Abstract
BackgroundThe homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells.ResultsBy introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1 kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.1 kb bovine sex-determining region Y (SRY)-green fluorescent protein (GFP) template were achieved in Bos taurus zygotes. Embryos that developed to the blastocyst stage were screened for GFP, and nine were transferred to recipient cows resulting in a live phenotypically normal bull calf. Genomic analyses revealed no wildtype sequence at the H11 target site, but rather a 26 bp insertion allele, and a complex 38 kb knock-in allele with seven copies of the SRY-GFP template and a single copy of the donor plasmid backbone. An additional minor 18 kb allele was detected that looks to be a derivative of the 38 kb allele resulting from the deletion of an inverted repeat of four copies of the SRY-GFP template.ConclusionThe allelic heterogeneity in this biallelic knock-in calf appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, NHEJ, and rearrangement following editing of the gRNA target site in the donor template.This study illustrates the potential to produce targeted gene knock-in animals by direct cytoplasmic injection of bovine embryos with gRNA/Cas9, although further optimization is required to ensure a precise single-copy gene integration event.
Highlights
The homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins
We found that the use of a homology-mediated end joining (HMEJ) repair template to target an insertion to the X chromosome increased the knock-in frequency in bovine embryos as compared to a traditional HR template [9], and that more than a third of knock-in blastocysts analyzed were non-mosaic with precise integrations [10]
200 in vitro fertilized bovine zygotes were microinjected with gRNA/Cas9 ribonucleoprotein complex and HMEJ-template at 6 h post insemination, which is prior to the initiation of zygote DNA replication at 11–15 hpi
Summary
The homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. Attempts have been made to increase the efficiency of performing targeted gene insertions utilizing the homologous recombination (HR) pathway [4], which is primarily restricted to actively dividing cells (S/G2-phase) and only becomes highly active towards the end of the first round of DNA replication [5]. These have been largely unsuccessful in bovine embryos [6], and often result in mosaic animals. Blunt end ligation of cleaved donor template by homology independent insertion was observed, more frequently in male than female embryos, but no integration of the donor plasmid backbone was ever detected [10]
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