RS-1 enhances CRISPR-mediated targeted knock-in in bovine embryos

RS-1 enhances CRISPR-mediated targeted knock-in in bovine embryos

8 Pág.

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Main Authors: Lamas Toranzo, Ismael, Martínez-Moro, Álvaro, O'Callaghan, Elena, Millán de la Blanca, María Gemma, Sánchez, José María, Lonergan, P., Bermejo Álvarez, Pablo
Other Authors: Ministerio de Economía y Competitividad (España)
Format: artículo biblioteca
Language:English
Published: John Wiley & Sons 2020-05-01
Subjects:DNA repair, RS-1, Bovine, Embryo, Gene editing,
Online Access:http://hdl.handle.net/10261/340982
http://dx.doi.org/10.13039/100012818
http://dx.doi.org/10.13039/501100001602
http://dx.doi.org/10.13039/501100003329
http://dx.doi.org/10.13039/501100000780
https://api.elsevier.com/content/abstract/scopus_id/85082522173
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spelling dig-inia-es-10261-3409822024-05-16T20:36:50Z RS-1 enhances CRISPR-mediated targeted knock-in in bovine embryos Lamas Toranzo, Ismael Martínez-Moro, Álvaro O'Callaghan, Elena Millán de la Blanca, María Gemma Sánchez, José María Lonergan, P. Bermejo Álvarez, Pablo Ministerio de Economía y Competitividad (España) European Commission Science Foundation Ireland Comunidad de Madrid Lamas Toranzo, Ismael [0000-0002-7790-2649] Martínez-Moro, Álvaro [0000-0003-1981-1295] O'Callaghan, Elena [0000-0002-3055-0842] Millán de la Blanca, María Gemma [0000-0002-2810-8510] Sánchez, José María [0000-0003-3889-2342] Lonergan, P. [0000-0001-9500-057X] Bermejo Álvarez, Pablo [0000-0001-9907-2626] DNA repair RS-1 Bovine Embryo Gene editing 8 Pág. Targeted knock-in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)-assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining. The objective of this study was to explore whether CRISPR-assisted targeted KI rates can be improved in bovine embryos by exposure to the HDR enhancer RS-1. In vitro produced zygotes were injected with CRISPR components (300 ng/µl Cas9 messenger RNA and 100 ng/µl single guide RNA against a noncoding region) and a single-stranded DNA (ssDNA) repair template (100 ng/µl). ssDNA template contained a 6 bp XbaI site insert, allowing targeted KI detection by restriction analysis, flanked by 50 bp homology arms. Following microinjection, zygotes were exposed to 0, 3.75, or 7.5 µM RS-1 for 24 hr. No differences were noted between groups in terms of development or genome edition rates. However, targeted KI rates were doubled in the group exposed to 7.5 µM RS-1 compared to the others (52.8% vs. 25% and 23.1%, for 7.5, 0, and 3.75 µM, respectively). In conclusion, transient exposure to 7.5 µM RS-1 enhances targeted KI rates resulting in approximately half of the embryos containing the intended mutation, hence allowing direct KI generation in embryos. This study was supported by grants from the Spanish Ministry of Science and Competitiveness (AGL2014‐58739‐R and AGL2017‐84908‐R to PBA), the European Research Council (Starting Grant 757886‐ELONGAN to PBA) and Science Foundation Ireland (13/IA/1983 to PL). ILT was funded by an FPI scholarship from the Spanish Ministry of Science and Competitiveness (BES2015‐072774) and AMM by an industrial doctorate scholarship from Madrid Region Government (IND2017/BIO7748). Peer reviewed 2023-12-20T08:21:15Z 2023-12-20T08:21:15Z 2020-05-01 artículo http://purl.org/coar/resource_type/c_6501 Molecular Reproduction and Development 87(5): 542-549 (2020) 1040-452X http://hdl.handle.net/10261/340982 10.1002/mrd.23341 1098-2795 http://dx.doi.org/10.13039/100012818 http://dx.doi.org/10.13039/501100001602 http://dx.doi.org/10.13039/501100003329 http://dx.doi.org/10.13039/501100000780 32227559 2-s2.0-85082522173 https://api.elsevier.com/content/abstract/scopus_id/85082522173 en #PLACEHOLDER_PARENT_METADATA_VALUE# #PLACEHOLDER_PARENT_METADATA_VALUE# #PLACEHOLDER_PARENT_METADATA_VALUE# #PLACEHOLDER_PARENT_METADATA_VALUE# #PLACEHOLDER_PARENT_METADATA_VALUE# info:eu-repo/grantAgreement/MINECO//AGL2014-58739-R/ES/DESARROLLO DE LA TECNOLOGIA CRISPR PARA LA MODIFICACION GENICA EN ESPECIES DE INTERES GANADERO/ info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/AGL2017-84908-R/ES/ESTUDIO MOLECULAR DE FALLOS REPRODUCTIVOS EN ESPECIES GANADERAS MEDIANTE LA TECNOLOGIA CRISPR/ info:eu-repo/grantAgreement/EC//757886 info:eu-repo/grantAgreement/MINECO//BES-2015-072774/ES/BES-2015-072774/ S2017/BIO7748 Departamento de Reproducción Animal​  Publisher's version https://doi.org/10.1002/mrd.23341 Sí open application/pdf John Wiley & Sons
institution INIA ES
collection DSpace
country España
countrycode ES
component Bibliográfico
access En linea
databasecode dig-inia-es
tag biblioteca
region Europa del Sur
libraryname Biblioteca del INIA España
language English
topic DNA repair
RS-1
Bovine
Embryo
Gene editing
DNA repair
RS-1
Bovine
Embryo
Gene editing
spellingShingle DNA repair
RS-1
Bovine
Embryo
Gene editing
DNA repair
RS-1
Bovine
Embryo
Gene editing
Lamas Toranzo, Ismael
Martínez-Moro, Álvaro
O'Callaghan, Elena
Millán de la Blanca, María Gemma
Sánchez, José María
Lonergan, P.
Bermejo Álvarez, Pablo
RS-1 enhances CRISPR-mediated targeted knock-in in bovine embryos
description 8 Pág.
author2 Ministerio de Economía y Competitividad (España)
author_facet Ministerio de Economía y Competitividad (España)
Lamas Toranzo, Ismael
Martínez-Moro, Álvaro
O'Callaghan, Elena
Millán de la Blanca, María Gemma
Sánchez, José María
Lonergan, P.
Bermejo Álvarez, Pablo
format artículo
topic_facet DNA repair
RS-1
Bovine
Embryo
Gene editing
author Lamas Toranzo, Ismael
Martínez-Moro, Álvaro
O'Callaghan, Elena
Millán de la Blanca, María Gemma
Sánchez, José María
Lonergan, P.
Bermejo Álvarez, Pablo
author_sort Lamas Toranzo, Ismael
title RS-1 enhances CRISPR-mediated targeted knock-in in bovine embryos
title_short RS-1 enhances CRISPR-mediated targeted knock-in in bovine embryos
title_full RS-1 enhances CRISPR-mediated targeted knock-in in bovine embryos
title_fullStr RS-1 enhances CRISPR-mediated targeted knock-in in bovine embryos
title_full_unstemmed RS-1 enhances CRISPR-mediated targeted knock-in in bovine embryos
title_sort rs-1 enhances crispr-mediated targeted knock-in in bovine embryos
publisher John Wiley & Sons
publishDate 2020-05-01
url http://hdl.handle.net/10261/340982
http://dx.doi.org/10.13039/100012818
http://dx.doi.org/10.13039/501100001602
http://dx.doi.org/10.13039/501100003329
http://dx.doi.org/10.13039/501100000780
https://api.elsevier.com/content/abstract/scopus_id/85082522173
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AT bermejoalvarezpablo rs1enhancescrisprmediatedtargetedknockininbovineembryos
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