Multi-locus CRISPRi targeting with a single truncated guide RNA
Abstract A critical goal in functional genomics is evaluating which non-coding elements contribute to gene expression, cellular function, and disease. Functional characterization remains a challenge due to the abundance and complexity of candidate elements. Here, we develop a CRISPRi-based approach...
Saved in:
| Main Authors: | , , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2025-02-01
|
| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-56144-x |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1823861811493994496 |
|---|---|
| author | Molly M. Moore Siddarth Wekhande Robbyn Issner Alejandro Collins Anna J. Cruz Yanjing V. Liu Nauman Javed Salvador Casaní-Galdón Jason D. Buenrostro Charles B. Epstein Eugenio Mattei John G. Doench Bradley E. Bernstein Noam Shoresh Fadi J. Najm |
| author_facet | Molly M. Moore Siddarth Wekhande Robbyn Issner Alejandro Collins Anna J. Cruz Yanjing V. Liu Nauman Javed Salvador Casaní-Galdón Jason D. Buenrostro Charles B. Epstein Eugenio Mattei John G. Doench Bradley E. Bernstein Noam Shoresh Fadi J. Najm |
| author_sort | Molly M. Moore |
| collection | DOAJ |
| description | Abstract A critical goal in functional genomics is evaluating which non-coding elements contribute to gene expression, cellular function, and disease. Functional characterization remains a challenge due to the abundance and complexity of candidate elements. Here, we develop a CRISPRi-based approach for multi-locus screening of putative transcription factor binding sites with a single truncated guide. A truncated guide with hundreds of sequence match sites can reliably disrupt enhancer activity, which expands the targeting scope of CRISPRi while maintaining repressive efficacy. We screen over 13,000 possible CTCF binding sites with 24 guides at 10 nucleotides in spacer length. These truncated guides direct CRISPRi-mediated deposition of repressive H3K9me3 marks and disrupt transcription factor binding at most sequence match target sites. This approach can be a valuable screening step for testing transcription factor binding motifs or other repeated genomic sequences and is easily implemented with existing tools. |
| format | Article |
| id | doaj-art-698680dfb1144e32a6875737033bd7b6 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-698680dfb1144e32a6875737033bd7b62025-02-09T12:44:55ZengNature PortfolioNature Communications2041-17232025-02-0116111210.1038/s41467-025-56144-xMulti-locus CRISPRi targeting with a single truncated guide RNAMolly M. Moore0Siddarth Wekhande1Robbyn Issner2Alejandro Collins3Anna J. Cruz4Yanjing V. Liu5Nauman Javed6Salvador Casaní-Galdón7Jason D. Buenrostro8Charles B. Epstein9Eugenio Mattei10John G. Doench11Bradley E. Bernstein12Noam Shoresh13Fadi J. Najm14Gene Regulation Observatory, Broad Institute of MIT and HarvardGene Regulation Observatory, Broad Institute of MIT and HarvardGene Regulation Observatory, Broad Institute of MIT and HarvardGene Regulation Observatory, Broad Institute of MIT and HarvardGene Regulation Observatory, Broad Institute of MIT and HarvardGenetic Perturbation Platform, Broad Institute of MIT and HarvardGene Regulation Observatory, Broad Institute of MIT and HarvardGene Regulation Observatory, Broad Institute of MIT and HarvardGene Regulation Observatory, Broad Institute of MIT and HarvardGene Regulation Observatory, Broad Institute of MIT and HarvardGene Regulation Observatory, Broad Institute of MIT and HarvardGenetic Perturbation Platform, Broad Institute of MIT and HarvardGene Regulation Observatory, Broad Institute of MIT and HarvardGene Regulation Observatory, Broad Institute of MIT and HarvardGene Regulation Observatory, Broad Institute of MIT and HarvardAbstract A critical goal in functional genomics is evaluating which non-coding elements contribute to gene expression, cellular function, and disease. Functional characterization remains a challenge due to the abundance and complexity of candidate elements. Here, we develop a CRISPRi-based approach for multi-locus screening of putative transcription factor binding sites with a single truncated guide. A truncated guide with hundreds of sequence match sites can reliably disrupt enhancer activity, which expands the targeting scope of CRISPRi while maintaining repressive efficacy. We screen over 13,000 possible CTCF binding sites with 24 guides at 10 nucleotides in spacer length. These truncated guides direct CRISPRi-mediated deposition of repressive H3K9me3 marks and disrupt transcription factor binding at most sequence match target sites. This approach can be a valuable screening step for testing transcription factor binding motifs or other repeated genomic sequences and is easily implemented with existing tools.https://doi.org/10.1038/s41467-025-56144-x |
| spellingShingle | Molly M. Moore Siddarth Wekhande Robbyn Issner Alejandro Collins Anna J. Cruz Yanjing V. Liu Nauman Javed Salvador Casaní-Galdón Jason D. Buenrostro Charles B. Epstein Eugenio Mattei John G. Doench Bradley E. Bernstein Noam Shoresh Fadi J. Najm Multi-locus CRISPRi targeting with a single truncated guide RNA Nature Communications |
| title | Multi-locus CRISPRi targeting with a single truncated guide RNA |
| title_full | Multi-locus CRISPRi targeting with a single truncated guide RNA |
| title_fullStr | Multi-locus CRISPRi targeting with a single truncated guide RNA |
| title_full_unstemmed | Multi-locus CRISPRi targeting with a single truncated guide RNA |
| title_short | Multi-locus CRISPRi targeting with a single truncated guide RNA |
| title_sort | multi locus crispri targeting with a single truncated guide rna |
| url | https://doi.org/10.1038/s41467-025-56144-x |
| work_keys_str_mv | AT mollymmoore multilocuscrispritargetingwithasingletruncatedguiderna AT siddarthwekhande multilocuscrispritargetingwithasingletruncatedguiderna AT robbynissner multilocuscrispritargetingwithasingletruncatedguiderna AT alejandrocollins multilocuscrispritargetingwithasingletruncatedguiderna AT annajcruz multilocuscrispritargetingwithasingletruncatedguiderna AT yanjingvliu multilocuscrispritargetingwithasingletruncatedguiderna AT naumanjaved multilocuscrispritargetingwithasingletruncatedguiderna AT salvadorcasanigaldon multilocuscrispritargetingwithasingletruncatedguiderna AT jasondbuenrostro multilocuscrispritargetingwithasingletruncatedguiderna AT charlesbepstein multilocuscrispritargetingwithasingletruncatedguiderna AT eugeniomattei multilocuscrispritargetingwithasingletruncatedguiderna AT johngdoench multilocuscrispritargetingwithasingletruncatedguiderna AT bradleyebernstein multilocuscrispritargetingwithasingletruncatedguiderna AT noamshoresh multilocuscrispritargetingwithasingletruncatedguiderna AT fadijnajm multilocuscrispritargetingwithasingletruncatedguiderna |