Engineered transcription-associated Cas9 targeting in eukaryotic cells
Abstract DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid...
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Nature Portfolio
2024-11-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-024-54629-9 |
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| author | Gregory W. Goldberg Manjunatha Kogenaru Sarah Keegan Max A. B. Haase Larisa Kagermazova Mauricio A. Arias Kenenna Onyebeke Samantha Adams Daniel K. Beyer David Fenyö Marcus B. Noyes Jef D. Boeke |
| author_facet | Gregory W. Goldberg Manjunatha Kogenaru Sarah Keegan Max A. B. Haase Larisa Kagermazova Mauricio A. Arias Kenenna Onyebeke Samantha Adams Daniel K. Beyer David Fenyö Marcus B. Noyes Jef D. Boeke |
| author_sort | Gregory W. Goldberg |
| collection | DOAJ |
| description | Abstract DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid sequence specific but function constitutively; thus, they provide intrinsic spatial control over DNA targeting activities but naturally lack temporal control. Here we show that engineered Cas9 fusion proteins which bind to nascent RNAs near a protospacer can facilitate spatiotemporal coupling between transcription and DNA targeting at that protospacer: Transcription-associated Cas9 Targeting (TraCT). Engineered TraCT is enabled in eukaryotic yeast or human cells when suboptimal PAM interactions limit basal activity and when one or more nascent RNA substrates are still tethered to the actively transcribed target DNA in cis. Using yeast, we further show that this phenomenon can be applied for selective editing at one of two identical targets in distinct gene loci, or, in diploid allelic loci that are differentially transcribed. Our work demonstrates that temporal control over Cas9’s targeting activity at specific DNA sites may be engineered without modifying Cas9’s core domains and guide RNA components or their expression levels. More broadly, it establishes co-transcriptional RNA binding as a cis-acting mechanism that can conditionally stimulate CRISPR-Cas DNA targeting in eukaryotic cells. |
| format | Article |
| id | doaj-art-3bdeec378a054518868d5fd4d1eac2b8 |
| institution | OA Journals |
| issn | 2041-1723 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-3bdeec378a054518868d5fd4d1eac2b82025-08-20T02:08:26ZengNature PortfolioNature Communications2041-17232024-11-0115111910.1038/s41467-024-54629-9Engineered transcription-associated Cas9 targeting in eukaryotic cellsGregory W. Goldberg0Manjunatha Kogenaru1Sarah Keegan2Max A. B. Haase3Larisa Kagermazova4Mauricio A. Arias5Kenenna Onyebeke6Samantha Adams7Daniel K. Beyer8David Fenyö9Marcus B. Noyes10Jef D. Boeke11Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone HealthInstitute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone HealthInstitute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone HealthInstitute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone HealthInstitute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone HealthCourant Institute of Mathematical Sciences, New York UniversityInstitute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone HealthInstitute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone HealthInstitute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone HealthInstitute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone HealthInstitute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone HealthInstitute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone HealthAbstract DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid sequence specific but function constitutively; thus, they provide intrinsic spatial control over DNA targeting activities but naturally lack temporal control. Here we show that engineered Cas9 fusion proteins which bind to nascent RNAs near a protospacer can facilitate spatiotemporal coupling between transcription and DNA targeting at that protospacer: Transcription-associated Cas9 Targeting (TraCT). Engineered TraCT is enabled in eukaryotic yeast or human cells when suboptimal PAM interactions limit basal activity and when one or more nascent RNA substrates are still tethered to the actively transcribed target DNA in cis. Using yeast, we further show that this phenomenon can be applied for selective editing at one of two identical targets in distinct gene loci, or, in diploid allelic loci that are differentially transcribed. Our work demonstrates that temporal control over Cas9’s targeting activity at specific DNA sites may be engineered without modifying Cas9’s core domains and guide RNA components or their expression levels. More broadly, it establishes co-transcriptional RNA binding as a cis-acting mechanism that can conditionally stimulate CRISPR-Cas DNA targeting in eukaryotic cells.https://doi.org/10.1038/s41467-024-54629-9 |
| spellingShingle | Gregory W. Goldberg Manjunatha Kogenaru Sarah Keegan Max A. B. Haase Larisa Kagermazova Mauricio A. Arias Kenenna Onyebeke Samantha Adams Daniel K. Beyer David Fenyö Marcus B. Noyes Jef D. Boeke Engineered transcription-associated Cas9 targeting in eukaryotic cells Nature Communications |
| title | Engineered transcription-associated Cas9 targeting in eukaryotic cells |
| title_full | Engineered transcription-associated Cas9 targeting in eukaryotic cells |
| title_fullStr | Engineered transcription-associated Cas9 targeting in eukaryotic cells |
| title_full_unstemmed | Engineered transcription-associated Cas9 targeting in eukaryotic cells |
| title_short | Engineered transcription-associated Cas9 targeting in eukaryotic cells |
| title_sort | engineered transcription associated cas9 targeting in eukaryotic cells |
| url | https://doi.org/10.1038/s41467-024-54629-9 |
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