Off-target effect of engineered AsCas12f variants
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE211600 | GEO | 2023/04/24
REPOSITORIES: GEO
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Project description:Five different sgRNAs targeting mCherry gene (wild-type, without a spacer, and three truncated variants) were used to investigate the off-target effects of CRISPR interference.
2023-04-04 | GSE222051 | GEO
Project description:Off-target effect of engineered AsCas12f variants
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Project description:The Detect-seq provides an unbiased method for genome-wide identification of CBE induced off-targets inside of cells. Purpose: Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, its genome-wide specificity remains poorly understood, precluding its therapeutic applications. Unbiased tools are urgently needed to comprehensively evaluate CBE off-targets at the genome-wide level. Methods: The genomic DNA from CBE transfected cells are processed with fragmentation, endogenous d5fC protection, damage repair, biotin-dUTP and 5fdCTP labeling, and pull-down steps. After the sequencing library preparation, the FASTQ files are produced by the Illumina HiSeq XTen platform. To guarantee the reproducibility, each case of the experiment is present with two biological replicates. Results: Through Detect-seq, we comprehensively profiled the genome-wide off-targets of CBE for several representative sgRNAs, and observed both weak, “random-like” off-targets as well as strong Cas9-dependent off-targets in different human cell lines. Cas9-dependent off-targets can be prevalent for several sgRNAs, and demonstrate a divergent degrees of sequence similarity to the sgRNA. These CBE-induced off-target sites differ greatly from those caused by Cas9 nuclease alone, suggesting inherently different properties among these genome editing tools. Targeted amplicon sequencing further corroborated the novel off-target sites by Detect-seq; quite a few sites showed editing ratio that is on the same order of magnitude or even comparable to the on-target sites. Detect-seq also revealed two unexpected types of Cas9-dependent off-targets, which are out-of-protospacer editing and target-strand editing. These novel off-targets were likely caused by an unstable structure at the PAM-distal side of the Cas9-sgRNA-DNA complex. Based upon such understanding to the off-target editing, we engineered several CBE variants bearing mutations in APOBEC1 and obtained improved CBEs with significantly reduced off-target editing activities. Detect-seq utilized a dU-mapping strategy to profile on-target and off-target editing events by CBE at the whole-genome level. We applied Detect-seq to off-target evaluation in HEK293T and MCF7 cells for BE4max with several frequently used sgRNAs: “VEGFA site 2”, “HEK293 site 4”, “EMX1”, and “RNF2”.
2021-04-08 | GSE151265 | GEO
Project description:The Detect-seq provides an unbiased method for genome-wide identification of CBE induced off-targets inside of cells. Purpose: Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, its genome-wide specificity remains poorly understood, precluding its therapeutic applications. Unbiased tools are urgently needed to comprehensively evaluate CBE off-targets at the genome-wide level. Methods: The genomic DNA from CBE transfected cells are processed with fragmentation, endogenous d5fC protection, damage repair, biotin-dUTP and 5fdCTP labeling, and pull-down steps. After the sequencing library preparation, the FASTQ files are produced by the Illumina HiSeq XTen platform. To guarantee the reproducibility, each case of the experiment is present with two biological replicates. Results: Through Detect-seq, we comprehensively profiled the genome-wide off-targets of CBE for several representative sgRNAs, and observed both weak, “random-like” off-targets as well as strong Cas9-dependent off-targets in different human cell lines. Cas9-dependent off-targets can be prevalent for several sgRNAs, and demonstrate a divergent degrees of sequence similarity to the sgRNA. These CBE-induced off-target sites differ greatly from those caused by Cas9 nuclease alone, suggesting inherently different properties among these genome editing tools. Targeted amplicon sequencing further corroborated the novel off-target sites by Detect-seq; quite a few sites showed editing ratio that is on the same order of magnitude or even comparable to the on-target sites. Detect-seq also revealed two unexpected types of Cas9-dependent off-targets, which are out-of-protospacer editing and target-strand editing. These novel off-targets were likely caused by an unstable structure at the PAM-distal side of the Cas9-sgRNA-DNA complex. Based upon such understanding to the off-target editing, we engineered several CBE variants bearing mutations in APOBEC1 and obtained improved CBEs with significantly reduced off-target editing activities.
2021-04-08 | GSE152907 | GEO
Project description:To determine Sox10-regulated genes in the oligodendroglial precursor cell line OLN93, CRISPR/Cas9-mediated genome editing was performed to inactivate Sox10. For CRISPR/Cas9-mediated Sox10 inactivation, two guide sequences (5’-GCTGCGAGCCAGCCCAGGGCC-3’ for clone A and 5’-GTTGCCCAATTCGCCG GGCCC-3’ for clone B) were separately cloned into pX330. Oln93 cells were co-transfected with pEGFP-N1 and the pX330 plasmids that express the respective guide RNA and SpCas9. Transfected GFP-positive cells were enriched by fluorescence activated cell-sorting and seeded at single cell density in 96-well plates. Homogeneously GFP-expressing clones were analysed for loss of Sox10 protein by immunocytochemistry. Clones without Sox10 protein were obtained with both guide RNAs. Sox10 inactivation was confirmed by Sanger sequencing of the Sox10 gene. RNA was isolated from one clone A and one clone B as well as two independent RNA samples of wildtype OLN93 cells and submitted to RNA-seq.
2019-12-15 | GSE136659 | GEO
Project description:Prime editor (PE) has been recently developed to induce efficient and precise on-target editing, whereas its guide RNA (gRNA)-independent off-target effects remain unknown. Here, we used whole-genome and whole-transcriptome sequencing to determine gRNA-independent off-target mutations in cells expanded from single colonies, in which PE generated precise editing at on-target sites. We found that PE triggered no observable gRNA-independent off-target mutation genome-wide or transcriptome-wide in transfected human cells, highlighting its high specificity.
2022-02-03 | GSE178112 | GEO
Project description:Prime editor (PE) has been recently developed to induce efficient and precise on-target editing, whereas its guide RNA (gRNA)-independent off-target effects remain unknown. Here, we used whole-genome and whole-transcriptome sequencing to determine gRNA-independent off-target mutations in cells expanded from single colonies, in which PE generated precise editing at on-target sites. We found that PE triggered no observable gRNA-independent off-target mutation genome-wide or transcriptome-wide in transfected human cells, highlighting its high specificity.
2022-02-03 | GSE178111 | GEO
Project description:The bacterial CRISPR-Cas9 system has been widely adapted for RNA-guided genome editing and gene regulation in diverse organisms yet its in vivo target specificity is poorly understood. Here we provide the first genome-wide binding maps of nuclease-deactivated Cas9 loaded with guide RNAs in mammalian cells. We find a 5-nucleotide seed region in the guide RNA targets Cas9 to thousands of sites in the genome. Chromatin accessibility limits binding to the other hundreds of thousands sites with matching seed sequences, and consequently 70% of off-target binding sites are associated with genes. U-rich seeds have low numbers of off-target sites limited by both low guide RNA abundance and scarcity of complimentary sites in accessible chromatin. Unexpectedly, off-target sites show little evidence of cleavage, supporting a two-state model reminiscent of eukaryotic RNAi machinery where a short seed match triggers binding but extensive pairing is required for cleavage. ChIP-seq of HA-dCas9 loaded with 4 sgRNAs (Phc1-sg1, Phc1-sg2, Nanog-sg2, and Nanog-sg3) in mouse, and 2 sgRNAs in human (EMX1-sg1 and EMX1-sg3)
2014-04-20 | E-GEOD-54745 | biostudies-arrayexpress
Project description:In mammals, double-stranded RNA (dsRNA) plays roles in sequence-specific RNA interference, sequence-independent interferon response, and RNA editing by adenosine deaminases. We have previously shown that long hairpin dsRNA expression in cultured cells does not activate the interferon response, it is poorly processed into siRNAs, and it is partially edited. Here, we demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits expression of co-transfected reporter plasmids but not expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent and independent of a cell type, transfection method, or dsRNA sequence. The inhibition occurs at the level of translation and is mediated by protein kinase R (PKR). PKR binds the expressed dsRNA, becomes phosphorylated and changes its distribution along polysome fractions. In conclusion, we demonstrate that expression from plasmids is selectively repressed if one of co transfected plasmids produces dsRNA. Our results highlight the importance of proper controls and careful interpretation of co-transfection experiments.
2013-12-27 | GSE46959 | GEO