{"id":{"repo_id":"trento","oai_identifier":"oai:iris.unitn.it:11572/363808"},"canonical_url":"https://search.dev.ndltd.org/etd/trento/oai:iris.unitn.it:11572/363808","repository":{"repo_id":"trento","name":"Università degli Studi di Trento","base_url":"https://iris.unitn.it/oai/request"},"display":{"title":"CRISPR-based approach enables targeted random nucleotide diversification in mammalian cells","abstract":"In vivo directed evolution opens the possibility to introduce genetic variation through targeted nucleotide diversification, leading to the discovery of new protein phenotypes that had been difficult to generate via rational mutagenesis. Tools for in vivo directed evolution have been mainly developed for bacterial systems, preventing the evolution of proteins in higher organisms in their cellular context. The only tools available for mammalian cells rely on the AID cytidine deaminases, able to promote only G and C nucleotide diversification. Here we present hEvolvR, a CRISPR/Cas-guided error-prone DNA polymerase specifically designed for in vivo directed evolution in mammalian systems. hEvolvR is capable to generate unbiased random mutagenesis in a targeted DNA locus. We thoroughly tested and optimized its use in mammalian cells by reverting a premature STOP codon in an eGFP reporter system. We successfully use hEvolvR to restore antibiotic resistance under selective pressure. Furthermore, hEvolvR allowed us to generate variants of SARS- CoV-2 Spike-PP protein with restored fusogenic activity as visualized in high throughput imaging screening.","abstract_html":"In vivo directed evolution opens the possibility to introduce genetic variation through targeted nucleotide diversification, leading to the discovery of new protein phenotypes that had been difficult to generate via rational mutagenesis. Tools for in vivo directed evolution have been mainly developed for bacterial systems, preventing the evolution of proteins in higher organisms in their cellular context. The only tools available for mammalian cells rely on the AID cytidine deaminases, able to promote only G and C nucleotide diversification. Here we present hEvolvR, a CRISPR/Cas-guided error-prone DNA polymerase specifically designed for in vivo directed evolution in mammalian systems. hEvolvR is capable to generate unbiased random mutagenesis in a targeted DNA locus. We thoroughly tested and optimized its use in mammalian cells by reverting a premature STOP codon in an eGFP reporter system. We successfully use hEvolvR to restore antibiotic resistance under selective pressure. Furthermore, hEvolvR allowed us to generate variants of SARS- CoV-2 Spike-PP protein with restored fusogenic activity as visualized in high throughput imaging screening.","abstract_has_math":false,"creators":["Aiello, Davide"],"institution":"Università degli studi di Trento","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["supervisore aggiunto: D. Arosio","Cereseto, Anna"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-01-13","date_published":"2023-01-13","updated_at":"2026-07-24T05:04:43Z","subjects":[],"languages":["eng"],"rights":["info:eu-repo/semantics/openAccess","license:Tutti i diritti riservati (All rights reserved)","license uri:iris.PRI01"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["http://dx.doi.org/10.15168/11572_363808","10.15168/11572_363808"],"render_values":[{"text":"http://dx.doi.org/10.15168/11572_363808","href":"http://dx.doi.org/10.15168/11572_363808","code":true},{"text":"10.15168/11572_363808","href":"https://doi.org/10.15168/11572_363808","code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/11572/363808","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["supervisore aggiunto: D. 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Tools for in vivo directed evolution have been mainly developed for bacterial systems, preventing the evolution of proteins in higher organisms in their cellular context. The only tools available for mammalian cells rely on the AID cytidine deaminases, able to promote only G and C nucleotide diversification. Here we present hEvolvR, a CRISPR/Cas-guided error-prone DNA polymerase specifically designed for in vivo directed evolution in mammalian systems. hEvolvR is capable to generate unbiased random mutagenesis in a targeted DNA locus. We thoroughly tested and optimized its use in mammalian cells by reverting a premature STOP codon in an eGFP reporter system. We successfully use hEvolvR to restore antibiotic resistance under selective pressure. Furthermore, hEvolvR allowed us to generate variants of SARS- CoV-2 Spike-PP protein with restored fusogenic activity as visualized in high throughput imaging screening."]},{"key":"dc:title","label":"Title","values":["CRISPR-based approach enables targeted random nucleotide diversification in mammalian cells"]}]}],"canonical_facts":{"dc:contributor":["supervisore aggiunto: D. Arosio","Aiello, Davide","Cereseto, Anna"],"dc:creator":["Aiello, Davide"],"dc:date":["2023-01-13"],"dc:description":["In vivo directed evolution opens the possibility to introduce genetic variation through targeted nucleotide diversification, leading to the discovery of new protein phenotypes that had been difficult to generate via rational mutagenesis. Tools for in vivo directed evolution have been mainly developed for bacterial systems, preventing the evolution of proteins in higher organisms in their cellular context. The only tools available for mammalian cells rely on the AID cytidine deaminases, able to promote only G and C nucleotide diversification. Here we present hEvolvR, a CRISPR/Cas-guided error-prone DNA polymerase specifically designed for in vivo directed evolution in mammalian systems. hEvolvR is capable to generate unbiased random mutagenesis in a targeted DNA locus. We thoroughly tested and optimized its use in mammalian cells by reverting a premature STOP codon in an eGFP reporter system. We successfully use hEvolvR to restore antibiotic resistance under selective pressure. Furthermore, hEvolvR allowed us to generate variants of SARS- CoV-2 Spike-PP protein with restored fusogenic activity as visualized in high throughput imaging screening."],"dc:identifier":["https://hdl.handle.net/11572/363808","http://dx.doi.org/10.15168/11572_363808","10.15168/11572_363808"],"dc:language":["eng"],"dc:publisher":["Università degli studi di Trento","place:TRENTO"],"dc:relation":["firstpage:1","lastpage:69","numberofpages:69","alleditors:supervisore aggiunto: D. Arosio"],"dc:rights":["info:eu-repo/semantics/openAccess","license:Tutti i diritti riservati (All rights reserved)","license uri:iris.PRI01"],"dc:title":["CRISPR-based approach enables targeted random nucleotide diversification in mammalian cells"],"dc:type":["info:eu-repo/semantics/doctoralThesis"]},"updated_at":"2026-07-24T05:04:43Z"}