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Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model
Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773193/ https://www.ncbi.nlm.nih.gov/pubmed/29346415 http://dx.doi.org/10.1371/journal.pone.0191503 |
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author | Jobst-Schwan, Tilman Schmidt, Johanna Magdalena Schneider, Ronen Hoogstraten, Charlotte A. Ullmann, Jeremy F. P. Schapiro, David Majmundar, Amar J. Kolb, Amy Eddy, Kaitlyn Shril, Shirlee Braun, Daniela A. Poduri, Annapurna Hildebrandt, Friedhelm |
author_facet | Jobst-Schwan, Tilman Schmidt, Johanna Magdalena Schneider, Ronen Hoogstraten, Charlotte A. Ullmann, Jeremy F. P. Schapiro, David Majmundar, Amar J. Kolb, Amy Eddy, Kaitlyn Shril, Shirlee Braun, Daniela A. Poduri, Annapurna Hildebrandt, Friedhelm |
author_sort | Jobst-Schwan, Tilman |
collection | PubMed |
description | Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest. |
format | Online Article Text |
id | pubmed-5773193 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-57731932018-01-26 Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model Jobst-Schwan, Tilman Schmidt, Johanna Magdalena Schneider, Ronen Hoogstraten, Charlotte A. Ullmann, Jeremy F. P. Schapiro, David Majmundar, Amar J. Kolb, Amy Eddy, Kaitlyn Shril, Shirlee Braun, Daniela A. Poduri, Annapurna Hildebrandt, Friedhelm PLoS One Research Article Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest. Public Library of Science 2018-01-18 /pmc/articles/PMC5773193/ /pubmed/29346415 http://dx.doi.org/10.1371/journal.pone.0191503 Text en © 2018 Jobst-Schwan et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Jobst-Schwan, Tilman Schmidt, Johanna Magdalena Schneider, Ronen Hoogstraten, Charlotte A. Ullmann, Jeremy F. P. Schapiro, David Majmundar, Amar J. Kolb, Amy Eddy, Kaitlyn Shril, Shirlee Braun, Daniela A. Poduri, Annapurna Hildebrandt, Friedhelm Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model |
title | Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model |
title_full | Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model |
title_fullStr | Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model |
title_full_unstemmed | Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model |
title_short | Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model |
title_sort | acute multi-sgrna knockdown of keops complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773193/ https://www.ncbi.nlm.nih.gov/pubmed/29346415 http://dx.doi.org/10.1371/journal.pone.0191503 |
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