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Identification and mitigation of pervasive off-target activity in CRISPR-Cas9 screens for essential non-coding elements

ABSTRACT: Pooled CRISPR-Cas9 screens have recently emerged as a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we conducted multiple genome-scale CRISPR screens for essential CTCF loop anchors in the human K562 erythroid cell line. Surprisingly, the primary drivers of apparent ``hits'' in this screen were single guide RNAs (sgRNAs) with low sequence specificity. After removing these confounders, we found that no CTCF loop anchors among the ones we screened are essential for cell growth in culture. We also observed analogous effects in independent non-coding screens densely tiling regulatory elements and genomic neighborhoods near previously known essential genes. Strikingly, we found that low-specificity guides also result in strong confounding growth effects in screens employing epigenetic perturbations that do not cause DNA damage, such as CRISPRi and CRISPRa. Remarkably, the set of confounded guides is distinct for each perturbation mode. Promisingly, strict filtering of CRISPRi libraries using GuideScan-aggregate specificity scores removed these confounded sgRNAs and allowed for the identification of essential enhancers, which we validated extensively. Our stduy presents the first genome-scale functional characterization of CTCF binding sites in the human genome, while also identifying the limitations on and outlining the future prospects for the detailed functional dissection of regulatory elements in the genome using Cas9.

ORGANISM(S): Homo sapiens

PROVIDER: GSE131349 | GEO | 2019/07/01

REPOSITORIES: GEO

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Project description:Using 4C-Seq experimental procedure we have characterized, in cultured chicken lymphoid and erythroid cells, genome-wide patterns of spatial contacts of several CpG islands scattered along the chromosome 14. A clear tendency for interaction of CpG islands present within the same and different chromosomes has been observed. Accordingly, preferential spatial contacts between Sp1 binding motifs, and other GC-rich genomic elements including DNA sequence motifs capable to form G-quadruplexes were demonstrated. On the other hand, an anchor placed in gene/CpG islands-poor area was found to form spatial contacts with other gene/CpG islands-poor areas within chromosome 14 and other chromosomes. These results corroborate the two compartments model of interphase chromosome spatial organization and suggest that clustering of CpG islands harboring promoters and origins of DNA replication constitutes an important determinant of the 3D organization of eukaryotic genome in the cell nucleus. Using ChIP-Seq experimental procedure we have mapped genome-wide the CTCF deposition sites in chicken lymphoid and erythroid cells subjected to the 4C analysis. A good correlation between the density of these sites and the level of 4C signals was observed for the anchors located in CpG islands. It is thus possible that CTCF contributes to the clustering of CpG islands revealed in our experiments. We applied 4C-seq to map long-range interactions of a CpG island harboring promoter of a housekeeping gene NPRL3 on a genome-wide scale in DT40 and HD3 cell lines in chicken (Gallus gallus). Two replicates per cell line were sequenced in paired-end mode with a depth of 45-64 million reads. We next performed additional 4C experiments on HD3 cells with different 4C anchors. In three experiments anchors were placed on different CpG island on the chromosome 14 that have demonstrated a strong interaction with the NPRL3 anchor (anchors near TSR3, TRAP1, PPL genes). In the forth experiment (anchor GENE-Des) the anchor was placed in a gene-poor area that did not interact with the NPRL3 promoter. The libraries for all anchors were pooled and sequenced in paired-end mode with a total depth of 75 million reads. Anchor NPRL3: two replicates for HD3 and two replicates for DT40 cell lines. Other anchors (TSR3, GENE-Des, TRAP1, PPL): two replicates, pooled library

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Identification and mitigation of pervasive off-target activity in CRISPR-Cas9 screens for essential non-coding elements

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