Cargando…
Oligo targeting for profiling drug resistance mutations in the parasitic trypanosomatids
Trypanosomatids cause the neglected tropical diseases, sleeping sickness, Chagas disease and the leishmaniases. Studies on these lethal parasites would be further facilitated by new and improved genetic technologies. Scalable precision editing methods, for example, could be used to improve our under...
Autores principales: | , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2022
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9371896/ https://www.ncbi.nlm.nih.gov/pubmed/35524555 http://dx.doi.org/10.1093/nar/gkac319 |
_version_ | 1784767261976821760 |
---|---|
author | Altmann, Simone Rico, Eva Carvalho, Sandra Ridgway, Melanie Trenaman, Anna Donnelly, Hannah Tinti, Michele Wyllie, Susan Horn, David |
author_facet | Altmann, Simone Rico, Eva Carvalho, Sandra Ridgway, Melanie Trenaman, Anna Donnelly, Hannah Tinti, Michele Wyllie, Susan Horn, David |
author_sort | Altmann, Simone |
collection | PubMed |
description | Trypanosomatids cause the neglected tropical diseases, sleeping sickness, Chagas disease and the leishmaniases. Studies on these lethal parasites would be further facilitated by new and improved genetic technologies. Scalable precision editing methods, for example, could be used to improve our understanding of potential mutations associated with drug resistance, a current priority given that several new anti-trypanosomal drugs, with known targets, are currently in clinical development. We report the development of a simple oligo targeting method for rapid and precise editing of priority drug targets in otherwise wild type trypanosomatids. In Trypanosoma brucei, approx. 50-b single-stranded oligodeoxynucleotides were optimal, multiple base edits could be incorporated, and editing efficiency was substantially increased when mismatch repair was suppressed. Resistance-associated edits were introduced in T. brucei cyclin dependent kinase 12 (CRK12, L(482)F) or cleavage and polyadenylation specificity factor 3 (N(232)H), in the Trypanosoma cruzi proteasome β5 subunit (G(208)S), or in Leishmania donovani CRK12 (G(572)D). We further implemented oligo targeting for site saturation mutagenesis, targeting codon G(492) in T. brucei CRK12. This approach, combined with amplicon sequencing for codon variant scoring, revealed fourteen resistance conferring G(492) edits encoding six distinct amino acids. The outputs confirm on-target drug activity, reveal a variety of resistance-associated mutations, and facilitate rapid assessment of potential impacts on drug efficacy. |
format | Online Article Text |
id | pubmed-9371896 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-93718962022-08-12 Oligo targeting for profiling drug resistance mutations in the parasitic trypanosomatids Altmann, Simone Rico, Eva Carvalho, Sandra Ridgway, Melanie Trenaman, Anna Donnelly, Hannah Tinti, Michele Wyllie, Susan Horn, David Nucleic Acids Res Methods Online Trypanosomatids cause the neglected tropical diseases, sleeping sickness, Chagas disease and the leishmaniases. Studies on these lethal parasites would be further facilitated by new and improved genetic technologies. Scalable precision editing methods, for example, could be used to improve our understanding of potential mutations associated with drug resistance, a current priority given that several new anti-trypanosomal drugs, with known targets, are currently in clinical development. We report the development of a simple oligo targeting method for rapid and precise editing of priority drug targets in otherwise wild type trypanosomatids. In Trypanosoma brucei, approx. 50-b single-stranded oligodeoxynucleotides were optimal, multiple base edits could be incorporated, and editing efficiency was substantially increased when mismatch repair was suppressed. Resistance-associated edits were introduced in T. brucei cyclin dependent kinase 12 (CRK12, L(482)F) or cleavage and polyadenylation specificity factor 3 (N(232)H), in the Trypanosoma cruzi proteasome β5 subunit (G(208)S), or in Leishmania donovani CRK12 (G(572)D). We further implemented oligo targeting for site saturation mutagenesis, targeting codon G(492) in T. brucei CRK12. This approach, combined with amplicon sequencing for codon variant scoring, revealed fourteen resistance conferring G(492) edits encoding six distinct amino acids. The outputs confirm on-target drug activity, reveal a variety of resistance-associated mutations, and facilitate rapid assessment of potential impacts on drug efficacy. Oxford University Press 2022-05-07 /pmc/articles/PMC9371896/ /pubmed/35524555 http://dx.doi.org/10.1093/nar/gkac319 Text en © The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Altmann, Simone Rico, Eva Carvalho, Sandra Ridgway, Melanie Trenaman, Anna Donnelly, Hannah Tinti, Michele Wyllie, Susan Horn, David Oligo targeting for profiling drug resistance mutations in the parasitic trypanosomatids |
title | Oligo targeting for profiling drug resistance mutations in the parasitic trypanosomatids |
title_full | Oligo targeting for profiling drug resistance mutations in the parasitic trypanosomatids |
title_fullStr | Oligo targeting for profiling drug resistance mutations in the parasitic trypanosomatids |
title_full_unstemmed | Oligo targeting for profiling drug resistance mutations in the parasitic trypanosomatids |
title_short | Oligo targeting for profiling drug resistance mutations in the parasitic trypanosomatids |
title_sort | oligo targeting for profiling drug resistance mutations in the parasitic trypanosomatids |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9371896/ https://www.ncbi.nlm.nih.gov/pubmed/35524555 http://dx.doi.org/10.1093/nar/gkac319 |
work_keys_str_mv | AT altmannsimone oligotargetingforprofilingdrugresistancemutationsintheparasitictrypanosomatids AT ricoeva oligotargetingforprofilingdrugresistancemutationsintheparasitictrypanosomatids AT carvalhosandra oligotargetingforprofilingdrugresistancemutationsintheparasitictrypanosomatids AT ridgwaymelanie oligotargetingforprofilingdrugresistancemutationsintheparasitictrypanosomatids AT trenamananna oligotargetingforprofilingdrugresistancemutationsintheparasitictrypanosomatids AT donnellyhannah oligotargetingforprofilingdrugresistancemutationsintheparasitictrypanosomatids AT tintimichele oligotargetingforprofilingdrugresistancemutationsintheparasitictrypanosomatids AT wylliesusan oligotargetingforprofilingdrugresistancemutationsintheparasitictrypanosomatids AT horndavid oligotargetingforprofilingdrugresistancemutationsintheparasitictrypanosomatids |