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Nucleic Acids Res. 2014 Dec 16;42(22):13778-87. doi: 10.1093/nar/gku1102. Epub 2014 Nov 15.

Landscape of target:guide homology effects on Cas9-mediated cleavage.

Author information

1
Department of Genetics, Stanford University, Stanford, CA 94305, USA xuhua@stanford.edu.
2
Department of Pathology, Stanford University, Stanford, CA 94305, USA.
3
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
4
Department of Genetics, Stanford University, Stanford, CA 94305, USA Department of Pathology, Stanford University, Stanford, CA 94305, USA Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110, USA xuhua@stanford.edu.

Abstract

To study target sequence specificity, selectivity, and reaction kinetics of Streptococcus pyogenes Cas9 activity, we challenged libraries of random variant targets with purified Cas9::guide RNA complexes in vitro. Cleavage kinetics were nonlinear, with a burst of initial activity followed by slower sustained cleavage. Consistent with other recent analyses of Cas9 sequence specificity, we observe considerable (albeit incomplete) impairment of cleavage for targets mutated in the PAM sequence or in 'seed' sequences matching the proximal 8 bp of the guide. A second target region requiring close homology was located at the other end of the guide::target duplex (positions 13-18 relative to the PAM). Sequences flanking the guide+PAM region had measurable (albeit modest) effects on cleavage. In addition, the first-base Guanine constraint commonly imposed by gRNA expression systems has little effect on overall cleavage efficiency. Taken together, these studies provide an in vitro understanding of the complexities of Cas9-gRNA interaction and cleavage beyond the general paradigm of site determination based on the 'seed' sequence and PAM.

PMID:
25399416
PMCID:
PMC4267615
DOI:
10.1093/nar/gku1102
[Indexed for MEDLINE]
Free PMC Article

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