Dataset Information

Refined sgRNA efficacy prediction improves large- and small-scale CRISPR-Cas9 applications.

ABSTRACT: Genome editing with the CRISPR-Cas9 system has enabled unprecedented efficacy for reverse genetics and gene correction approaches. While off-target effects have been successfully tackled, the effort to eliminate variability in sgRNA efficacies-which affect experimental sensitivity-is in its infancy. To address this issue, studies have analyzed the molecular features of highly active sgRNAs, but independent cross-validation is lacking. Utilizing fluorescent reporter knock-out assays with verification at selected endogenous loci, we experimentally quantified the target efficacies of 430 sgRNAs. Based on this dataset we tested the predictive value of five recently-established prediction algorithms. Our analysis revealed a moderate correlation (r = 0.04 to r = 0.20) between the predicted and measured activity of the sgRNAs, and modest concordance between the different algorithms. We uncovered a strong PAM-distal GC-content-dependent activity, which enabled the exclusion of inactive sgRNAs. By deriving nine additional predictive features we generated a linear model-based discrete system for the efficient selection (r = 0.4) of effective sgRNAs (CRISPRater). We proved our algorithms' efficacy on small and large external datasets, and provide a versatile combined on- and off-target sgRNA scanning platform. Altogether, our study highlights current issues and efforts in sgRNA efficacy prediction, and provides an easily-applicable discrete system for selecting efficient sgRNAs.

SUBMITTER: Labuhn M

PROVIDER: S-EPMC5814880 | biostudies-other | 2018 Feb

REPOSITORIES: biostudies-other

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Refined sgRNA efficacy prediction improves large- and small-scale CRISPR-Cas9 applications.

Labuhn Maurice M Adams Felix F FF Ng Michelle M Knoess Sabine S Schambach Axel A Charpentier Emmanuelle M EM Schwarzer Adrian A Mateo Juan L JL Klusmann Jan-Henning JH Heckl Dirk D

Nucleic acids research 20180201 3

Genome editing with the CRISPR-Cas9 system has enabled unprecedented efficacy for reverse genetics and gene correction approaches. While off-target effects have been successfully tackled, the effort to eliminate variability in sgRNA efficacies-which affect experimental sensitivity-is in its infancy. To address this issue, studies have analyzed the molecular features of highly active sgRNAs, but independent cross-validation is lacking. Utilizing fluorescent reporter knock-out assays with verifica ...[more]

PMID: 29267886

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Project description:BackgroundLarge-scale genetic screening using CRISPR-Cas9 technology has emerged as a powerful approach to uncover and validate gene functions. The ability to control the timing of genetic perturbation during CRISPR screens will facilitate precise dissection of dynamic and complex biological processes. Here, we report the optimization of a drug-inducible CRISPR-Cas9 system that allows high-throughput gene interrogation with a temporal control.ResultsWe designed multiple drug-inducible sgRNA expression vectors and measured their activities using an EGFP gene disruption assay in 11 human and mouse cell lines. The optimal design allows for a tight and inducible control of gene knockout in vitro, and in vivo during a seven-week-long experiment following hematopoietic reconstitution in mice. We next performed parallel genome-wide loss-of-function screens using the inducible and constitutive CRISPR-Cas9 systems. In proliferation-based dropout screens, these two approaches have similar performance in discriminating essential and nonessential genes. In a more challenging phenotypic assay that requires cytokine stimulation and cell staining, we observed similar sensitivity of the constitutive and drug-induced screening approaches in detecting known hits. Importantly, we demonstrate minimal leakiness of our inducible CRISPR screening platforms in the absence of chemical inducers in large-scale settings.ConclusionsIn this study, we have developed a drug-inducible CRISPR-Cas9 system that shows high cleavage efficiency upon induction but low background activity. Using this system, we have achieved inducible gene disruption in a wide range of cell types both in vitro and in vivo. For the first time, we present a systematic side-by-side comparison of constitutive and drug-inducible CRISPR-Cas9 platforms in large-scale functional screens. We demonstrate the tightness and efficiency of our drug-inducible CRISPR-Cas9 system in genome-wide pooled screening. Our design increases the versatility of CRISPR-based genetic screening and represents a significant upgrade on existing functional genomics toolbox.

Dataset Information

Refined sgRNA efficacy prediction improves large- and small-scale CRISPR-Cas9 applications.

Publications

Refined sgRNA efficacy prediction improves large- and small-scale CRISPR-Cas9 applications.

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