Simplified pipelines for genetic engineering of mammalian embryos by CRISPR-Cas9 electroporation†.

ABSTRACT: Gene editing technologies, such as CRISPR-Cas9, have important applications in mammalian embryos for generating novel animal models in biomedical research and lines of livestock with enhanced production traits. However, the lack of methods for efficient introduction of gene editing reagents into zygotes of various species and the need for surgical embryo transfer in mice have been technical barriers of widespread use. Here, we described methodologies that overcome these limitations for embryos of mice, cattle, and pigs. Using mutation of the Nanos2 gene as a readout, we refined electroporation parameters with preassembled sgRNA-Cas9 RNPs for zygotes of all three species without the need for zona pellucida dissolution that led to high-efficiency INDEL edits. In addition, we optimized cultur

SUBMITTER: Miao D 

PROVIDER: S-EPMC6614578 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature