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Cold Spring Harb Protoc. 2016 May 2;2016(5). doi: 10.1101/pdb.prot090183.

Characterizing Cas9 Protospacer-Adjacent Motifs with High-Throughput Sequencing of Library Depletion Experiments.

Author information

1
Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, Massachusetts 02115;
2
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.
3
Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, Massachusetts 02115; Department of Genetics, Harvard Medical School, New Research Building, Boston, Massachusetts 02115;

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.

PMID:
27140916
DOI:
10.1101/pdb.prot090183
[Indexed for MEDLINE]

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