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. 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 Nishimasu  1 , Xi Shi  2   3 , Soh Ishiguro  4   5   6 , Linyi Gao  2   7 , Seiichi Hirano  8 , Sae Okazaki  8 , Taichi Noda  9 , 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 Tanaka  4 , Motoaki Seki  4 , Hisato Hirano  8 , Hiroyuki Aburatani  4 , Ryuichiro Ishitani  8 , Masahito Ikawa  9   11   12 , Nozomu Yachie  8   4   5   6 , Feng Zhang  2   3   7   10 , Osamu Nureki  1
Affiliations

Engineered CRISPR-Cas9 nuclease with expanded targeting space

Hiroshi Nishimasu et al. Science. .

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.

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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.
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.
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.
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.
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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References

    1. Jinek M et al., Science 337, 816–821 (2012). - PMC - PubMed
    1. Cong L et al., Science 339, 819–823 (2013). - PMC - PubMed
    1. Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA, Nature 507, 62–67 (2014). - PMC - PubMed
    1. Kleinstiver BP et al., Nature 523, 481–485 (2015). - PMC - PubMed
    1. Ran FA et al., Nature 520, 186–191 (2015). - PMC - PubMed

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