Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

0

Saturating mutagenesis with assembled CRISPR-Cas9 ribonucleoprotein complexes.


ABSTRACT: 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.

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Danio rerio

SUBMITTER: Mark Robinson 

PROVIDER: E-MTAB-4143 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

Similar Datasets

2014-05-18 | E-GEOD-55887 | biostudies-arrayexpress
2014-06-18 | E-GEOD-58511 | biostudies-arrayexpress
| PRJEB12161 | ENA
2017-12-25 | E-MTAB-6310 | biostudies-arrayexpress
2017-12-15 | E-MTAB-6357 | biostudies-arrayexpress
2017-12-15 | E-MTAB-6358 | biostudies-arrayexpress
2017-12-15 | E-MTAB-6356 | biostudies-arrayexpress
2017-12-15 | E-MTAB-6355 | biostudies-arrayexpress
2021-07-26 | E-MTAB-9948 | biostudies-arrayexpress
2024-10-17 | PXD037398 | Pride