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Cas9:sgRNA in vitro selection libraries

ABSTRACT: Cas9:sgRNA in vitro selection libraries

PROVIDER: PRJNA212837 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR942784_1.fastq.gz	Fastqsanger.gz
	SRR942784_2.fastq.gz	Fastqsanger.gz
	SRR942787_1.fastq.gz	Fastqsanger.gz
	SRR942787_2.fastq.gz	Fastqsanger.gz
	SRR942789_1.fastq.gz	Fastqsanger.gz

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Pervasive Cas9 expression driven by mammalian promoter activity in E. coli affects representation of CRISPR sgRNA libraries

Project description:CRISPR–Cas9 screening relies on uniform representation of single-guide RNA (sgRNA) libraries to enable accurate gene discovery. However, technical biases during library preparation can compromise screen performance. Here we show that commonly used “all-in-one” CRISPR vectors expressing both Cas9 and sgRNAs drive unintended Cas9 protein expression in Escherichia coli during plasmid amplification. This bacterial Cas9 expression causes guide-specific toxicity, leading to selective loss of sgRNAs and highly skewed library representation. We demonstrate that this effect occurs across multiple bacterial strains and affects both targeted and genome-wide libraries, including widely used human CRISPR libraries. Mechanistically, toxicity is driven by Cas9 expression rather than plasmid size and is only partially alleviated by catalytically inactive Cas9. Importantly, replacing the EF-1α promoter with a mouse phosphoglycerate kinase promoter suppresses Cas9 expression in bacteria while preserving genome editing efficiency in mammalian cells and restoring sgRNA uniformity. These findings identify a previously unrecognized source of bias in CRISPR library preparation and provide a practical solution to improve screening fidelity.

2026-03-17 | GSE324219 | GEO

Pervasive Cas9 expression driven by mammalian promoter activity in E. coli affects representation of CRISPR sgRNA libraries [II]

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Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish (Run6)

Project description:Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish. Each loci has a control, where no Cas9/sgRNA were injected. This is amplicon sequencing with Illumina, after PCR amplification. Data was processed with ampliCan R package version 1.1.1.

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Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish (Run 9)

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Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish (Run 10)

2017-12-15 | E-MTAB-6358 | biostudies-arrayexpress

Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish (Run 8)

2017-12-15 | E-MTAB-6356 | biostudies-arrayexpress

Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish (Run 7)

2017-12-15 | E-MTAB-6355 | biostudies-arrayexpress

Genome-wide identification of CRISPR/Cas9 off-targets in human genome

Project description:By a robust unbiased ChIP-seq approach, we demonstrated that CRISPR/Cas9 had crRNA-specific off-target binding activities in human genome. However, most of those binding off-targets could not be efficiently cleaved both in vivo and in vitro which suggested the cleavage off-target activity of CRISPR/Cas9 in human genome is very limited. We provided a valuable tool to further investigate the molecular mechanism of CRISPR/Cas9 and to optimize its in vivo targeting sgRNA binding sites were identified with ChipSeq by using GFP antibody (there are 2 replicates for egfa-t1 sgRNA,emx1 sgRNA and control without sgRNA in Hek293T cells, one egfa-t1 sgRNA,emx1 sgRNA and control without sgRNA in HeLaS3 cells)

2014-06-18 | E-GEOD-58511 | biostudies-arrayexpress

Pervasive Cas9 expression driven by mammalian promoter activity in E. coli affects representation of CRISPR sgRNA libraries

Project description:Pervasive Cas9 expression driven by mammalian promoter activity in E. coli affects representation of CRISPR sgRNA libraries

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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.

2016-05-01 | E-MTAB-4143 | biostudies-arrayexpress

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