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Saturating mutagenesis with assembled CRISPR-Cas9 ribonucleoprotein complexes.

ABSTRACT: Saturating mutagenesis with assembled CRISPR-Cas9 ribonucleoprotein complexes.

PROVIDER: PRJEB12161 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR1193520_1.fastq.gz	Fastqsanger.gz
	ERR1193520_2.fastq.gz	Fastqsanger.gz
	ERR1193521_1.fastq.gz	Fastqsanger.gz
	ERR1193521_2.fastq.gz	Fastqsanger.gz
	ERR1193516_1.fastq.gz	Fastqsanger.gz

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Saturating mutagenesis with assembled CRISPR-Cas9 ribonucleoprotein complexes.

Project 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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