Thursday, May 22, 2014
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Uracil enrichment in the
seed region of single guide RNAs (sgRNAs) decreases off-target binding by the
clustered, regularly interspaced
short palindromic repeats (CRISPR)-Cas9
Cell culture studies
suggest sgRNA sequence composition influences Cas9 binding specificity. In
mouse embryonic stem cells stably expressing Cas9 and transfected with
sgRNAs, ChIP-seq experiments revealed thousands of binding sites for the Cas9
protein. In the cells, genome editing was detected at 4 of 4 on-target sites
and only 1 of 295 potential off-target sites for 4 sgRNAs. In the cells,
sgRNAs with uracil-rich seed regions or downstream regions were expressed at
lower levels and bound to fewer off-target sites than sgRNAs without uracil
enrichment at the sites. Next steps could include establishing guidelines for
engineering uracil-rich sgRNAs to improve editing-site selectivity.
SciBX 7(20); doi:10.1038/scibx.2014.599
Published online May 22, 2014
Patent and licensing status
Wu, X. et al. Nat.
Biotechnol.; published online April 20, 2014;
Contact: Phillip A. Sharp, Massachusetts Institute of
Technology, Cambridge, Mass.
Contact: Feng Zhang, Broad Institute of MIT and Harvard, Cambridge,
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