Degenerate sequence-based CRISPR diagnostic for Crimean-Congo hemorrhagic fever virus.
CRISPR (clustered regularly interspaced short palindromic repeats), an ancient defense mechanism used by prokaryotes to cleave nucleic acids from invading viruses and plasmids, is currently being harnessed by researchers worldwide to develop new point-of-need diagnostics. In CRISPR diagnostics, a CR...
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ftdoajarticles:oai:doaj.org/article:25d4992587604365b80906f3358f5ac2 2023-05-15T15:16:03+02:00 Degenerate sequence-based CRISPR diagnostic for Crimean-Congo hemorrhagic fever virus. Hongzhao Li Alexander Bello Greg Smith Dominic M S Kielich James E Strong Bradley S Pickering 2022-03-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0010285 https://doaj.org/article/25d4992587604365b80906f3358f5ac2 EN eng Public Library of Science (PLoS) https://doi.org/10.1371/journal.pntd.0010285 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0010285 https://doaj.org/article/25d4992587604365b80906f3358f5ac2 PLoS Neglected Tropical Diseases, Vol 16, Iss 3, p e0010285 (2022) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2022 ftdoajarticles https://doi.org/10.1371/journal.pntd.0010285 2022-12-31T08:10:51Z CRISPR (clustered regularly interspaced short palindromic repeats), an ancient defense mechanism used by prokaryotes to cleave nucleic acids from invading viruses and plasmids, is currently being harnessed by researchers worldwide to develop new point-of-need diagnostics. In CRISPR diagnostics, a CRISPR RNA (crRNA) containing a "spacer" sequence that specifically complements with the target nucleic acid sequence guides the activation of a CRISPR effector protein (Cas13a, Cas12a or Cas12b), leading to collateral cleavage of RNA or DNA reporters and enormous signal amplification. CRISPR function can be disrupted by some types of sequence mismatches between the spacer and target, according to previous studies. This poses a potential challenge in the detection of variable targets such as RNA viruses with a high degree of sequence diversity, since mismatches can result from target variations. To cover viral diversity, we propose in this study that during crRNA synthesis mixed nucleotide types (degenerate sequences) can be introduced into the spacer sequence positions corresponding to viral sequence variations. We test this crRNA design strategy in the context of the Cas13a-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) technology for detection of Crimean-Congo hemorrhagic fever virus (CCHFV), a biosafety level 4 pathogen with wide geographic distribution and broad sequence variability. The degenerate-sequence CRISPR diagnostic proves functional, sensitive, specific and rapid. It detects within 30-40 minutes 1 copy/μl of viral RNA from CCHFV strains representing all clades, and from more recently identified strains with new mutations in the CRISPR target region. Also importantly, it shows no cross-reactivity with a variety of CCHFV-related viruses. This proof-of-concept study demonstrates that the degenerate sequence-based CRISPR diagnostic is a promising tool of choice for effective detection of highly variable viral pathogens. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 16 3 e0010285 |
institution |
Open Polar |
collection |
Directory of Open Access Journals: DOAJ Articles |
op_collection_id |
ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Hongzhao Li Alexander Bello Greg Smith Dominic M S Kielich James E Strong Bradley S Pickering Degenerate sequence-based CRISPR diagnostic for Crimean-Congo hemorrhagic fever virus. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
CRISPR (clustered regularly interspaced short palindromic repeats), an ancient defense mechanism used by prokaryotes to cleave nucleic acids from invading viruses and plasmids, is currently being harnessed by researchers worldwide to develop new point-of-need diagnostics. In CRISPR diagnostics, a CRISPR RNA (crRNA) containing a "spacer" sequence that specifically complements with the target nucleic acid sequence guides the activation of a CRISPR effector protein (Cas13a, Cas12a or Cas12b), leading to collateral cleavage of RNA or DNA reporters and enormous signal amplification. CRISPR function can be disrupted by some types of sequence mismatches between the spacer and target, according to previous studies. This poses a potential challenge in the detection of variable targets such as RNA viruses with a high degree of sequence diversity, since mismatches can result from target variations. To cover viral diversity, we propose in this study that during crRNA synthesis mixed nucleotide types (degenerate sequences) can be introduced into the spacer sequence positions corresponding to viral sequence variations. We test this crRNA design strategy in the context of the Cas13a-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) technology for detection of Crimean-Congo hemorrhagic fever virus (CCHFV), a biosafety level 4 pathogen with wide geographic distribution and broad sequence variability. The degenerate-sequence CRISPR diagnostic proves functional, sensitive, specific and rapid. It detects within 30-40 minutes 1 copy/μl of viral RNA from CCHFV strains representing all clades, and from more recently identified strains with new mutations in the CRISPR target region. Also importantly, it shows no cross-reactivity with a variety of CCHFV-related viruses. This proof-of-concept study demonstrates that the degenerate sequence-based CRISPR diagnostic is a promising tool of choice for effective detection of highly variable viral pathogens. |
format |
Article in Journal/Newspaper |
author |
Hongzhao Li Alexander Bello Greg Smith Dominic M S Kielich James E Strong Bradley S Pickering |
author_facet |
Hongzhao Li Alexander Bello Greg Smith Dominic M S Kielich James E Strong Bradley S Pickering |
author_sort |
Hongzhao Li |
title |
Degenerate sequence-based CRISPR diagnostic for Crimean-Congo hemorrhagic fever virus. |
title_short |
Degenerate sequence-based CRISPR diagnostic for Crimean-Congo hemorrhagic fever virus. |
title_full |
Degenerate sequence-based CRISPR diagnostic for Crimean-Congo hemorrhagic fever virus. |
title_fullStr |
Degenerate sequence-based CRISPR diagnostic for Crimean-Congo hemorrhagic fever virus. |
title_full_unstemmed |
Degenerate sequence-based CRISPR diagnostic for Crimean-Congo hemorrhagic fever virus. |
title_sort |
degenerate sequence-based crispr diagnostic for crimean-congo hemorrhagic fever virus. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2022 |
url |
https://doi.org/10.1371/journal.pntd.0010285 https://doaj.org/article/25d4992587604365b80906f3358f5ac2 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 16, Iss 3, p e0010285 (2022) |
op_relation |
https://doi.org/10.1371/journal.pntd.0010285 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0010285 https://doaj.org/article/25d4992587604365b80906f3358f5ac2 |
op_doi |
https://doi.org/10.1371/journal.pntd.0010285 |
container_title |
PLOS Neglected Tropical Diseases |
container_volume |
16 |
container_issue |
3 |
container_start_page |
e0010285 |
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1766346366324310016 |