Dramatic Changes in the Ratio of Homologous Recombination to Nonhomologous DNA-End Joining in Oocytes and Early Embryos ofXenopus laevis

Abstract

We have developed a versatile plasmid vector (pReco<r) for recombination studies. When linearized and introduced into the cells of interest, pReco-allows the simultaneous determination of the relative frequencies of homologous recombination versus nonhomologous DMA-end joining (also termed end-to-end joining), the latter an example of illegitimate recombination processes. As a system we made use of stage VI oocytes and fertilized eggs of the African clawed frog Xenopus laevis, which were previously described to support homologous recombination and DMA-end joining, respectively. Extending these earlier findings, we show that oocytes yield >80% of the homologously recombined product, whereas in eggs a highly efficient DMA-end joining activity predominates Both reactions, homologous recombination and DMA-end joining, are shown to occur quickly, with the majority of the respective products being formed within the first 20 minutes of incubation under optimal conditions. In fertilized eggs, up to 50% of all injected linear DMA molecules are recircularized by DMA-end joining. With high amounts of injected DMA per fertilized egg, DMA-end joining is reduced, presumably due to competition for essential factors, and homologous recombination becomes readily detectable.As there is a sequence of rapid cleavage divisions after fertilization of the egg, the fast and highly efficient DMA-end joining, even though it is error-prone at the junction site, seems to be best suited to cope 1 present address: Institut für Zellbiologie, Swiss Federal Institute of Technology, Zürich-Hönggerberg, CH-8093 Zürich, Switzerland with DMA double-strand breaks that might occur in the genome during early embryogenesis. On the other hand, the long-lived oocytes seem to repair DMA double-strand breaks via homologous recombination. This latter property may be exploited both in Xenopus and in other organisms to achieve homologous integration of exogenous DMA into germ cells for gene targeting.

Simian Virus 40 Strains with Novel Properties Generated by Replacing the Viral Enhancer with Synthetic Oligonucleotides

“…Even though the oligonucleotides had 5= protruding "sticky ends," these were not necessarily used by the cell. For example, in head-to-tail or head-to-head fusions of oligonucleotides where the ends did not match each other, the new enhancers were most likely assembled by a trimming and ligation process, termed nonhomologous end joining (NHEJ) (5,11,15,30,34). An extensively trimmed junction between the XbaI site of the enhancerless SV40 and an 18-bp repeat oligonucleotide is depicted in Fig.…”

High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus

“…Since Xenopus oocytes have a higher potential for homologous recombination than fertilized embryos (Hagmann et al, 1996), we next tested whether the host transfer method could be used for efficient HDR-mediated knock-in. We targeted the C-terminus of X. laevis Ctnnb1 (β-catenin), a key cytoskeletal protein and effector of the canonical Wnt pathway, because previous studies have shown that addition of epitope tags to the C-terminus do not affect the function of the resulting fusion protein ( Fig.…”

“…Since it has been reported that inhibition of DNA ligase activity enhances the rate of HDR over NHEJ in a cell type-specific and context-dependent manner (Chu et al, 2015;Hagmann et al, 1996;Maruyama et al, 2015;Song et al, 2016), we tested whether this could be used to further improve HDR-mediated knock-in efficiency in oocytes. Consistent with this hypothesis, addition of the DNA ligase inhibitor SCR-7 (5 µM) to oocyte culture medium after injection of CRISPR components into oocytes increased the rate of successful knock-in for both ctnnb1 and vangl2 from an average of 7.4% to 22% (Fig.…”

“…Here we have developed a novel method that utilizes CRISPR/Cas9 gene editing in Xenopus oocytes, which have higher HDR activity than embryos (Hagmann et al, 1996). Isolated oocytes injected with CRISPR/Cas9 reagents and recombinant DNA are cultured in vitro for several days in the presence of DNA ligase inhibitor to increase HDR activity.…”

Ku-Dependent Nonhomologous DNA End Joining in Xenopus Egg Extracts

“…Both processes were studied in vivo by microinjection of DNA as well as in vitro in extracts derived from various stages of oogenesis and early embryogenesis (12,18,26). In oocytes, homologous recombination is the prevalent mechanism for the joining of two linear DNA molecules and NHEJ is virtually undetectable.…”