Engineered CRISPR-Cas9 nuclease with expanded targeting space

The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineer...

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Published in:	Science (American Association for the Advancement of Science) Vol. 361; no. 6408; p. 1259
Main Authors:	Nishimasu, Hiroshi, Shi, Xi, Ishiguro, Soh, Gao, Linyi, Hirano, Seiichi, Okazaki, Sae, Noda, Taichi, Abudayyeh, Omar O, Gootenberg, Jonathan S, Mori, Hideto, Oura, Seiya, Holmes, Benjamin, Tanaka, Mamoru, Seki, Motoaki, Hirano, Hisato, Aburatani, Hiroyuki, Ishitani, Ryuichiro, Ikawa, Masahito, Yachie, Nozomu, Zhang, Feng, Nureki, Osamu
Format:	Journal Article
Language:	English
Published:	United States 21.09.2018
Subjects:	Bacterial Proteins - chemistry Bacterial Proteins - genetics CRISPR-Associated Protein 9 CRISPR-Cas Systems Crystallography, X-Ray Endonucleases - chemistry Endonucleases - genetics Gene Editing HEK293 Cells Humans Protein Engineering
ISSN:	1095-9203, 1095-9203
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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 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.
AbstractList	The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used 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. 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.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.
Author	Shi, Xi Nishimasu, Hiroshi Gao, Linyi Holmes, Benjamin Mori, Hideto Nureki, Osamu Oura, Seiya Ishitani, Ryuichiro Seki, Motoaki Abudayyeh, Omar O Tanaka, Mamoru Ishiguro, Soh Ikawa, Masahito Okazaki, Sae Noda, Taichi Gootenberg, Jonathan S Aburatani, Hiroyuki Hirano, Seiichi Hirano, Hisato Yachie, Nozomu Zhang, Feng
Author_xml	– sequence: 1 givenname: Hiroshi orcidid: 0000-0002-2505-1416 surname: Nishimasu fullname: Nishimasu, Hiroshi email: nisimasu@bs.s.u-tokyo.ac.jp, nureki@bs.s.u-tokyo.ac.jp organization: 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 – sequence: 2 givenname: Xi orcidid: 0000-0002-7691-2239 surname: Shi fullname: Shi, Xi organization: McGovern Institute for Brain Research, Cambridge, MA 02139, USA – sequence: 3 givenname: Soh orcidid: 0000-0002-2826-2470 surname: Ishiguro fullname: Ishiguro, Soh organization: Graduate School of Media and Governance, Keio University, 5322 Endo, Fujisawa, Kanagawa 252-0882, Japan – sequence: 4 givenname: Linyi surname: Gao fullname: Gao, Linyi organization: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA – sequence: 5 givenname: Seiichi surname: Hirano fullname: Hirano, Seiichi organization: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan – sequence: 6 givenname: Sae surname: Okazaki fullname: Okazaki, Sae organization: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan – sequence: 7 givenname: Taichi orcidid: 0000-0003-0260-7861 surname: Noda fullname: Noda, Taichi organization: Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan – sequence: 8 givenname: Omar O orcidid: 0000-0002-7979-3220 surname: Abudayyeh fullname: Abudayyeh, Omar O organization: Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA – sequence: 9 givenname: Jonathan S orcidid: 0000-0003-2757-2175 surname: Gootenberg fullname: Gootenberg, Jonathan S organization: Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA – sequence: 10 givenname: Hideto orcidid: 0000-0003-4849-0056 surname: Mori fullname: Mori, Hideto organization: Graduate School of Media and Governance, Keio University, 5322 Endo, Fujisawa, Kanagawa 252-0882, Japan – sequence: 11 givenname: Seiya orcidid: 0000-0002-7606-732X surname: Oura fullname: Oura, Seiya organization: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan – sequence: 12 givenname: Benjamin orcidid: 0000-0002-1444-445X surname: Holmes fullname: Holmes, Benjamin organization: McGovern Institute for Brain Research, Cambridge, MA 02139, USA – sequence: 13 givenname: Mamoru orcidid: 0000-0001-8984-0257 surname: Tanaka fullname: Tanaka, Mamoru organization: Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan – sequence: 14 givenname: Motoaki orcidid: 0000-0003-4207-1408 surname: Seki fullname: Seki, Motoaki organization: Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan – sequence: 15 givenname: Hisato surname: Hirano fullname: Hirano, Hisato organization: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan – sequence: 16 givenname: Hiroyuki orcidid: 0000-0003-0438-1544 surname: Aburatani fullname: Aburatani, Hiroyuki organization: Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan – sequence: 17 givenname: Ryuichiro orcidid: 0000-0002-4136-5685 surname: Ishitani fullname: Ishitani, Ryuichiro organization: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan – sequence: 18 givenname: Masahito orcidid: 0000-0001-9859-6217 surname: Ikawa fullname: Ikawa, Masahito organization: Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan – sequence: 19 givenname: Nozomu orcidid: 0000-0003-1582-6027 surname: Yachie fullname: Yachie, Nozomu organization: Graduate School of Media and Governance, Keio University, 5322 Endo, Fujisawa, Kanagawa 252-0882, Japan – sequence: 20 givenname: Feng orcidid: 0000-0003-0178-7995 surname: Zhang fullname: Zhang, Feng organization: Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA – sequence: 21 givenname: Osamu surname: Nureki fullname: Nureki, Osamu email: nisimasu@bs.s.u-tokyo.ac.jp, nureki@bs.s.u-tokyo.ac.jp organization: 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
BackLink	https://www.ncbi.nlm.nih.gov/pubmed/30166441$$D View this record in MEDLINE/PubMed
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Snippet	The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Cas9 enzyme (SpCas9) requires an NGG... The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Streptococcus pyogenes Cas9 enzyme...
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SubjectTerms	Bacterial Proteins - chemistry Bacterial Proteins - genetics CRISPR-Associated Protein 9 CRISPR-Cas Systems Crystallography, X-Ray Endonucleases - chemistry Endonucleases - genetics Gene Editing HEK293 Cells Humans Protein Engineering
Title	Engineered CRISPR-Cas9 nuclease with expanded targeting space
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