title: Characterizing Cas9 Protospacer-Adjacent Motifs with High-Throughput Sequencing of Library Depletion Experiments.
abstract: This protocol outlines a general approach for characterizing the protospacer-adjacent motifs (PAMs) of Cas9 orthologs. It uses a three-plasmid system: One plasmid carries Cas9 and its tracrRNA, a second targeting vector contains the spacer and repeat, and the third plasmid encodes the targeted sequence (as the protospacer) with varying PAM sequences. It leverages the Cas9 nuclease activity to cleave and destroy plasmids that bear a compatible PAM. The level of depletion of a library of targeted plasmids after Cas9-mediated selection can then be assessed by deep sequencing to reveal candidate PAMs for downstream validation.
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, Massachusetts 02115;
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Braff
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, Massachusetts 02115; Department of Genetics, Harvard Medical School, New Research Building, Boston, Massachusetts 02115; Program in Medical Engineering and Medical Physics, Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
authors.forename: Stephanie J
authors.identifier: 0000-0003-4203-2505
authors.initials: SJ
authors.lastname: Yaung
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, Massachusetts 02115;
authors.forename: Kevin M
authors.identifier: 0000-0001-8797-3945
authors.initials: KM
authors.lastname: Esvelt
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, Massachusetts 02115; Department of Genetics, Harvard Medical School, New Research Building, Boston, Massachusetts 02115;
authors.forename: George M
authors.initials: GM
authors.lastname: Church
mesh_terms: D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism*; D005820:Genetic Testing; D010957:Plasmids; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D004720:Endonucleases; D000094704:RNA, Guide, CRISPR-Cas Systems