The genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis

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dc.citation.issue1
dc.citation.volume22
dc.contributor.authorJivanji S
dc.contributor.authorHarland C
dc.contributor.authorCole S
dc.contributor.authorBrophy B
dc.contributor.authorGarrick D
dc.contributor.authorSnell R
dc.contributor.authorLittlejohn M
dc.contributor.authorLaible G
dc.coverage.spatialEngland
dc.date.accessioned2024-04-17T01:24:09Z
dc.date.accessioned2024-07-25T06:48:32Z
dc.date.available2021-06-17
dc.date.available2024-04-17T01:24:09Z
dc.date.available2024-07-25T06:48:32Z
dc.date.issued2021-06-17
dc.description.abstractBACKGROUND: Animal health and welfare are at the forefront of public concern and the agricultural sector is responding by prioritising the selection of welfare-relevant traits in their breeding schemes. In some cases, welfare-enhancing traits such as horn-status (i.e., polled) or diluted coat colour, which could enhance heat tolerance, may not segregate in breeds of primary interest, highlighting gene-editing tools such as the CRISPR-Cas9 technology as an approach to rapidly introduce variation into these populations. A major limitation preventing the acceptance of CRISPR-Cas9 mediated gene-editing, however, is the potential for off-target mutagenesis, which has raised concerns about the safety and ultimate applicability of this technology. Here, we present a clone-based study design that has allowed a detailed investigation of off-target and de novo mutagenesis in a cattle line bearing edits in the PMEL gene for diluted coat-colour. RESULTS: No off-target events were detected from high depth whole genome sequencing performed in precursor cell-lines and resultant calves cloned from those edited and non-edited cell lines. Long molecule sequencing at the edited site and plasmid-specific PCRs did not reveal structural variations and/or plasmid integration events in edited samples. Furthermore, an in-depth analysis of de novo mutations across the edited and non-edited cloned calves revealed that the mutation frequency and spectra were unaffected by editing status. Cells in culture, however, appeared to have a distinct mutation signature where de novo mutations were predominantly C > A mutations, and in cloned calves they were predominantly T > G mutations, deviating from the expected excess of C > T mutations. CONCLUSIONS: We found no detectable CRISPR-Cas9 associated off-target mutations in the gene-edited cells or calves derived from the gene-edited cell line. Comparison of de novo mutation in two gene-edited calves and three non-edited control calves did not reveal a higher mutation load in any one group, gene-edited or control, beyond those anticipated from spontaneous mutagenesis. Cell culture and somatic cell nuclear transfer cloning processes contributed the major source of contrast in mutational profile between samples.
dc.description.confidentialfalse
dc.edition.edition17 June 2021
dc.format.pagination457-
dc.identifier.author-urlhttps://www.ncbi.nlm.nih.gov/pubmed/34139989
dc.identifier.citationJivanji S, Harland C, Cole S, Brophy B, Garrick D, Snell R, Littlejohn M, Laible G. (2021). The genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis.. BMC Genomics. 22. 1. (pp. 457-).
dc.identifier.doi10.1186/s12864-021-07804-x
dc.identifier.eissn1471-2164
dc.identifier.elements-typejournal-article
dc.identifier.issn1471-2164
dc.identifier.numberARTN 457
dc.identifier.pii10.1186/s12864-021-07804-x
dc.identifier.urihttps://mro.massey.ac.nz/handle/10179/70928
dc.languageeng
dc.publisherBioMed Central Ltd
dc.publisher.urihttps://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-021-07804-x
dc.relation.isPartOfBMC Genomics
dc.rights(c) 2021 The Author/s
dc.rightsCC BY 4.0
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subjectAnimals
dc.subjectCRISPR-Cas Systems
dc.subjectCattle
dc.subjectClustered Regularly Interspaced Short Palindromic Repeats
dc.subjectGene Editing
dc.subjectGenome
dc.subjectMutagenesis
dc.subjectMutation
dc.titleThe genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis
dc.typeJournal article
pubs.elements-id446828
pubs.organisational-groupOther
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