Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation

Abstract

We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for targeting nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) and activation (CRISPRa). We use this algorithm to design next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes. A CRISPRi screen for essential genes in K562 cells demonstrates that the large majority of sgRNAs are highly active. We also find CRISPRi does not exhibit any detectable non-specific toxicity recently observed with CRISPR nuclease approaches. Precision-recall analysis shows that we detect over 90% of essential genes with minimal false positives using a compact 5 sgRNA/gene library. Our results establish CRISPRi and CRISPRa as premier tools for loss- or gain-of-function studies and provide a general strategy for identifying Cas9 target sites.DOI: http://dx.doi.org/10.7554/eLife.19760.001

Challenges and progress in interpretation of non-coding genetic variants associated with human disease

“…The principles of such screening assays have been well demonstrated by several studies performing Genome-Scale CRISPR Knock-Out. 76,77 Recently, Horlbeck et al 78 described an enhancer-version of the assay by utilizing dCas9 activator and inhibitor. In addition to the throughput, another concern, when performing genome editing, is the choice of a suitable model.…”

CRISPR/Cas9-Based Engineering of the Epigenome

“…Of note, it is recently demonstrated that (d)Cas9 binding to the target DNA may be affected by nucleosome occupancy (Horlbeck et al, 2016b). This observation has led to improved gRNA libraries for gene activation or repression (Horlbeck et al, 2016a). However, it remains unclear to which degree this steric impediment may be circumvented by the use of multiple repressors or activators (e.g.…”

Mammalian Transcription Factor Networks: Recent Advances in Interrogating Biological Complexity

“…Several CRISPR gRNA libraries have recently been published (Sanjana et al, 2014, Tzelepis et al, 2016, Horlbeck et al, 2016), for both genetic deletion and transcriptional activation/repression. As with any screening method, it will be important to develop appropriate readouts and/or reporters for these assays.…”