Scientific Understanding of Consciousness
Consciousness as an Emergent Property of Thalamocortical Activity

Genome-Editing with Rationally Engineered Cas9 Nucleases


Science  01 Jan 2016: Vol. 351, Issue 6268, pp. 84-88

Rationally engineered Cas9 nucleases with improved specificity

Ian M. Slaymaker,,

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.

McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Graduate Program in Biophysics, Harvard Medical School, Boston, MA 02115, USA.

Harvard-MIT Division of Health Sciences and Technology, Harvard Medical School, Boston, MA 02115, USA.


The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that “enhanced specificity” SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.

The RNA-guided endonuclease Cas9 from microbial clustered regularly interspaced short palindromic repeat (CRISPR)Cas adaptive immune systems is a powerful tool for genome editing in eukaryotic cells. However, the nuclease activity of Cas9 can be triggered even when there is imperfect complementarity between the RNA guide sequence and an off-target genomic site, particularly if mismatches are distal to the protospacer adjacent motif (PAM), a short stretch of nucleotides required for target selection. These off-target effects pose a challenge for genome-editing applications. Here, we report the structure-guided engineering of Streptococcus pyogenes Cas9 (SpCas9) to improve its DNA targeting specificity.

We have demonstrated through structure-guided design that neutralization of positive charges in the nt-groove can dramatically decrease off-target indel formation while preserving on-target activity. These data show that eSpCas9(1.1) can be used to increase the specificity of genome-editing applications. Future structure-guided interrogation of Cas9 binding and cleavage mechanism will likely enable further optimization of the CRISPR-Cas9 genome-editing toolbox.

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