Approach

Summary

Licensing status

Publication and contact information

Drug platforms

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 platform

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 unavailable

Wu, X. et al. Nat. Biotechnol.; published online April 20, 2014;
doi:10.1038/nbt.2889
Contact: Phillip A. Sharp, Massachusetts Institute of Technology, Cambridge, Mass.
e-mail:

sharppa@mit.edu
Contact: Feng Zhang, Broad Institute of MIT and Harvard, Cambridge, Mass.
e-mail:

zhang@broadinstitute.org