Functional metagenomic screening for anti-CRISPR acitvity

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. The 39 insert sequences can be found at Biostudies repository under accession number S-BSST226.