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Extant, multidomain proteins are hypothesized to be the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by\n                    <jats:underline>i<\/jats:underline>\n                    terative\n                    <jats:underline>s<\/jats:underline>\n                    ize-\n                    <jats:underline>e<\/jats:underline>\n                    xclusion and\n                    <jats:underline>r<\/jats:underline>\n                    ecombination (MISER) for comprehensively making all possible deletions of a protein. Using MISER, we generate a deletion landscape for the CRISPR protein Cas9. We find that the catalytically-dead\n                    <jats:italic>Streptococcus pyogenes<\/jats:italic>\n                    Cas9 can tolerate large single deletions in the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.\n                  <\/jats:p>","DOI":"10.1038\/s41467-021-25992-8","type":"journal-article","created":{"date-parts":[[2021,9,27]],"date-time":"2021-09-27T06:20:00Z","timestamp":1632723600000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":51,"title":["Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules"],"prefix":"10.1038","volume":"12","author":[{"given":"Arik","family":"Shams","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sean A.","family":"Higgins","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9545-5723","authenticated-orcid":false,"given":"Christof","family":"Fellmann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8943-1330","authenticated-orcid":false,"given":"Thomas G.","family":"Laughlin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin L.","family":"Oakes","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rachel","family":"Lew","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shin","family":"Kim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Lukarska","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0130-4789","authenticated-orcid":false,"given":"Madeline","family":"Arnold","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brett T.","family":"Staahl","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9161-999X","authenticated-orcid":false,"given":"Jennifer A.","family":"Doudna","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0042-2257","authenticated-orcid":false,"given":"David F.","family":"Savage","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,9,27]]},"reference":[{"key":"25992_CR1","doi-asserted-by":"publisher","first-page":"14658","DOI":"10.1073\/pnas.95.25.14658","volume":"95","author":"SA Teichmann","year":"1998","unstructured":"Teichmann, S. 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J.A.D., D.F.S., S.A.H., and B.L.O. are listed as inventors. S.A.H. is an employee of Scribe Therapeutics. B.L.O. and B.T.S. are co-founders and employees of Scribe Therapeutics. C.F. is a co-founder of Mirimus, Inc. J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, Intellia Therapeutics, Scribe Therapeutics, and Mammoth Biosciences. J.A.D. is a scientific advisory board member of Caribous Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Synthego, Metagenomi, Mammoth Biosciences, and Inari. J.A.D. is a member of the board of directors at Driver and Johnson & Johnson. D.F.S. is a co-founder of Scribe Therapeutics and a scientific advisory board member of Scribe Therapeutics and Mammoth Biosciences. All the other authors declare no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"5664"}}