Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes

The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAMs, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not...

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Autores: Vicencio, Jeremy, Sánchez-Bolaños, Carlos, Moreno-Sánchez, Ismael, Brena, David, Vejnar, Charles E., Kuthar, Dmytro, Ruiz-López, Miguel, Cots-Ponjoan, Mariona, Rubio Valle, Alejandro, Rodrigo Melero, Natalia, Crespo Cuadrado, Jesús, Carolis, Carlos, Pérez-Pulido, Antonio J., Giraldez, Antonio J., Kleinstiver, Benjamin P., Cerón, Julián, Moreno Mateos, Miguel A.
Tipo de recurso: artículo
Fecha de publicación:2022
País:España
Institución:Universidad Pablo de Olavide (UPO)
Repositorio:RIO. Repositorio Institucional Olavide
Idioma:inglés
OAI Identifier:oai:rio.upo.es:10433/26116
Acceso en línea:https://hdl.handle.net/10433/26116
Access Level:acceso abierto
Palabra clave:CRISPR-Cas
Genome editing
Zebrafish
Nematodes
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spelling Genome editing in animals with minimal PAM CRISPR-Cas9 enzymesVicencio, JeremySánchez-Bolaños, CarlosMoreno-Sánchez, IsmaelBrena, DavidVejnar, Charles E.Kuthar, DmytroRuiz-López, MiguelCots-Ponjoan, MarionaRubio Valle, AlejandroRodrigo Melero, NataliaCrespo Cuadrado, JesúsCarolis, CarlosPérez-Pulido, Antonio J.Giraldez, Antonio J.Kleinstiver, Benjamin P.Cerón, JuliánMoreno Mateos, Miguel A.CRISPR-CasGenome editingZebrafishNematodesThe requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAMs, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals. Here we study the nuclease activity of SpG and SpRY by targeting 40 sites in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm could help to predict SpG and SpRY targets with high activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.Nature20262026-02-1720222022-05-1220222022-05-12journal articlehttp://purl.org/coar/resource_type/c_6501VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttps://hdl.handle.net/10433/26116reponame:RIO. Repositorio Institucional Olavideinstname:Universidad Pablo de Olavide (UPO)InglésengAgencia Estatal de Investigación http://dx.doi.org/10.13039/501100011033 Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020 PGC2018-097260-B-I00 NUEVAS APLICACIONES DEL SISTEMA CRISPR-CAS IN VIVO PARA IDENTIFICAR FACTORES INVOLUCRADOS EN EL DESARROLLO TEMPRANO DE VERTEBRADOSopen accesshttp://purl.org/coar/access_right/c_abf2Attribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:rio.upo.es:10433/261162026-06-13T12:46:27Z
dc.title.none.fl_str_mv Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes
title Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes
spellingShingle Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes
Vicencio, Jeremy
CRISPR-Cas
Genome editing
Zebrafish
Nematodes
title_short Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes
title_full Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes
title_fullStr Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes
title_full_unstemmed Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes
title_sort Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes
dc.creator.none.fl_str_mv Vicencio, Jeremy
Sánchez-Bolaños, Carlos
Moreno-Sánchez, Ismael
Brena, David
Vejnar, Charles E.
Kuthar, Dmytro
Ruiz-López, Miguel
Cots-Ponjoan, Mariona
Rubio Valle, Alejandro
Rodrigo Melero, Natalia
Crespo Cuadrado, Jesús
Carolis, Carlos
Pérez-Pulido, Antonio J.
Giraldez, Antonio J.
Kleinstiver, Benjamin P.
Cerón, Julián
Moreno Mateos, Miguel A.
author Vicencio, Jeremy
author_facet Vicencio, Jeremy
Sánchez-Bolaños, Carlos
Moreno-Sánchez, Ismael
Brena, David
Vejnar, Charles E.
Kuthar, Dmytro
Ruiz-López, Miguel
Cots-Ponjoan, Mariona
Rubio Valle, Alejandro
Rodrigo Melero, Natalia
Crespo Cuadrado, Jesús
Carolis, Carlos
Pérez-Pulido, Antonio J.
Giraldez, Antonio J.
Kleinstiver, Benjamin P.
Cerón, Julián
Moreno Mateos, Miguel A.
author_role author
author2 Sánchez-Bolaños, Carlos
Moreno-Sánchez, Ismael
Brena, David
Vejnar, Charles E.
Kuthar, Dmytro
Ruiz-López, Miguel
Cots-Ponjoan, Mariona
Rubio Valle, Alejandro
Rodrigo Melero, Natalia
Crespo Cuadrado, Jesús
Carolis, Carlos
Pérez-Pulido, Antonio J.
Giraldez, Antonio J.
Kleinstiver, Benjamin P.
Cerón, Julián
Moreno Mateos, Miguel A.
author2_role author
author
author
author
author
author
author
author
author
author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv
dc.subject.none.fl_str_mv CRISPR-Cas
Genome editing
Zebrafish
Nematodes
topic CRISPR-Cas
Genome editing
Zebrafish
Nematodes
description The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAMs, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals. Here we study the nuclease activity of SpG and SpRY by targeting 40 sites in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm could help to predict SpG and SpRY targets with high activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.
publishDate 2022
dc.date.none.fl_str_mv 2022
2022-05-12
2022
2022-05-12
2026
2026-02-17
dc.type.none.fl_str_mv journal article
http://purl.org/coar/resource_type/c_6501
VoR
http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://hdl.handle.net/10433/26116
url https://hdl.handle.net/10433/26116
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.relation.none.fl_str_mv Agencia Estatal de Investigación http://dx.doi.org/10.13039/501100011033 Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020 PGC2018-097260-B-I00 NUEVAS APLICACIONES DEL SISTEMA CRISPR-CAS IN VIVO PARA IDENTIFICAR FACTORES INVOLUCRADOS EN EL DESARROLLO TEMPRANO DE VERTEBRADOS
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Nature
publisher.none.fl_str_mv Nature
dc.source.none.fl_str_mv reponame:RIO. Repositorio Institucional Olavide
instname:Universidad Pablo de Olavide (UPO)
instname_str Universidad Pablo de Olavide (UPO)
reponame_str RIO. Repositorio Institucional Olavide
collection RIO. Repositorio Institucional Olavide
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869412609401290752
score 15,812429

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