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Joint single-cell profiling of Cas9 edits and transcriptomes reveals on- and off-target effects on gene expression

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Homo sapiens

PROVIDER: GSE314191 | GEO | 2025/12/18

REPOSITORIES: GEO

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Joint single-cell profiling of Cas9 edits and transcriptomes reveals on- and off-target effects on gene expression (rhAmpSeq)

Project description:A longstanding barrier in genome engineering with CRISPR-Cas9 has been the inability to characterize editing outcomes at single-cell resolution. We present "Superb-seq", a new scRNA-seq method that measures on-target and off-target genome edits and associated transcriptomes. In contrast to Perturb-seq that captures single-cell guide information, Superb-seq directly captures single-cell edit profiles by T7 in situ transcription. We demonstrate and validate this method through two Superb-seq experiments in 30,000 cells of three cell types, amplicon sequencing (rhAmpSeq), and bulk RNA-seq. Superb-seq uses off-the-shelf kits, standard laboratory equipment, and requires no virus, which will improve CRISPR screens in diverse tissues and functional characterization of CRISPR therapeutics.

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Joint single-cell profiling of Cas9 edits and transcriptomes reveals on- and off-target effects on gene expression (RNA-seq)

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Joint single-cell profiling of Cas9 edits and transcriptomes reveals on- and off-target effects on gene expression (Superb-seq)

Project description:A longstanding barrier in genome engineering with CRISPR-Cas9 has been the inability to measure Cas9 edit outcomes at single-cell resolution. Here we present Superb-seq, a new technology for joint measurement of on-target and off-target Cas9 edits and transcriptomes by single-cell RNA sequencing. In contrast to Perturb-seq methods that read each cell’s guide RNA, Superb-seq directly reads Cas9 edits by leveraging T7 in situ transcription. We performed Superb-seq on 9,500 K562 cells, targeting four chromatin remodeler genes with seven guide RNAs. Superb-seq identified 11,891 edit events in 6,230 edited cells at all seven on-target sites and at 36 off-target sites. One notable off-target edit fell within the first intron of the deubiquitinase gene USP9X, which decreased its expression and perturbed the expression of downstream genes. Superb-seq uses off-the-shelf kits, standard equipment, and requires no virus, which will enable CRISPR screens in virus-intolerant cell types and functional characterization of off-target events.

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Joint single-cell profiling of Cas9 edits and transcriptomes reveals on- and off-target effects on gene expression

Project description:Joint single-cell profiling of Cas9 edits and transcriptomes reveals on- and off-target effects on gene expression

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Joint single-cell profiling of Cas9 edits and transcriptomes reveals on- and off-target effects on gene expression (rhAmpSeq)

Project description:Joint single-cell profiling of Cas9 edits and transcriptomes reveals on- and off-target effects on gene expression (rhAmpSeq)

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Project description:Joint single-cell profiling of Cas9 edits and transcriptomes reveals on- and off-target effects on gene expression (RNA-seq)

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Project description:This SuperSeries is composed of the SubSeries listed below.

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