A CRISPR/Cas9-based enhancement of high-throughput single-cell transcriptomics
Abstract Single-cell RNA-seq (scRNAseq) struggles to capture the cellular heterogeneity of transcripts within individual cells due to the prevalence of highly abundant and ubiquitous transcripts, which can obscure the detection of biologically distinct transcripts expressed up to several orders of m...
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Nature Portfolio
2025-05-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-59880-2 |
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| author | Amitabh C. Pandey Jon Bezney Dante DeAscanis Ethan B. Kirsch Farin Ahmed Austin Crinklaw Kumari Sonal Choudhary Tony Mandala Jeffrey Deason Jasmin S. Hamidi Azeem Siddique Sridhar Ranganathan Keith Brown Jon Armstrong Steven Head Phillip Ordoukhanian Lars M. Steinmetz Eric J. Topol |
| author_facet | Amitabh C. Pandey Jon Bezney Dante DeAscanis Ethan B. Kirsch Farin Ahmed Austin Crinklaw Kumari Sonal Choudhary Tony Mandala Jeffrey Deason Jasmin S. Hamidi Azeem Siddique Sridhar Ranganathan Keith Brown Jon Armstrong Steven Head Phillip Ordoukhanian Lars M. Steinmetz Eric J. Topol |
| author_sort | Amitabh C. Pandey |
| collection | DOAJ |
| description | Abstract Single-cell RNA-seq (scRNAseq) struggles to capture the cellular heterogeneity of transcripts within individual cells due to the prevalence of highly abundant and ubiquitous transcripts, which can obscure the detection of biologically distinct transcripts expressed up to several orders of magnitude lower levels. To address this challenge, here we introduce single-cell CRISPRclean (scCLEAN), a molecular method that globally recomposes scRNAseq libraries, providing a benefit that cannot be recapitulated with deeper sequencing. scCLEAN utilizes the programmability of CRISPR/Cas9 to target and remove less than 1% of the transcriptome while redistributing approximately half of reads, shifting the focus toward less abundant transcripts. We experimentally apply scCLEAN to both heterogeneous immune cells and homogenous vascular smooth muscle cells to demonstrate its ability to uncover biological signatures in different biological contexts. We further emphasize scCLEAN’s versatility by applying it to a third-generation sequencing method, single-cell MAS-Seq, to increase transcript-level detection and discovery. Here we show the possible utility of scCLEAN across a wide array of human tissues and cell types, indicating which contexts this technology proves beneficial and those in which its application is not advisable. |
| format | Article |
| id | doaj-art-51e543890af94b83b1e79f45bb11c46a |
| institution | OA Journals |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-51e543890af94b83b1e79f45bb11c46a2025-08-20T01:53:14ZengNature PortfolioNature Communications2041-17232025-05-0116111710.1038/s41467-025-59880-2A CRISPR/Cas9-based enhancement of high-throughput single-cell transcriptomicsAmitabh C. Pandey0Jon Bezney1Dante DeAscanis2Ethan B. Kirsch3Farin Ahmed4Austin Crinklaw5Kumari Sonal Choudhary6Tony Mandala7Jeffrey Deason8Jasmin S. Hamidi9Azeem Siddique10Sridhar Ranganathan11Keith Brown12Jon Armstrong13Steven Head14Phillip Ordoukhanian15Lars M. Steinmetz16Eric J. Topol17Section of Cardiology, Tulane Heart and Vascular Institute, Department of Medicine, Tulane University School of MedicineGenomics Core Facility, The Scripps Research InstituteJumpcode GenomicsDepartment of Molecular Medicine, Scripps Research Translational Institute, The Scripps Research InstituteGenomics Core Facility, The Scripps Research InstituteJumpcode GenomicsJumpcode GenomicsGenomics Core Facility, The Scripps Research InstituteJumpcode GenomicsDepartment of Molecular Medicine, Scripps Research Translational Institute, The Scripps Research InstituteJumpcode GenomicsJumpcode GenomicsJumpcode GenomicsJumpcode GenomicsGenomics Core Facility, The Scripps Research InstituteGenomics Core Facility, The Scripps Research InstituteDepartment of Genetics, Stanford University School of MedicineDepartment of Molecular Medicine, Scripps Research Translational Institute, The Scripps Research InstituteAbstract Single-cell RNA-seq (scRNAseq) struggles to capture the cellular heterogeneity of transcripts within individual cells due to the prevalence of highly abundant and ubiquitous transcripts, which can obscure the detection of biologically distinct transcripts expressed up to several orders of magnitude lower levels. To address this challenge, here we introduce single-cell CRISPRclean (scCLEAN), a molecular method that globally recomposes scRNAseq libraries, providing a benefit that cannot be recapitulated with deeper sequencing. scCLEAN utilizes the programmability of CRISPR/Cas9 to target and remove less than 1% of the transcriptome while redistributing approximately half of reads, shifting the focus toward less abundant transcripts. We experimentally apply scCLEAN to both heterogeneous immune cells and homogenous vascular smooth muscle cells to demonstrate its ability to uncover biological signatures in different biological contexts. We further emphasize scCLEAN’s versatility by applying it to a third-generation sequencing method, single-cell MAS-Seq, to increase transcript-level detection and discovery. Here we show the possible utility of scCLEAN across a wide array of human tissues and cell types, indicating which contexts this technology proves beneficial and those in which its application is not advisable.https://doi.org/10.1038/s41467-025-59880-2 |
| spellingShingle | Amitabh C. Pandey Jon Bezney Dante DeAscanis Ethan B. Kirsch Farin Ahmed Austin Crinklaw Kumari Sonal Choudhary Tony Mandala Jeffrey Deason Jasmin S. Hamidi Azeem Siddique Sridhar Ranganathan Keith Brown Jon Armstrong Steven Head Phillip Ordoukhanian Lars M. Steinmetz Eric J. Topol A CRISPR/Cas9-based enhancement of high-throughput single-cell transcriptomics Nature Communications |
| title | A CRISPR/Cas9-based enhancement of high-throughput single-cell transcriptomics |
| title_full | A CRISPR/Cas9-based enhancement of high-throughput single-cell transcriptomics |
| title_fullStr | A CRISPR/Cas9-based enhancement of high-throughput single-cell transcriptomics |
| title_full_unstemmed | A CRISPR/Cas9-based enhancement of high-throughput single-cell transcriptomics |
| title_short | A CRISPR/Cas9-based enhancement of high-throughput single-cell transcriptomics |
| title_sort | crispr cas9 based enhancement of high throughput single cell transcriptomics |
| url | https://doi.org/10.1038/s41467-025-59880-2 |
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