Off-Target Activity in CRISPR Screens of the Non-Coding Genome

The non-coding genome makes up 98% of human genetic sequence and contains most trait-associated variants found by GWAS. However, we lack functional characterization of non-coding elements, so the consequence of variation in these elements is difficult to predict. Now, many researchers are using CRISPR-Cas9 to edit or epigenetically modify non-coding regions to test their function in high-throughput pooled screens.

Here, we report that this approach can be confounded by CRISPR-Cas9 off-targets, at least when screening for essential non-coding elements. Further, we find that CRISPRi and CRISPRa non-coding screens are also vulnerable to a bias for low-specificity sgRNAs. In one example, a CRISPR screen for essential CTCF-bound chromatin loop anchors returned many more off-target artifacts than true hits. Lastly, we observe that sgRNA libraries targeting the non-coding genome are less specific than gene-targeting libraries due to their design constraints. Non-coding perturbation studies should include careful consideration and mitigation of the off-target effects that arise from using low-specificity sgRNA.