Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9

  • Nat Biotechnol. 2016 Feb;34(2):184-191. doi: 10.1038/nbt.3437.
John G Doench  #  1 ,  Nicolo Fusi  #  2 ,  Meagan Sullender  #  1 ,  Mudra Hegde  #  1 ,  Emma W Vaimberg  #  1 ,  Katherine F Donovan  1 ,  Ian Smith  1 ,  Zuzana Tothova  1  3 ,  Craig Wilen  4 ,  Robert Orchard  4 ,  Herbert W Virgin  4 ,  Jennifer Listgarten  #  2 ,  David E Root  1
Affiliations
  • 1. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
  • 2. Microsoft Research New England, Cambridge, Massachusetts, USA.
  • 3. Dana Farber Cancer Institute, Division of Hematologic Malignancies, Boston, Massachusetts, USA.
  • 4. Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, Missouri, USA.
  • # Contributed equally.
PMID: 26780180 DOI: 10.1038/nbt.3437
Abstract

CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), one can reprogram Cas9 to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.