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ACS Synth Biol. 2017 Jun 16;6(6):1103-1113. doi: 10.1021/acssynbio.7b00050. Epub 2017 Mar 20.

The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9.

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Institute of Molecular Biophysics, ‡Department of Chemistry and Biochemistry, Florida State University , Tallahassee, Florida 32306, United States.


Cas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be complementary to the guide region, typically 20-nucleotides in length, of the Cas9-bound guide RNA, and adjacent to a short Cas9-specific element called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition of the protospacer- and PAM-interaction with Cas9 will enable development of versatile and safe Cas9-based technology. We report identification and biochemical characterization of Cas9 from Acidothermus cellulolyticus (AceCas9). AceCas9 depends on a 5'-NNNCC-3' PAM and is more efficient in cleaving negative supercoils than relaxed DNA. Kinetic as well as in vivo activity assays reveal that AceCas9 achieves optimal activity when combined with a guide RNA containing a 24-nucleotide complementarity region. The cytosine-specific, DNA topology-sensitive, and extended guide-dependent properties of AceCas9 may be explored for specific genome editing applications.


CRISPR; Cas9; PAM; RNA-guided DNA cleavage

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