Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos

Abstract The CRISPR/Cas9 genome editing tool has the potential to improve the livestock breeding industry by allowing for the introduction of desirable traits. Although an efficient and targeted tool, the CRISPR/Cas9 system can have some drawbacks, including off-target mutations and mosaicism, parti...

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Autores principales:	Sadie L. Hennig, Joseph R. Owen, Jason C. Lin, Amy E. Young, Pablo J. Ross, Alison L. Van Eenennaam, James D. Murray
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a638503bf58644bba5b9de2748db09bb
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spelling	oai:doaj.org-article:a638503bf58644bba5b9de2748db09bb2021-12-02T13:34:10ZEvaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos10.1038/s41598-020-78264-82045-2322https://doaj.org/article/a638503bf58644bba5b9de2748db09bb2020-12-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-78264-8https://doaj.org/toc/2045-2322Abstract The CRISPR/Cas9 genome editing tool has the potential to improve the livestock breeding industry by allowing for the introduction of desirable traits. Although an efficient and targeted tool, the CRISPR/Cas9 system can have some drawbacks, including off-target mutations and mosaicism, particularly when used in developing embryos. Here, we introduced genome editing reagents into single-cell bovine embryos to compare the effect of Cas9 mRNA and protein on the mutation efficiency, level of mosaicism, and evaluate potential off-target mutations utilizing next generation sequencing. We designed guide-RNAs targeting three loci (POLLED, H11, and ZFX) in the bovine genome and saw a significantly higher rate of mutation in embryos injected with Cas9 protein (84.2%) vs. Cas9 mRNA (68.5%). In addition, the level of mosaicism was higher in embryos injected with Cas9 mRNA (100%) compared to those injected with Cas9 protein (94.2%), with little to no unintended off-target mutations detected. This study demonstrated that the use of gRNA/Cas9 ribonucleoprotein complex resulted in a high editing efficiency at three different loci in bovine embryos and decreased levels of mosaicism relative to Cas9 mRNA. Additional optimization will be required to further reduce mosaicism to levels that make single-step embryo editing in cattle commercially feasible.Sadie L. HennigJoseph R. OwenJason C. LinAmy E. YoungPablo J. RossAlison L. Van EenennaamJames D. MurrayNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-9 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Sadie L. Hennig Joseph R. Owen Jason C. Lin Amy E. Young Pablo J. Ross Alison L. Van Eenennaam James D. Murray Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos
description	Abstract The CRISPR/Cas9 genome editing tool has the potential to improve the livestock breeding industry by allowing for the introduction of desirable traits. Although an efficient and targeted tool, the CRISPR/Cas9 system can have some drawbacks, including off-target mutations and mosaicism, particularly when used in developing embryos. Here, we introduced genome editing reagents into single-cell bovine embryos to compare the effect of Cas9 mRNA and protein on the mutation efficiency, level of mosaicism, and evaluate potential off-target mutations utilizing next generation sequencing. We designed guide-RNAs targeting three loci (POLLED, H11, and ZFX) in the bovine genome and saw a significantly higher rate of mutation in embryos injected with Cas9 protein (84.2%) vs. Cas9 mRNA (68.5%). In addition, the level of mosaicism was higher in embryos injected with Cas9 mRNA (100%) compared to those injected with Cas9 protein (94.2%), with little to no unintended off-target mutations detected. This study demonstrated that the use of gRNA/Cas9 ribonucleoprotein complex resulted in a high editing efficiency at three different loci in bovine embryos and decreased levels of mosaicism relative to Cas9 mRNA. Additional optimization will be required to further reduce mosaicism to levels that make single-step embryo editing in cattle commercially feasible.
format	article
author	Sadie L. Hennig Joseph R. Owen Jason C. Lin Amy E. Young Pablo J. Ross Alison L. Van Eenennaam James D. Murray
author_facet	Sadie L. Hennig Joseph R. Owen Jason C. Lin Amy E. Young Pablo J. Ross Alison L. Van Eenennaam James D. Murray
author_sort	Sadie L. Hennig
title	Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos
title_short	Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos
title_full	Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos
title_fullStr	Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos
title_full_unstemmed	Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos
title_sort	evaluation of mutation rates, mosaicism and off target mutations when injecting cas9 mrna or protein for genome editing of bovine embryos
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/a638503bf58644bba5b9de2748db09bb
work_keys_str_mv	AT sadielhennig evaluationofmutationratesmosaicismandofftargetmutationswheninjectingcas9mrnaorproteinforgenomeeditingofbovineembryos AT josephrowen evaluationofmutationratesmosaicismandofftargetmutationswheninjectingcas9mrnaorproteinforgenomeeditingofbovineembryos AT jasonclin evaluationofmutationratesmosaicismandofftargetmutationswheninjectingcas9mrnaorproteinforgenomeeditingofbovineembryos AT amyeyoung evaluationofmutationratesmosaicismandofftargetmutationswheninjectingcas9mrnaorproteinforgenomeeditingofbovineembryos AT pablojross evaluationofmutationratesmosaicismandofftargetmutationswheninjectingcas9mrnaorproteinforgenomeeditingofbovineembryos AT alisonlvaneenennaam evaluationofmutationratesmosaicismandofftargetmutationswheninjectingcas9mrnaorproteinforgenomeeditingofbovineembryos AT jamesdmurray evaluationofmutationratesmosaicismandofftargetmutationswheninjectingcas9mrnaorproteinforgenomeeditingofbovineembryos
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Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos

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