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| Status |
Public on Mar 05, 2024 |
| Title |
Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations (amplicon) |
| Organism |
Homo sapiens |
| Experiment type |
Other
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| Summary |
Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting 1-3 genomic sites per cell. We engineer an Acidaminococcus Cas12a (AsCas12a) variant, multiplexed transcriptional interference AsCas12a (multiAsCas12a), that incorporates R1226A, a mutation that stabilizes the ribonucleoprotein:DNA complex via DNA nicking. The multiAsCas12a-KRAB fusion improves CRISPRi activity over DNase-dead AsCas12a-KRAB fusions, often rescuing the activities of lentivirally delivered CRISPR RNAs (crRNA) that are inactive when used with the latter. multiAsCas12a-KRAB supports CRISPRi using 6-plex crRNA arrays in high-throughput pooled screens. Using multiAsCas12a-KRAB, we discover enhancer elements and dissect the combinatorial function of cis-regulatory elements in human cells. These results instantiate a group testing framework for efficiently surveying numerous combinations of chromatin perturbations for biological discovery and engineering.
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| Overall design |
Analysis of indel frequencies by short-read Illumina sequencing of PCR amplicons surrounding genomic sites targeted by Cas12a variant fusion proteins using CRISPR RNAs (crRNA).
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| Contributor(s) |
Hsiung CC, Wilson CM, Sambold NA, Dai R, Chen Q, Misiukiewicz S, Arab A, Teyssier N, O'Loughlin T, Cofsky JC, Gilbert LA |
| Citation(s) |
38760567 |
| |
| Submission date |
Mar 04, 2024 |
| Last update date |
Jun 04, 2024 |
| Contact name |
Chris Hsiung |
| Organization name |
Arc Institute
|
| Lab |
Luke Gilbert Lab
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| Street address |
3181 Porter Dr
|
| City |
Palo Alto |
| State/province |
CA |
| ZIP/Postal code |
94304 |
| Country |
USA |
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| Platforms (1) |
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| Samples (19)
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| This SubSeries is part of SuperSeries: |
| GSE260832 |
Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations |
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| Relations |
| BioProject |
PRJNA1083589 |
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