TY - JOUR
T1 - CRISPR-dCas9 based DNA detection scheme for diagnostics in resource-limited settings
AU - Bengtson, Michel
AU - Bharadwaj, Mitasha
AU - Franch, Oskar
AU - van der Torre, Jaco
AU - Meerdink, Veronique
AU - Schallig, Henk
AU - Dekker, Cees
N1 - Funding Information: We thank F. Hol for discussions that initiated this project, and T. Abeel for bioinformatics support. We acknowledge the Delft Global Initiative for financial support. O.F. thanks the Carlsberg foundation for financial support. C D. was funded by The Netherlands Organization of Scientific Research (NWO/ OCW) as part of the NanoFront and Basyc Programs. Publisher Copyright: © 2022 The Royal Society of Chemistry.
PY - 2022/2/7
Y1 - 2022/2/7
N2 - Nucleic-acid detection is crucial for basic research as well as for applications in medicine such as diagnostics. In resource-limited settings, however, most DNA-detection diagnostic schemes are inapplicable since they rely on expensive machinery, electricity, and trained personnel. Here, we present an isothermal DNA detection scheme for the diagnosis of pathogenic DNA in resource-limited settings. DNA was extracted from urine and blood samples using two different instrument-free methods, and amplified using Recombinase Polymerase Amplification with a sensitivity of <10 copies of DNA within 15 minutes. Target DNA was bound by dCas9/sgRNA that was labelled with a DNA oligomer to subsequently induce Rolling Circle Amplification. This second amplification step produced many copies of a G-quadruplex DNA structure that facilitates a colorimetric readout that is visible to the naked eye. This isothermal DNA-detection scheme can be performed at temperatures between 20-45 °C. As an example of the applicability of the approach, we isothermally (23 °C) detected DNA from a parasite causing visceral leishmaniasis that was spiked into buffer and resulted in a sensitivity of at least 1 zeptomole. For proof of principle, DNA spiked into blood was coupled to the CRISPR-dCas9-based detection scheme yielding a colorimetric readout visible to the naked eye. Given the versatility of the guide-RNA programmability of targets, we envision that this DNA detection scheme can be adapted to detect any DNA with minimal means, which facilitates applications such as point-of-care diagnostics in resource-limited settings.
AB - Nucleic-acid detection is crucial for basic research as well as for applications in medicine such as diagnostics. In resource-limited settings, however, most DNA-detection diagnostic schemes are inapplicable since they rely on expensive machinery, electricity, and trained personnel. Here, we present an isothermal DNA detection scheme for the diagnosis of pathogenic DNA in resource-limited settings. DNA was extracted from urine and blood samples using two different instrument-free methods, and amplified using Recombinase Polymerase Amplification with a sensitivity of <10 copies of DNA within 15 minutes. Target DNA was bound by dCas9/sgRNA that was labelled with a DNA oligomer to subsequently induce Rolling Circle Amplification. This second amplification step produced many copies of a G-quadruplex DNA structure that facilitates a colorimetric readout that is visible to the naked eye. This isothermal DNA-detection scheme can be performed at temperatures between 20-45 °C. As an example of the applicability of the approach, we isothermally (23 °C) detected DNA from a parasite causing visceral leishmaniasis that was spiked into buffer and resulted in a sensitivity of at least 1 zeptomole. For proof of principle, DNA spiked into blood was coupled to the CRISPR-dCas9-based detection scheme yielding a colorimetric readout visible to the naked eye. Given the versatility of the guide-RNA programmability of targets, we envision that this DNA detection scheme can be adapted to detect any DNA with minimal means, which facilitates applications such as point-of-care diagnostics in resource-limited settings.
UR - http://www.scopus.com/inward/record.url?scp=85124056054&partnerID=8YFLogxK
U2 - https://doi.org/10.1039/d1nr06557b
DO - https://doi.org/10.1039/d1nr06557b
M3 - Article
C2 - 35044397
SN - 2040-3364
VL - 14
SP - 1885
EP - 1895
JO - Nanoscale
JF - Nanoscale
IS - 5
ER -