Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasite Schistosoma mansoni [version 1; peer review: 2 approved]
Background. At least 250 million people worldwide suffer from schistosomiasis, caused by Schistosoma worms. Genome sequences for several Schistosoma species are available, including a high-quality annotated reference for Schistosoma mansoni. There is a pressing need to develop a reliable functional...
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| Format: | Article |
| Language: | English |
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Wellcome
2020-07-01
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| Series: | Wellcome Open Research |
| Online Access: | https://wellcomeopenresearch.org/articles/5-178/v1 |
| _version_ | 1818342022159794176 |
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| author | Geetha Sankaranarayanan Avril Coghlan Patrick Driguez Magda E. Lotkowska Mandy Sanders Nancy Holroyd Alan Tracey Matthew Berriman Gabriel Rinaldi |
| author_facet | Geetha Sankaranarayanan Avril Coghlan Patrick Driguez Magda E. Lotkowska Mandy Sanders Nancy Holroyd Alan Tracey Matthew Berriman Gabriel Rinaldi |
| author_sort | Geetha Sankaranarayanan |
| collection | DOAJ |
| description | Background. At least 250 million people worldwide suffer from schistosomiasis, caused by Schistosoma worms. Genome sequences for several Schistosoma species are available, including a high-quality annotated reference for Schistosoma mansoni. There is a pressing need to develop a reliable functional toolkit to translate these data into new biological insights and targets for intervention. CRISPR-Cas9 was recently demonstrated for the first time in S. mansoni, to produce somatic mutations in the omega-1 (ω1) gene. Methods. We employed CRISPR-Cas9 to introduce somatic mutations in a second gene, SULT-OR, a sulfotransferase expressed in the parasitic stages of S. mansoni, in which mutations confer resistance to the drug oxamniquine. A 262-bp PCR product spanning the region targeted by the gRNA against SULT-OR was amplified, and mutations identified in it by high-throughput sequencing. Results. We found that 0.3-2.0% of aligned reads from CRISPR-Cas9-treated adult worms showed deletions spanning the predicted Cas9 cut site, compared to 0.1-0.2% for sporocysts, while deletions were extremely rare in eggs. The most common deletion observed in adults and sporocysts was a 34 bp-deletion directly upstream of the predicted cut site, but rarer deletions reaching as far as 102 bp upstream of the cut site were also detected. The CRISPR-Cas9-induced deletions, if homozygous, are predicted to cause resistance to oxamniquine by producing frameshifts, ablating SULT-OR transcription, or leading to mRNA degradation via the nonsense-mediated mRNA decay pathway. However, no SULT-OR knock down at the mRNA level was observed, presumably because the cells in which CRISPR-Cas9 did induce mutations represented a small fraction of all cells expressing SULT-OR. Conclusions. Further optimisation of CRISPR-Cas protocols for different developmental stages and particular cell types, including germline cells, will contribute to the generation of a homozygous knock-out in any gene of interest, and in particular the SULT-OR gene to derive an oxamniquine-resistant stable transgenic line. |
| first_indexed | 2024-12-13T16:08:04Z |
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| id | doaj.art-962673fb93c94d0f9736f6df15d2ed9e |
| institution | Directory Open Access Journal |
| issn | 2398-502X |
| language | English |
| last_indexed | 2024-12-13T16:08:04Z |
| publishDate | 2020-07-01 |
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| series | Wellcome Open Research |
| spelling | doaj.art-962673fb93c94d0f9736f6df15d2ed9e2022-12-21T23:39:00ZengWellcomeWellcome Open Research2398-502X2020-07-01510.12688/wellcomeopenres.16031.117586Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasite Schistosoma mansoni [version 1; peer review: 2 approved]Geetha Sankaranarayanan0Avril Coghlan1Patrick Driguez2Magda E. Lotkowska3Mandy Sanders4Nancy Holroyd5Alan Tracey6Matthew Berriman7Gabriel Rinaldi8Wellcome Sanger Institute, Hinxton, CB10 1SA, UKWellcome Sanger Institute, Hinxton, CB10 1SA, UKWellcome Sanger Institute, Hinxton, CB10 1SA, UKWellcome Sanger Institute, Hinxton, CB10 1SA, UKWellcome Sanger Institute, Hinxton, CB10 1SA, UKWellcome Sanger Institute, Hinxton, CB10 1SA, UKWellcome Sanger Institute, Hinxton, CB10 1SA, UKWellcome Sanger Institute, Hinxton, CB10 1SA, UKWellcome Sanger Institute, Hinxton, CB10 1SA, UKBackground. At least 250 million people worldwide suffer from schistosomiasis, caused by Schistosoma worms. Genome sequences for several Schistosoma species are available, including a high-quality annotated reference for Schistosoma mansoni. There is a pressing need to develop a reliable functional toolkit to translate these data into new biological insights and targets for intervention. CRISPR-Cas9 was recently demonstrated for the first time in S. mansoni, to produce somatic mutations in the omega-1 (ω1) gene. Methods. We employed CRISPR-Cas9 to introduce somatic mutations in a second gene, SULT-OR, a sulfotransferase expressed in the parasitic stages of S. mansoni, in which mutations confer resistance to the drug oxamniquine. A 262-bp PCR product spanning the region targeted by the gRNA against SULT-OR was amplified, and mutations identified in it by high-throughput sequencing. Results. We found that 0.3-2.0% of aligned reads from CRISPR-Cas9-treated adult worms showed deletions spanning the predicted Cas9 cut site, compared to 0.1-0.2% for sporocysts, while deletions were extremely rare in eggs. The most common deletion observed in adults and sporocysts was a 34 bp-deletion directly upstream of the predicted cut site, but rarer deletions reaching as far as 102 bp upstream of the cut site were also detected. The CRISPR-Cas9-induced deletions, if homozygous, are predicted to cause resistance to oxamniquine by producing frameshifts, ablating SULT-OR transcription, or leading to mRNA degradation via the nonsense-mediated mRNA decay pathway. However, no SULT-OR knock down at the mRNA level was observed, presumably because the cells in which CRISPR-Cas9 did induce mutations represented a small fraction of all cells expressing SULT-OR. Conclusions. Further optimisation of CRISPR-Cas protocols for different developmental stages and particular cell types, including germline cells, will contribute to the generation of a homozygous knock-out in any gene of interest, and in particular the SULT-OR gene to derive an oxamniquine-resistant stable transgenic line.https://wellcomeopenresearch.org/articles/5-178/v1 |
| spellingShingle | Geetha Sankaranarayanan Avril Coghlan Patrick Driguez Magda E. Lotkowska Mandy Sanders Nancy Holroyd Alan Tracey Matthew Berriman Gabriel Rinaldi Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasite Schistosoma mansoni [version 1; peer review: 2 approved] Wellcome Open Research |
| title | Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasite Schistosoma mansoni [version 1; peer review: 2 approved] |
| title_full | Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasite Schistosoma mansoni [version 1; peer review: 2 approved] |
| title_fullStr | Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasite Schistosoma mansoni [version 1; peer review: 2 approved] |
| title_full_unstemmed | Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasite Schistosoma mansoni [version 1; peer review: 2 approved] |
| title_short | Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasite Schistosoma mansoni [version 1; peer review: 2 approved] |
| title_sort | large crispr cas induced deletions in the oxamniquine resistance locus of the human parasite schistosoma mansoni version 1 peer review 2 approved |
| url | https://wellcomeopenresearch.org/articles/5-178/v1 |
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