RT Journal Article
SR Electronic
T1 Evaluation of Mosaicism and Off Target Mutations in CRISPR-Mediated Genome Edited Bovine Embryos
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.06.04.134759
DO 10.1101/2020.06.04.134759
A1 Sadie L. Hennig
A1 Joseph R. Owen
A1 Jason C. Lin
A1 Amy E. Young
A1 Pablo J. Ross
A1 Alison L. Van Eenennaam
A1 James D. Murray
YR 2020
UL http://biorxiv.org/content/early/2020/06/04/2020.06.04.134759.abstract
AB 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 demonstrates that the use of Cas9 protein, rather than Cas9 mRNA, results in a higher editing efficiency in bovine embryos while lowering the level of mosaicism. However, further optimization must be carried out for the CRISPR/Cas9 system to become feasible for single-step embryo editing in a commercial system.Competing Interest StatementThe authors have declared no competing interest.