Bidirectional
Degradation of DNA Cleavage Products
Catalyzed by CRISPR/Cas9
Version 2 2018-02-20, 23:43
Version 1 2018-02-19, 22:44
Posted on 2018-02-20 - 23:43
Since
the initial characterization of Streptococcus pyogenes CRISPR/Cas9 as a powerful gene-editing tool, it has been widely
accepted that Cas9 generates blunt-ended DNA products by concerted
cleavage of the target (tDNA) and non-target (ntDNA) strands three
nucleotides away from the protospacer adjacent motif (PAM) by HNH
and RuvC nuclease active sites, respectively. Following initial DNA
cleavage, RuvC catalyzes 3′→5′ degradation of
the ntDNA resulting in DNA products of various lengths. Here, we found
that Cas9 selects multiple sites for initial ntDNA cleavage and preferentially
generates staggered-ended DNA products containing single-nucleotide
5′-overhangs. We also quantitatively evaluated 3′→5′
post-cleavage trimming (PCT) activity of RuvC to find that ntDNA degradation
continues up to the −10 position on the PAM distal DNA product
and is kinetically significant when compared to extremely slow DNA
product release. We also discovered a previously unidentified 5′→3′
PCT activity of RuvC which can shorten the PAM proximal ntDNA product
by precisely one nucleotide with a comparable rate as the 3′→5′
PCT activity. Taken together, our results demonstrate that RuvC-catalyzed
PCT ultimately generates DNA fragments with heterogeneous ends following
initial DNA cleavage including a PAM proximal fragment with a blunt
end and a PAM distal fragment with a staggered-end, 3′-recessed
on the ntDNA strand. These kinetic and biochemical findings underline
the importance of temporal control of Cas9 during gene-editing experiments
and help explain the patterns of nucleotide insertions at sites of
Cas9-catalyzed gene modification in vivo.
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Stephenson, Anthony
A.; Raper, Austin T.; Suo, Zucai (2018). Bidirectional
Degradation of DNA Cleavage Products
Catalyzed by CRISPR/Cas9. ACS Publications. Collection. https://doi.org/10.1021/jacs.7b13050
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AUTHORS (3)
AS
Anthony
A. Stephenson
AR
Austin T. Raper
ZS
Zucai Suo