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Functional metagenomic screening for anti-CRISPR acitvity

ABSTRACT: Using a synthetic genetic circuit we developed a high-throughput approach to discover anti-CRISPR genes from metagenomic libraries based on their functional activity rather than sequence homology or genetic context. We identified eleven DNA fragments from soil, animal and human metagenomes that circumvent Streptococcus pyogenes Cas9 activity in our selection strain. We transformed different metagenomic libraries into our selection strain and then selected the libraries in inhibitory concentrations of chloramphenicol (30 µg ml-1) and collected the clones that appeared on the selective plates. Metagenomic inserts were extracted from pooled clones on selection plates, barcoded per library and sequenced using nanopore technology to obtain full inserts. Note that this submission is linked to European Nucleotide Archive Study ID PRJEB29470, where the sequences of the four anti-CRISPRs discovered in this study have been submitted.

SUBMITTER: Maria-Anna Misiakou

PROVIDER: S-BSST226 | biostudies-other |

SECONDARY ACCESSION(S): PRJEB29470

REPOSITORIES: biostudies-other

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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.

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Functional metagenomic screening for anti-CRISPR acitvity

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