Fig. 1: PAM identification by PAM-DOSE.
From: Versatile and efficient genome editing with Neisseria cinerea Cas9
a Schematic showing the CRISPR/Cas loci of the strains encoding the Nme1Cas9, Nme2Cas9, and NcCas9 orthologs. Red arrows denote tracrRNA transcription initiation sites. Percent identities of Nme2Cas9 and NcCas9 with Nme1Cas9 are 86% and 94%, respectively. b Schematic showing the PAM-DOSE assay for characterization of PAM sequences recognized by Cas9s. An 8 N library of PAM sequences were introduced into the constructs. In the presence of a functional PAM, cleavage-mediated tdTomato cassette excision was performed, which led to the expression of EGFP. After Cas9 cleavage, cleaved products were PCR amplified and subjected to deep sequencing to identify PAM sequences. c The sequence logo showing the PAM specificity of Nme1Cas9, Nme2Cas9, and NcCas9 was obtained using deep-sequencing data.
