Continuous evolution of SpCas9 variants compatible with non-G PAMs

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data-test="authors-list" data-component-authors-activator="authors-list"><li class="c-article-author-list__item"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-Shannon_M_-Miller-Aff1-Aff2-Aff3" data-author-popup="auth-Shannon_M_-Miller-Aff1-Aff2-Aff3" data-author-search="Miller, Shannon M.">Shannon M. Miller</a><sup class="u-js-hide"><a href="#Aff1">1</a>,<a href="#Aff2">2</a>,<a href="#Aff3">3</a></sup><sup class="u-js-hide"> <a href="#na1">na1</a></sup>, </li><li class="c-article-author-list__item"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-Tina-Wang-Aff1-Aff2-Aff3" data-author-popup="auth-Tina-Wang-Aff1-Aff2-Aff3" data-author-search="Wang, Tina">Tina Wang</a><sup class="u-js-hide"><a href="#Aff1">1</a>,<a href="#Aff2">2</a>,<a href="#Aff3">3</a></sup><sup class="u-js-hide"> <a href="#na1">na1</a></sup>, </li><li class="c-article-author-list__item c-article-author-list__item--hide-small-screen"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-Peyton_B_-Randolph-Aff1-Aff2-Aff3" data-author-popup="auth-Peyton_B_-Randolph-Aff1-Aff2-Aff3" data-author-search="Randolph, Peyton B.">Peyton B. Randolph</a><span class="u-js-hide"> <a class="js-orcid" href="http://orcid.org/0000-0001-6385-364X"><span class="u-visually-hidden">ORCID: </span>orcid.org/0000-0001-6385-364X</a></span><sup class="u-js-hide"><a href="#Aff1">1</a>,<a href="#Aff2">2</a>,<a href="#Aff3">3</a></sup>, </li><li class="c-article-author-list__item c-article-author-list__item--hide-small-screen"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-Mandana-Arbab-Aff1-Aff2-Aff3" data-author-popup="auth-Mandana-Arbab-Aff1-Aff2-Aff3" data-author-search="Arbab, Mandana">Mandana Arbab</a><sup class="u-js-hide"><a href="#Aff1">1</a>,<a href="#Aff2">2</a>,<a href="#Aff3">3</a></sup>, </li><li class="c-article-author-list__item c-article-author-list__item--hide-small-screen"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-Max_W_-Shen-Aff1-Aff2-Aff3-Aff4" data-author-popup="auth-Max_W_-Shen-Aff1-Aff2-Aff3-Aff4" data-author-search="Shen, Max W.">Max W. Shen</a><sup class="u-js-hide"><a href="#Aff1">1</a>,<a href="#Aff2">2</a>,<a href="#Aff3">3</a>,<a href="#Aff4">4</a></sup>, </li><li class="c-article-author-list__item c-article-author-list__item--hide-small-screen"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-Tony_P_-Huang-Aff1-Aff2-Aff3" data-author-popup="auth-Tony_P_-Huang-Aff1-Aff2-Aff3" data-author-search="Huang, Tony P.">Tony P. Huang</a><sup class="u-js-hide"><a href="#Aff1">1</a>,<a href="#Aff2">2</a>,<a href="#Aff3">3</a></sup>, </li><li class="c-article-author-list__item c-article-author-list__item--hide-small-screen"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-Zaneta-Matuszek-Aff1-Aff2-Aff3-Aff5" data-author-popup="auth-Zaneta-Matuszek-Aff1-Aff2-Aff3-Aff5" data-author-search="Matuszek, Zaneta">Zaneta Matuszek</a><sup class="u-js-hide"><a href="#Aff1">1</a>,<a href="#Aff2">2</a>,<a href="#Aff3">3</a>,<a href="#Aff5">5</a></sup>, </li><li class="c-article-author-list__item c-article-author-list__item--hide-small-screen"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-Gregory_A_-Newby-Aff1-Aff2-Aff3" data-author-popup="auth-Gregory_A_-Newby-Aff1-Aff2-Aff3" data-author-search="Newby, Gregory A.">Gregory A. Newby</a><span class="u-js-hide"> <a class="js-orcid" href="http://orcid.org/0000-0001-7869-2615"><span class="u-visually-hidden">ORCID: </span>orcid.org/0000-0001-7869-2615</a></span><sup class="u-js-hide"><a href="#Aff1">1</a>,<a href="#Aff2">2</a>,<a href="#Aff3">3</a></sup>, </li><li class="c-article-author-list__item c-article-author-list__item--hide-small-screen"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-Holly_A_-Rees-Aff1-Aff2-Aff3" data-author-popup="auth-Holly_A_-Rees-Aff1-Aff2-Aff3" data-author-search="Rees, Holly A.">Holly A. Rees</a><sup class="u-js-hide"><a href="#Aff1">1</a>,<a href="#Aff2">2</a>,<a href="#Aff3">3</a></sup> & </li><li class="c-article-author-list__show-more" aria-label="Show all 10 authors for this article" title="Show all 10 authors for this article">…</li><li class="c-article-author-list__item"><a data-test="author-name" data-track="click" data-track-action="open author" data-track-label="link" href="#auth-David_R_-Liu-Aff1-Aff2-Aff3" data-author-popup="auth-David_R_-Liu-Aff1-Aff2-Aff3" data-author-search="Liu, David R." data-corresp-id="c1">David R. Liu<svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-mail-medium"></use></svg></a><span class="u-js-hide"> <a class="js-orcid" href="http://orcid.org/0000-0002-9943-7557"><span class="u-visually-hidden">ORCID: </span>orcid.org/0000-0002-9943-7557</a></span><sup class="u-js-hide"><a href="#Aff1">1</a>,<a href="#Aff2">2</a>,<a href="#Aff3">3</a></sup> </li></ul><button aria-expanded="false" class="c-article-author-list__button"><svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-chevron-down-medium"></use></svg><span>Show authors</span></button> <p class="c-article-info-details" data-container-section="info"> <a data-test="journal-link" href="/nbt" data-track="click" data-track-action="journal homepage" data-track-category="article body" data-track-label="link"><i data-test="journal-title">Nature Biotechnology</i></a> <b data-test="journal-volume"><span class="u-visually-hidden">volume</span> 38</b>, <span class="u-visually-hidden">pages </span>471–481 (<span data-test="article-publication-year">2020</span>)<a href="#citeas" class="c-article-info-details__cite-as u-hide-print" data-track="click" data-track-action="cite this article" data-track-label="link">Cite this article</a> </p> <div class="c-article-metrics-bar__wrapper u-clear-both"> <ul class="c-article-metrics-bar u-list-reset"> <li class=" c-article-metrics-bar__item" data-test="access-count"> <p class="c-article-metrics-bar__count">21k <span class="c-article-metrics-bar__label">Accesses</span></p> </li> <li class="c-article-metrics-bar__item" data-test="altmetric-score"> <p class="c-article-metrics-bar__count">107 <span class="c-article-metrics-bar__label">Altmetric</span></p> </li> <li class="c-article-metrics-bar__item"> <p class="c-article-metrics-bar__details"><a href="/articles/s41587-020-0412-8/metrics" data-track="click" data-track-action="view metrics" data-track-label="link" rel="nofollow">Metrics <span class="u-visually-hidden">details</span></a></p> </li> </ul> </div> </header> <div class="u-js-hide" data-component="article-subject-links"> <h3 class="c-article__sub-heading">Subjects</h3> <ul class="c-article-subject-list"> <li class="c-article-subject-list__subject"><a href="/subjects/cas9-endonuclease" data-track="click" data-track-action="view subject" data-track-label="link">CRISPR-Cas9 genome editing</a></li><li class="c-article-subject-list__subject"><a href="/subjects/genetic-engineering" data-track="click" data-track-action="view subject" data-track-label="link">Genetic engineering</a></li> </ul> </div> </div> <div class="c-article-body"> <section aria-labelledby="Abs1" data-title="Abstract" lang="en"><div class="c-article-section" id="Abs1-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="Abs1">Abstract</h2><div class="c-article-section__content" id="Abs1-content"><p>The targeting scope of <i>Streptococcus pyogenes</i> Cas9 (SpCas9) and its engineered variants is largely restricted to protospacer-adjacent motif (PAM) sequences containing G bases. Here we report the evolution of three new SpCas9 variants that collectively recognize NRNH PAMs (where R is A or G and H is A, C or T) using phage-assisted non-continuous evolution, three new phage-assisted continuous evolution strategies for DNA binding and a secondary selection for DNA cleavage. The targeting capabilities of these evolved variants and SpCas9-NG were characterized in HEK293T cells using a library of 11,776 genomically integrated protospacer–sgRNA pairs containing all possible NNNN PAMs. The evolved variants mediated indel formation and base editing in human cells and enabled A•T-to-G•C base editing of a sickle cell anemia mutation using a previously inaccessible CACC PAM. These new evolved SpCas9 variants, together with previously reported variants, in principle enable targeting of most NR PAM sequences and substantially reduce the fraction of genomic sites that are inaccessible by Cas9-based methods.</p></div></div></section> <noscript> <div class="c-nature-box c-nature-box--side " data-component="entitlement-box"> <div class="js-access-button"> <a href="https://wayf.springernature.com?redirect_uri=https%3A%2F%2Fwww.nature.com%2Farticles%2Fs41587-020-0412-8" class="c-article__button" data-test="ra21"> <svg class="u-icon" width="18" height="18" aria-hidden="true" focusable="false"><use href="#icon-institution"></use></svg> <span class="c-article__button-text">Access through your institution</span> </a> </div> <div class="js-buy-button"> <a href="#access-options" class="c-article__button c-article__button--inverted" data-test="ra21"> <span>Buy or subscribe</span> </a> </div> </div> </noscript> <div class="js-context-bar-sticky-point-mobile" 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class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-1"><figure><figcaption><b id="Fig1" class="c-article-section__figure-caption" data-test="figure-caption-text">Fig. 1: PANCE of SpCas9 binding activity on non-G PAMs.</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><picture><source type="image/webp" srcset="//media.springernature.com/m312/springer-static/image/art%3A10.1038%2Fs41587-020-0412-8/MediaObjects/41587_2020_412_Fig1_HTML.png?as=webp"><img src="//media.springernature.com/m312/springer-static/image/art%3A10.1038%2Fs41587-020-0412-8/MediaObjects/41587_2020_412_Fig1_HTML.png" alt="" loading="lazy" width="312" height="282"></picture></div></div></figure></div><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-2"><figure><figcaption><b id="Fig2" class="c-article-section__figure-caption" data-test="figure-caption-text">Fig. 2: Selection improvement enables evolution of SpCas9 variants with robust activity on non-G PAMs.</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><picture><source type="image/webp" srcset="//media.springernature.com/m312/springer-static/image/art%3A10.1038%2Fs41587-020-0412-8/MediaObjects/41587_2020_412_Fig2_HTML.png?as=webp"><img src="//media.springernature.com/m312/springer-static/image/art%3A10.1038%2Fs41587-020-0412-8/MediaObjects/41587_2020_412_Fig2_HTML.png" alt="" loading="lazy" width="286" height="312"></picture></div></div></figure></div><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-3"><figure><figcaption><b id="Fig3" class="c-article-section__figure-caption" data-test="figure-caption-text">Fig. 3: Comprehensive characterization of PAM preferences using a genomically integrated human cell target sequence library.</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><picture><source type="image/webp" srcset="//media.springernature.com/m312/springer-static/image/art%3A10.1038%2Fs41587-020-0412-8/MediaObjects/41587_2020_412_Fig3_HTML.png?as=webp"><img src="//media.springernature.com/m312/springer-static/image/art%3A10.1038%2Fs41587-020-0412-8/MediaObjects/41587_2020_412_Fig3_HTML.png" alt="" loading="lazy" width="312" height="302"></picture></div></div></figure></div><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-4"><figure><figcaption><b id="Fig4" class="c-article-section__figure-caption" data-test="figure-caption-text">Fig. 4: Mammalian cell indel formation and DNA specificity of evolved Cas9 variants.</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><picture><source type="image/webp" srcset="//media.springernature.com/m312/springer-static/image/art%3A10.1038%2Fs41587-020-0412-8/MediaObjects/41587_2020_412_Fig4_HTML.png?as=webp"><img src="//media.springernature.com/m312/springer-static/image/art%3A10.1038%2Fs41587-020-0412-8/MediaObjects/41587_2020_412_Fig4_HTML.png" alt="" loading="lazy" width="312" height="283"></picture></div></div></figure></div><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-5"><figure><figcaption><b id="Fig5" class="c-article-section__figure-caption" data-test="figure-caption-text">Fig. 5: Mammalian cytosine and adenine base editing activity and scope of evolved variants and SpCas9-NG.</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><picture><source type="image/webp" srcset="//media.springernature.com/m312/springer-static/image/art%3A10.1038%2Fs41587-020-0412-8/MediaObjects/41587_2020_412_Fig5_HTML.png?as=webp"><img src="//media.springernature.com/m312/springer-static/image/art%3A10.1038%2Fs41587-020-0412-8/MediaObjects/41587_2020_412_Fig5_HTML.png" alt="" loading="lazy" width="312" height="191"></picture></div></div></figure></div><div class="c-article-section__figure js-c-reading-companion-figures-item" data-test="figure" data-container-section="figure" id="figure-6"><figure><figcaption><b id="Fig6" class="c-article-section__figure-caption" data-test="figure-caption-text">Fig. 6: Evolved SpCas9 variants enable correction of pathogenic SNPs with non-G PAMs.</b></figcaption><div class="c-article-section__figure-content"><div class="c-article-section__figure-item"><picture><source type="image/webp" srcset="//media.springernature.com/m312/springer-static/image/art%3A10.1038%2Fs41587-020-0412-8/MediaObjects/41587_2020_412_Fig6_HTML.png?as=webp"><img src="//media.springernature.com/m312/springer-static/image/art%3A10.1038%2Fs41587-020-0412-8/MediaObjects/41587_2020_412_Fig6_HTML.png" alt="" loading="lazy" width="312" height="283"></picture></div></div></figure></div> </div> <section aria-labelledby="inline-recommendations" data-title="Inline Recommendations" class="c-article-recommendations" data-track-component="inline-recommendations"> <h3 class="c-article-recommendations-title" id="inline-recommendations">Similar content being viewed by others</h3> <div class="c-article-recommendations-list"> <div class="c-article-recommendations-list__item"> <article class="c-article-recommendations-card" itemscope itemtype="http://schema.org/ScholarlyArticle"> <div class="c-article-recommendations-card__img"><img src="https://media.springernature.com/w215h120/springer-static/image/art%3A10.1038%2Fs41467-020-20650-x/MediaObjects/41467_2020_20650_Fig1_HTML.png" loading="lazy" alt=""></div> <div class="c-article-recommendations-card__main"> <h3 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data-track-label="10.1038/s41589-023-01427-x">Continuous directed evolution of a compact <i>Cj</i>Cas9 variant with broad PAM compatibility </a> </h3> <div class="c-article-meta-recommendations" data-test="recommendation-info"> <span class="c-article-meta-recommendations__item-type">Article</span> <span class="c-article-meta-recommendations__date">21 September 2023</span> </div> </div> </article> </div> </div> </section> <script> window.dataLayer = window.dataLayer || []; window.dataLayer.push({ recommendations: { recommender: 'semantic', model: 'specter', policy_id: 'NA', timestamp: 1732652915, embedded_user: 'null' } }); </script> <div> <section data-title="Data availability"><div class="c-article-section" id="data-availability-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="data-availability">Data availability</h2><div class="c-article-section__content" id="data-availability-content"> <p>HTS data were deposited in the Sequence Read Archive database (<a href="https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA596996">PRJNA596996</a>). Plasmids encoding SpCas9-NRRH, SpCas9-NRTH and SpCas9-NRCH nucleases, BE4 and ABE are available through Addgene. A subset of the selection plasmids used in this study are available through Addgene. Other materials are available from the corresponding authors upon reasonable request.</p> </div></div></section><section data-title="Code availability"><div class="c-article-section" id="code-availability-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="code-availability">Code availability</h2><div class="c-article-section__content" id="code-availability-content"> <p>Custom scripts used to analyze the human cell target site library are available at <a href="https://github.com/maxwshen-PAMvar-processing">https://github.com/maxwshen-PAMvar-processing</a>.</p> </div></div></section><div id="MagazineFulltextArticleBodySuffix"><section aria-labelledby="Bib1" data-title="References"><div class="c-article-section" id="Bib1-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="Bib1">References</h2><div class="c-article-section__content" id="Bib1-content"><div data-container-section="references"><ol class="c-article-references" data-track-component="outbound reference" data-track-context="references section"><li class="c-article-references__item js-c-reading-companion-references-item" data-counter="1."><p class="c-article-references__text" id="ref-CR1">Komor, A. 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Blum and A. Raguram for helpful discussions. This work was supported by National Institutes of Health grants R01 EB027793, U01 AI142756, RM1 HG009490 and R35 GM118062, the Howard Hughes Medical Institute, the Bill and Melinda Gates Foundation and the St. Jude Collaborative Research Consortium. S.M.M. and M.S. were supported by a National Science Foundation Graduate Research Fellowship. T.W. was supported by a Ruth L. Kirchstein National Research Service Awards Postdoctoral Fellowship (F32GM119228). M.A. was supported by an NWO Rubicon Fellowship. G.A.N was supported by a Helen Hay Whitney Postdoctoral Fellowship.</p></div></div></section><section aria-labelledby="author-information" data-title="Author information"><div class="c-article-section" id="author-information-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="author-information">Author information</h2><div class="c-article-section__content" id="author-information-content"><span class="c-article-author-information__subtitle u-visually-hidden" id="author-notes">Author notes</span><ol class="c-article-author-information__list"><li class="c-article-author-information__item" id="na1"><p>These authors contributed equally: Shannon M. Miller, Tina Wang.</p></li></ol><h3 class="c-article__sub-heading" id="affiliations">Authors and Affiliations</h3><ol class="c-article-author-affiliation__list"><li id="Aff1"><p class="c-article-author-affiliation__address">Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA</p><p class="c-article-author-affiliation__authors-list">Shannon M. Miller, Tina Wang, Peyton B. Randolph, Mandana Arbab, Max W. Shen, Tony P. Huang, Zaneta Matuszek, Gregory A. Newby, Holly A. Rees & David R. Liu</p></li><li id="Aff2"><p class="c-article-author-affiliation__address">Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA</p><p class="c-article-author-affiliation__authors-list">Shannon M. Miller, Tina Wang, Peyton B. Randolph, Mandana Arbab, Max W. Shen, Tony P. Huang, Zaneta Matuszek, Gregory A. Newby, Holly A. Rees & David R. Liu</p></li><li id="Aff3"><p class="c-article-author-affiliation__address">Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA</p><p class="c-article-author-affiliation__authors-list">Shannon M. Miller, Tina Wang, Peyton B. Randolph, Mandana Arbab, Max W. Shen, Tony P. Huang, Zaneta Matuszek, Gregory A. Newby, Holly A. Rees & David R. Liu</p></li><li id="Aff4"><p class="c-article-author-affiliation__address">Computational and Systems Biology Program, Massachusetts Institute of Technology, Cambridge, MA, USA</p><p class="c-article-author-affiliation__authors-list">Max W. Shen</p></li><li id="Aff5"><p class="c-article-author-affiliation__address">Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA</p><p class="c-article-author-affiliation__authors-list">Zaneta Matuszek</p></li></ol><div class="u-js-hide u-hide-print" data-test="author-info"><span class="c-article__sub-heading">Authors</span><ol class="c-article-authors-search u-list-reset"><li id="auth-Shannon_M_-Miller-Aff1-Aff2-Aff3"><span class="c-article-authors-search__title u-h3 js-search-name">Shannon M. 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Liu</span><div class="c-article-authors-search__list"><div class="c-article-authors-search__item c-article-authors-search__list-item--left"><a href="/search?author=David%20R.%20Liu" class="c-article-button" data-track="click" data-track-action="author link - publication" data-track-label="link" rel="nofollow">View author publications</a></div><div class="c-article-authors-search__item c-article-authors-search__list-item--right"><p class="search-in-title-js c-article-authors-search__text">You can also search for this author in <span class="c-article-identifiers"><a class="c-article-identifiers__item" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&term=David%20R.%20Liu" data-track="click" data-track-action="author link - pubmed" data-track-label="link" rel="nofollow">PubMed</a><span class="u-hide"> </span><a class="c-article-identifiers__item" href="http://scholar.google.co.uk/scholar?as_q=&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_sauthors=%22David%20R.%20Liu%22&as_publication=&as_ylo=&as_yhi=&as_allsubj=all&hl=en" data-track="click" data-track-action="author link - scholar" data-track-label="link" rel="nofollow">Google Scholar</a></span></p></div></div></li></ol></div><h3 class="c-article__sub-heading" id="contributions">Contributions</h3><p>S.M.M., T.W. and D.R.L. designed the research. S.M.M., T.W. and P.B.R. performed evolution. S.M.M., T.W., P.B.R. and T.P.H. characterized variants in bacteria. S.M.M., T.W., P.B.R., M.A. and Z.M. conducted human cell experiments. M.A. and M.S. designed and constructed the integrated target site human cell library. M.S. wrote custom software. S.M.M., T.W., M.A. and M.S. analyzed library data. S.M.M. and T.W. performed GUIDE-seq experiments. G.N. and H.A.R. designed and performed the sickle cell anemia site experiments. S.M.M., T.W., P.B.R. and T.P.H. designed and performed the HbS editing experiments. S.M.M., T.W. and D.R.L. wrote the manuscript.</p><h3 class="c-article__sub-heading" id="corresponding-author">Corresponding author</h3><p id="corresponding-author-list">Correspondence to <a id="corresp-c1" href="mailto:drliu@fas.harvard.edu">David R. Liu</a>.</p></div></div></section><section data-title="Ethics declarations"><div class="c-article-section" id="ethics-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="ethics">Ethics declarations</h2><div class="c-article-section__content" id="ethics-content"> <h3 class="c-article__sub-heading" id="FPar1">Competing interests</h3> <p>The authors declare competing financial interests: S.M.M., T.W. and D.R.L. have filed patent applications on this work. D.R.L. is a consultant and co-founder of Editas Medicine, Pairwise Plants, Beam Therapeutics and Prime Medicine, companies that use genome-editing technologies.</p> </div></div></section><section data-title="Additional information"><div class="c-article-section" id="additional-information-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="additional-information">Additional information</h2><div class="c-article-section__content" id="additional-information-content"><p><b>Publisher’s note</b> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></div></div></section><section data-title="Supplementary information"><div class="c-article-section" id="Sec40-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="Sec40">Supplementary information</h2><div class="c-article-section__content" id="Sec40-content"><div data-test="supplementary-info"><div id="figshareContainer" class="c-article-figshare-container" data-test="figshare-container"></div><div class="c-article-supplementary__item" data-test="supp-item" id="MOESM1"><h3 class="c-article-supplementary__title u-h3"><a class="print-link" data-track="click" data-track-action="view supplementary info" data-test="supp-info-link" data-track-label="supplementary information" href="https://static-content.springer.com/esm/art%3A10.1038%2Fs41587-020-0412-8/MediaObjects/41587_2020_412_MOESM1_ESM.pdf" data-supp-info-image="">Supplementary Information</a></h3><div class="c-article-supplementary__description" data-component="thumbnail-container"><p>Supplementary Figs. 1–8, Tables 1, 3 and 6–9, and Notes 1–4</p></div></div><div class="c-article-supplementary__item" data-test="supp-item" id="MOESM2"><h3 class="c-article-supplementary__title u-h3"><a class="print-link" data-track="click" data-track-action="view supplementary info" data-test="supp-info-link" data-track-label="reporting summary" href="https://static-content.springer.com/esm/art%3A10.1038%2Fs41587-020-0412-8/MediaObjects/41587_2020_412_MOESM2_ESM.pdf" data-supp-info-image="">Reporting Summary</a></h3></div><div class="c-article-supplementary__item" data-test="supp-item" id="MOESM3"><h3 class="c-article-supplementary__title u-h3"><a class="print-link" data-track="click" data-track-action="view supplementary info" data-test="supp-info-link" data-track-label="supplementary table 2" href="https://static-content.springer.com/esm/art%3A10.1038%2Fs41587-020-0412-8/MediaObjects/41587_2020_412_MOESM3_ESM.xlsx" data-supp-info-image="">Supplementary Table 2</a></h3><div class="c-article-supplementary__description" data-component="thumbnail-container"><p>Excel table of PACE mutations.</p></div></div><div class="c-article-supplementary__item" data-test="supp-item" id="MOESM4"><h3 class="c-article-supplementary__title u-h3"><a class="print-link" data-track="click" data-track-action="view supplementary info" data-test="supp-info-link" data-track-label="supplementary table 4" href="https://static-content.springer.com/esm/art%3A10.1038%2Fs41587-020-0412-8/MediaObjects/41587_2020_412_MOESM4_ESM.xlsx" data-supp-info-image="">Supplementary Table 4</a></h3><div class="c-article-supplementary__description" data-component="thumbnail-container"><p>Excel table of GUIDE-seq-identified off-target sites.</p></div></div><div class="c-article-supplementary__item" data-test="supp-item" id="MOESM5"><h3 class="c-article-supplementary__title u-h3"><a class="print-link" data-track="click" data-track-action="view supplementary info" data-test="supp-info-link" data-track-label="supplementary table 5" href="https://static-content.springer.com/esm/art%3A10.1038%2Fs41587-020-0412-8/MediaObjects/41587_2020_412_MOESM5_ESM.xlsx" data-supp-info-image="">Supplementary Table 5</a></h3><div class="c-article-supplementary__description" data-component="thumbnail-container"><p>Excel table of PAMvar library data on all sites.</p></div></div></div></div></div></section><section data-title="Rights and permissions"><div class="c-article-section" id="rightslink-section"><h2 class="c-article-section__title js-section-title js-c-reading-companion-sections-item" id="rightslink">Rights and permissions</h2><div class="c-article-section__content" id="rightslink-content"><p class="c-article-rights"><a data-track="click" data-track-action="view rights and permissions" data-track-label="link" href="https://s100.copyright.com/AppDispatchServlet?title=Continuous%20evolution%20of%20SpCas9%20variants%20compatible%20with%20non-G%20PAMs&author=Shannon%20M.%20Miller%20et%20al&contentID=10.1038%2Fs41587-020-0412-8&copyright=The%20Author%28s%29%2C%20under%20exclusive%20licence%20to%20Springer%20Nature%20America%2C%20Inc.&publication=1087-0156&publicationDate=2020-02-10&publisherName=SpringerNature&orderBeanReset=true">Reprints and permissions</a></p></div></div></section><section 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id="citeas">Cite this article</h3><p class="c-bibliographic-information__citation">Miller, S.M., Wang, T., Randolph, P.B. <i>et al.</i> Continuous evolution of SpCas9 variants compatible with non-G PAMs. <i>Nat Biotechnol</i> <b>38</b>, 471–481 (2020). https://doi.org/10.1038/s41587-020-0412-8</p><p class="c-bibliographic-information__download-citation u-hide-print"><a data-test="citation-link" data-track="click" data-track-action="download article citation" data-track-label="link" data-track-external="" rel="nofollow" href="https://citation-needed.springer.com/v2/references/10.1038/s41587-020-0412-8?format=refman&flavour=citation">Download citation<svg width="16" height="16" focusable="false" role="img" aria-hidden="true" class="u-icon"><use xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#icon-eds-i-download-medium"></use></svg></a></p><ul class="c-bibliographic-information__list" data-test="publication-history"><li class="c-bibliographic-information__list-item"><p>Received<span class="u-hide">: </span><span class="c-bibliographic-information__value"><time datetime="2019-09-27">27 September 2019</time></span></p></li><li class="c-bibliographic-information__list-item"><p>Accepted<span class="u-hide">: </span><span class="c-bibliographic-information__value"><time datetime="2020-01-07">07 January 2020</time></span></p></li><li class="c-bibliographic-information__list-item"><p>Published<span class="u-hide">: </span><span class="c-bibliographic-information__value"><time datetime="2020-02-10">10 February 2020</time></span></p></li><li class="c-bibliographic-information__list-item"><p>Issue Date<span class="u-hide">: </span><span class="c-bibliographic-information__value"><time datetime="2020-04">April 2020</time></span></p></li><li class="c-bibliographic-information__list-item c-bibliographic-information__list-item--full-width"><p><abbr title="Digital Object Identifier">DOI</abbr><span class="u-hide">: 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Continuous evolution of SpCas9 variants compatible with non-G PAMs | Nature Biotechnology