Engineered CRISPR-Cas9 nuclease with expanded targeting space

doi:10.1126/science.aas9129

. 2018 Sep 21;361(6408):1259-1262.

doi: 10.1126/science.aas9129. Epub 2018 Aug 30.

Engineered CRISPR-Cas9 nuclease with expanded targeting space

Hiroshi Nishimasu¹, Xi Shi^{2

3}, Soh Ishiguro^{4

5

6}, Linyi Gao^{2

7}, Seiichi Hirano⁸, Sae Okazaki⁸, Taichi Noda⁹, Omar O Abudayyeh^{2

3

10}, Jonathan S Gootenberg^{2

3

10}, Hideto Mori^{4

5

6}, Seiya Oura^{9

11}, Benjamin Holmes^{2

3}, Mamoru Tanaka⁴, Motoaki Seki⁴, Hisato Hirano⁸, Hiroyuki Aburatani⁴, Ryuichiro Ishitani⁸, Masahito Ikawa^{9

11

12}, Nozomu Yachie^{8

4

5

6}, Feng Zhang^{2

3

7

10}, Osamu Nureki¹

Affiliations

¹ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. nisimasu@bs.s.u-tokyo.ac.jp nureki@bs.s.u-tokyo.ac.jp.
² Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
³ McGovern Institute for Brain Research, Cambridge, MA 02139, USA.
⁴ Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
⁵ Institute for Advanced Biosciences, Keio University, 14-1 Baba-cho, Tsuruoka, Yamagata 997-0035, Japan.
⁶ Graduate School of Media and Governance, Keio University, 5322 Endo, Fujisawa, Kanagawa 252-0882, Japan.
⁷ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁸ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
⁹ Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
¹⁰ Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
¹¹ Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
¹² Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

PMID: 30166441
PMCID: PMC6368452
DOI: 10.1126/science.aas9129

Free PMC article

Engineered CRISPR-Cas9 nuclease with expanded targeting space

Hiroshi Nishimasu et al. Science. 2018.

Free PMC article

. 2018 Sep 21;361(6408):1259-1262.

doi: 10.1126/science.aas9129. Epub 2018 Aug 30.

Authors

Affiliations

¹ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. nisimasu@bs.s.u-tokyo.ac.jp nureki@bs.s.u-tokyo.ac.jp.
² Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
³ McGovern Institute for Brain Research, Cambridge, MA 02139, USA.
⁴ Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
⁵ Institute for Advanced Biosciences, Keio University, 14-1 Baba-cho, Tsuruoka, Yamagata 997-0035, Japan.
⁶ Graduate School of Media and Governance, Keio University, 5322 Endo, Fujisawa, Kanagawa 252-0882, Japan.
⁷ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁸ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
⁹ Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
¹⁰ Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
¹¹ Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
¹² Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

PMID: 30166441
PMCID: PMC6368452
DOI: 10.1126/science.aas9129

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Abstract

The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Streptococcus pyogenes Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third nucleobase is compensated by newly introduced non-base-specific interactions, thereby enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells.

Conflict of interest statement

Competing interests: H.N. is a scientific adviser for EdiGENE. F.Z. is a co-founder and scientific adviser for Editas Medicine, Pairwise Plants, Beam Therapeutics, and Arbor Biotechnologies. F.Z. serves as a director for Beam Therapeutics and Arbor Biotechnologies. O.N. is a co-founder, board member, and scientific adviser for EdiGENE. H.N., H.H., and O.N. have filed a patent application related to this work.

Figures

Fig. 1.. In vitro cleavage activity.

(A) SDS-polyacrylamide gel electrophoresis analysis of wild-type SpCas9, SpCas9-NG, and xCas9. (B, C, and E) In vitro DNA cleavage activities of wild-type SpCas9 (B), SpCas9-NG (C), and xCas9 (E) toward the TGN PAM targets. Data are means ± SD (n = 3). (D) PAM preference of SpCas9-NG.

Fig. 2.. Crystal structure of SpCas9-NG.

(A) Recognition of the PAM duplex. Arg1333 and the substituted residues are shown as stick models. (B) Non-base-specific interactions between the PAM duplex and the substituted residues. TS, target strand; NTS, nontarget strand.

Fig. 3.. Gene editing in human cells.

(A) Indel formation efficiencies of wild-type SpCas9, SpCas9-NG, and xCas9 at the 69 endogenous target sites in HEK293FT cells. Data are means ± SD (n = 3). (B) Summary of the editing efficiencies of SpCas9, SpCas9-NG, and xCas9. Medians and first and third quartiles are shown. In (A) and (B), 20% indel frequency is indicated by dashed lines. (C) Specificities of wild-type SpCas9, SpCas9-NG, and the enhanced-specificity versions of SpCas9 (ES) and SpCas9-NG (NG-ES). The off-target cleavages were evaluated by GUIDE-seq.

Fig. 4.. Base editing in human cells.

(A and B) C-to-T conversion efficiencies at the 20 endogenous target sites (Target-AID and Target-AID-NG) (A) and at the 12 poly-C-containing target sites (Target-AID, Target-AID-NG, and xCas9-BE4) (B) in HEK293Tcells. The experiments were performed at least twice, and similar results were obtained.

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Engineered CRISPR-Cas9 nuclease with expanded targeting space

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Engineered CRISPR-Cas9 nuclease with expanded targeting space

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