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

John G. Doench, Nicolo Fusi, Meagan Sullender, Mudra Hegde, Emma W. Vaimberg, Katherine F. Donovan, Ian Smith, Zuzana Tothova, Craig Wilen, Robert Orchard, Herbert W. Virgin, Jennifer Listgarten, David E. Root

Research output: Contribution to journalArticle

644 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)184-191
Number of pages8
JournalNature biotechnology
Volume34
Issue number2
DOIs
StatePublished - Feb 1 2016
Externally publishedYes

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Clustered Regularly Interspaced Short Palindromic Repeats
Guide RNA
RNA
Genes
Genome
Genomic Library
Human Genome
Libraries

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Molecular Medicine
  • Biomedical Engineering

Cite this

Doench, J. G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E. W., Donovan, K. F., ... Root, D. E. (2016). Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nature biotechnology, 34(2), 184-191. https://doi.org/10.1038/nbt.3437

Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. / Doench, John G.; Fusi, Nicolo; Sullender, Meagan; Hegde, Mudra; Vaimberg, Emma W.; Donovan, Katherine F.; Smith, Ian; Tothova, Zuzana; Wilen, Craig; Orchard, Robert; Virgin, Herbert W.; Listgarten, Jennifer; Root, David E.

In: Nature biotechnology, Vol. 34, No. 2, 01.02.2016, p. 184-191.

Research output: Contribution to journalArticle

Doench, JG, Fusi, N, Sullender, M, Hegde, M, Vaimberg, EW, Donovan, KF, Smith, I, Tothova, Z, Wilen, C, Orchard, R, Virgin, HW, Listgarten, J & Root, DE 2016, 'Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9', Nature biotechnology, vol. 34, no. 2, pp. 184-191. https://doi.org/10.1038/nbt.3437
Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nature biotechnology. 2016 Feb 1;34(2):184-191. https://doi.org/10.1038/nbt.3437
Doench, John G. ; Fusi, Nicolo ; Sullender, Meagan ; Hegde, Mudra ; Vaimberg, Emma W. ; Donovan, Katherine F. ; Smith, Ian ; Tothova, Zuzana ; Wilen, Craig ; Orchard, Robert ; Virgin, Herbert W. ; Listgarten, Jennifer ; Root, David E. / Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. In: Nature biotechnology. 2016 ; Vol. 34, No. 2. pp. 184-191.
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