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

Autor: Jivanji S; School of Agriculture and Environment, Massey University, Palmerston North, New Zealand. swati.jivanji.1@uni.massey.ac.nz., Harland C; Livestock Improvement Corporation, Newstead, New Zealand., Cole S; AgResearch, Ruakura Research Centre, Hamilton, New Zealand., Brophy B; AgResearch, Ruakura Research Centre, Hamilton, New Zealand., Garrick D; School of Agriculture and Environment, Massey University, Palmerston North, New Zealand., Snell R; School of Biological Sciences, University of Auckland, Auckland, New Zealand., Littlejohn M; School of Agriculture and Environment, Massey University, Palmerston North, New Zealand.; Livestock Improvement Corporation, Newstead, New Zealand., Laible G; AgResearch, Ruakura Research Centre, Hamilton, New Zealand.; School of Medical Sciences, University of Auckland, Auckland, New Zealand.; Maurice Wilkins Centre for Molecular Biodiscovery, Auckland, New Zealand.
Jazyk: angličtina
Zdroj: BMC genomics [BMC Genomics] 2021 Jun 17; Vol. 22 (1), pp. 457. Date of Electronic Publication: 2021 Jun 17.
DOI: 10.1186/s12864-021-07804-x
Abstrakt: Background: 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.
Databáze: MEDLINE
Externí odkaz: