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SRX23829838: GSM8125122: CD55_parental-gCH27; Homo sapiens; OTHER
1 ILLUMINA (Illumina MiSeq) run: 256,716 spots, 35.9M bases, 10.6Mb downloads

External Id: GSM8125122_r1
Submitted by: Luke Gilbert Lab, Arc Institute
Study: Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations (amplicon)
show Abstracthide Abstract
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. 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).
Sample: CD55_parental-gCH27
SAMN40253978 • SRS20651884 • All experiments • All runs
Organism: Homo sapiens
Library:
Name: GSM8125122
Instrument: Illumina MiSeq
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: For analysis at the CD55 and CD81 loci, genomic DNA was isolated using NucleoSpin Blood (Macherey-Nagel, Catalog no. 740951.50). For analysis at the KIT locus, cell lystates were generated using QuickExtract DNA Solution (Lucigen) and directly subjected to PCR amplification. For analysis of CD55 and CD81 loci, PCRs for loci of interest were run using Amplicon-EZ (Genewiz) partial Illumina adapters and amplicons were processed using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Catalog no. 740609.250). Paired end (2 x 250 bp) sequencing was completed at GENEWIZ (Azenta Life Sciences). For analysis of the KIT locus, cell lysates were subjected to 15 cycles of PCR to introduce Illumina sequencing primer binding sites and 0-8 staggered bases to ensure library diversity. After reaction clean-up using ExoSAP-IT kit (Thermo Fisher 78201), an additional 15 cycles of PCR was used to introduce unique dual indices and Illumina P5 and P7 adaptors. Libraries were pooled and purified by SPRIselect magnetic beads before paired-end sequencing using an Illumina MiSeq. Sequencing primer binding sites, unique dual indices (from Illumina TruSeq kits), P5 and P7 adaptor sequences are from Illumina Adaptor Sequences Document # 1000000002694 v16. PCR amplicon sequencing
Runs: 1 run, 256,716 spots, 35.9M bases, 10.6Mb
Run# of Spots# of BasesSizePublished
SRR28217719256,71635.9M10.6Mb2024-03-05

ID:
32131635

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