biomedical and healthCAre Data Discovery Index Ecosystem
biomedical and healthCAre Data Discovery Index Ecosystem
| Title: | Saturating mutagenesis with assembled CRISPR-Cas9 ribonucleoprotein complexes
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| dateReleased: |
05-01-2016
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| description: |
The CRISPR-Cas9 system enables efficient sequence-specific mutagenesis for creating germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we established in vitro-assembled, fluorescent Cas9-sgRNA RNPs in stabilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. Sequence analysis of targeted loci in individual embryos reveals highly efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis reveals preliminary loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show efficient targeting of functional non-coding elements in gene-regulatory regions using saturating mutagenesis towards uncovering functional control elements in transgenic reporters and endogenous genes. Our results suggest that in vitro assembled, fluorescent Cas9-sgRNA RNPs provide a rapid reverse-genetics tool for direct and scalable loss-of-function studies beyond zebrafish applications.
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| privacy: |
not applicable
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| aggregation: |
instance of dataset
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| ID: |
E-MTAB-4143
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| refinement: |
raw
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| dateSubmitted: |
05-01-2015
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| keywords: |
functional genomics
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| dateModified: |
12-15-2015
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| creators: |
Mark Robinson
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| availability: |
available
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| types: |
gene expression
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| name: |
Danio rerio
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| accessURL: | https://www.ebi.ac.uk/arrayexpress/files/E-MTAB-4143/E-MTAB-4143.raw.1.zip |
| storedIn: |
ArrayExpress
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| qualifier: |
gzip compressed
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| format: |
TXT
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| accessType: |
download
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| authentication: |
none
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| authorization: |
none
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| accessURL: | https://www.ebi.ac.uk/arrayexpress/files/E-MTAB-4143/E-MTAB-4143.processed.1.zip |
| storedIn: |
ArrayExpress
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| qualifier: |
gzip compressed
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| format: |
TXT
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| accessType: |
download
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| authentication: |
none
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| authorization: |
none
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| abbreviation: |
EBI
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| homePage: | http://www.ebi.ac.uk/ |
| ID: |
SCR:004727
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| name: |
European Bioinformatics Institute
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| homePage: | https://www.ebi.ac.uk/arrayexpress/ |
| ID: |
SCR:002964
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| name: |
ArrayExpress
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