|
 Curently online :
50 |
|
 Total visitors :
7668282 |
|
|
The genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis
Saturday, 2021/06/26 | 05:48:31
|
|
Swati Jivanji, Chad Harland, Sally Cole, Brigid Brophy, Dorian Garrick, Russell Snell, Mathew Littlejohn & Götz Laible BMC Genomics volume 22, Article number: 457 (2021) BackgroundAnimal 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. ResultsNo 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. ConclusionsWe 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.
See: https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-021-07804-x
Figure 3: Absence of editing plasmid-specific fragments in genomic DNA extracted from the parental cell line (BEF2), the gene-edited cell clone CC14, DNA sent away for WGS of CC14 (CC14*), and genomic DNA extracted from cloned calves B071, 1805, and 1802. Each PCR reaction contained two sets of primers and BEF2 spiked in with 0.14pg of plasmid DNA was used as the positive control. (a) Primer pair designed to amplify bovine BTA2:110,817,757 − 110,818,275 bp (519 bp), and another designed to amplify CRISPR-Cas9 expression plasmid-specific region 6,263-7,019 bp (757 bp); (b) Primer pair designed to amplify bovine BTA2:110,817,757 − 110,818,275 bp (519 bp product), and another to amplify plasmid-specific region 6,939-7,628 bp (690 bp). (a) and (b) have been cropped, and full-length gels are presented in Supplementary Figure 2. |
Back
Print
View: 188
|
|
[ Other News ]___________________________________________________
|
(121).png "The genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis")
