{"id":545856,"date":"2018-10-26T11:55:37","date_gmt":"2018-10-26T18:55:37","guid":{"rendered":"https:\/\/www.microsoft.com\/en-us\/research\/?post_type=msr-research-item&#038;p=545856"},"modified":"2018-10-26T11:55:37","modified_gmt":"2018-10-26T18:55:37","slug":"orthologous-crispr-cas9-enzymes-for-combinatorial-genetic-screens","status":"publish","type":"msr-research-item","link":"https:\/\/www.microsoft.com\/en-us\/research\/publication\/orthologous-crispr-cas9-enzymes-for-combinatorial-genetic-screens\/","title":{"rendered":"Orthologous CRISPR\u2013Cas9 enzymes for combinatorial genetic screens"},"content":{"rendered":"<p>Combinatorial genetic screening using CRISPR\u2013Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from <i>Staphylococcus aureus<\/i> and <i>Streptococcus pyogenes<\/i>. We used machine learning to establish <i>S. aureus<\/i> Cas9 sgRNA design rules and paired <i>S. aureus<\/i> Cas9 with <i>S. pyogenes<\/i> Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach also enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with <i>TP53<\/i>. The \u201cBig Papi\u201d (paired <i>aureus<\/i> and <i>pyogenes<\/i> for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.<\/p>\n<span id=\"label-external-link\" class=\"sr-only\" aria-hidden=\"true\">Opens in a new tab<\/span>","protected":false},"excerpt":{"rendered":"<p>Combinatorial genetic screening using CRISPR\u2013Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes. We [&hellip;]<\/p>\n","protected":false},"featured_media":0,"template":"","meta":{"msr-url-field":"","msr-podcast-episode":"","msrModifiedDate":"","msrModifiedDateEnabled":false,"ep_exclude_from_search":false,"footnotes":""},"msr-content-type":[3],"msr-research-highlight":[],"research-area":[13553],"msr-publication-type":[193715],"msr-product-type":[],"msr-focus-area":[],"msr-platform":[],"msr-download-source":[],"msr-field-of-study":[],"msr-conference":[],"msr-journal":[],"msr-impact-theme":[],"msr-pillar":[],"msr_publishername":"","msr_edition":"","msr_affiliation":"","msr_published_date":"2017-10-18","msr_host":"","msr_duration":"","msr_version":"","msr_speaker":"","msr_other_contributors":"","msr_booktitle":"","msr_pages_string":"","msr_chapter":"","msr_isbn":"","msr_journal":"nature biotechnology","msr_volume":"36","msr_number":"","msr_editors":"","msr_series":"","msr_issue":"","msr_organization":"","msr_how_published":"","msr_notes":"","msr_highlight_text":"","msr_release_tracker_id":"","msr_original_fields_of_study":"","msr_download_urls":"","msr_external_url":"","msr_secondary_video_url":"","msr_longbiography":"","msr_microsoftintellectualproperty":1,"msr_main_download":"","msr_publicationurl":"","msr_doi":"","msr_publication_uploader":[{"type":"url","viewUrl":"false","id":"false","title":"https:\/\/www.nature.com\/articles\/nbt.4048","label_id":"243109","label":0}],"msr_related_uploader":"","msr_attachments":[],"msr-author-ordering":[{"type":"user_nicename","value":"Nicolo Fusi","user_id":31829,"rest_url":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/microsoft-research\/v1\/researchers?person=Nicolo Fusi"},{"type":"text","value":"et al","user_id":0,"rest_url":false}],"msr_impact_theme":[],"msr_research_lab":[199565],"msr_event":[],"msr_group":[703342],"msr_project":[],"publication":[],"video":[],"download":[],"msr_publication_type":"article","_links":{"self":[{"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/msr-research-item\/545856"}],"collection":[{"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/msr-research-item"}],"about":[{"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/types\/msr-research-item"}],"version-history":[{"count":1,"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/msr-research-item\/545856\/revisions"}],"predecessor-version":[{"id":545859,"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/msr-research-item\/545856\/revisions\/545859"}],"wp:attachment":[{"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/media?parent=545856"}],"wp:term":[{"taxonomy":"msr-content-type","embeddable":true,"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/msr-content-type?post=545856"},{"taxonomy":"msr-research-highlight","embeddable":true,"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/msr-research-highlight?post=545856"},{"taxonomy":"msr-research-area","embeddable":true,"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/research-area?post=545856"},{"taxonomy":"msr-publication-type","embeddable":true,"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/msr-publication-type?post=545856"},{"taxonomy":"msr-product-type","embeddable":true,"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/msr-product-type?post=545856"},{"taxonomy":"msr-focus-area","embeddable":true,"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/msr-focus-area?post=545856"},{"taxonomy":"msr-platform","embeddable":true,"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/msr-platform?post=545856"},{"taxonomy":"msr-download-source","embeddable":true,"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/msr-download-source?post=545856"},{"taxonomy":"msr-field-of-study","embeddable":true,"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/msr-field-of-study?post=545856"},{"taxonomy":"msr-conference","embeddable":true,"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/msr-conference?post=545856"},{"taxonomy":"msr-journal","embeddable":true,"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/msr-journal?post=545856"},{"taxonomy":"msr-impact-theme","embeddable":true,"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/msr-impact-theme?post=545856"},{"taxonomy":"msr-pillar","embeddable":true,"href":"https:\/\/www.microsoft.com\/en-us\/research\/wp-json\/wp\/v2\/msr-pillar?post=545856"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}