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

By Max A. Horlbeck, Luke A. Gilbert, Jacqueline E. Villalta, Britt Adamson, Ryan A. Pak, Yuwen Chen, Alexander P. Fields, C. Park, J. Corn, M. Kampmann and 1 more. eLife, 2016

795 citations recorded by the source corpus.

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

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/6ef86d48461ec98339f7935d5dc069083efbc912

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Citations in the corpus

A multiplexed single-cell CRISPR screening platform enables systematic dissection of the unfolded protein response (2016, methodology): “CRISPRi also has advantages when scaling to high-order combinations relative to CRISPR cutting, as genetic variability during indel formation and nonspecific toxicity due to DNA cutting both increase with the number of cut sites (Boettcher and McManus, 2015; Horlbeck et al., 2016; Wang et al., 2015)”
Genome-wide Programmable Transcriptional Memory by CRISPR-based Epigenome Editing (2021, methodology): “We designed a sgRNA library that targets over 20,000 protein-coding genes and includes 1,000 non-targeting sgRNAs (Horlbeck et al., 2016a; Replogle et al., 2020).”
CRISPRi-based genome-scale identification of functional long non-coding RNA loci in human cells (2016, positive): “S7A). sgRNAs outside of this 1-kb window around the TSS, which would not be expected to affect transcription of the major PVT1 isoform (23), showed no consistent impact on growth; this finding implies that the observed pro-growth phenotype is mediated by transcriptional interference.”